USE OF THE GENES IN THE HOG, Ras AND cAMP PATHWAY FOR TREATMENT OF FUNGAL INFECTION

Abstract

Provided herein are uses of genes for HOG, Ras and cAMP signal transduction pathways to treat fungal infection. To regulate the HOG pathway of Cryptococcus neoformans, roles of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 genes were investigated to find that a biosynthesis level of ergosterol is increased when these genes are inhibited. When the genes are inhibited, a large amount of ergosterol is distributed on a fungal cell membrane. Accordingly, since there are many working points of an ergosterol-binding antifungal agent, an efficiency of the ergosterol-binding antifungal agent can be considerably improved. To regulate the Ras and cAMP pathways of Cryptococcus neoformans, roles of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 genes were investigated to find that a sensitivity to a polyene- or azole-based drug is increased when these genes are inhibited. Therefore, an antifungal pharmaceutical composition including an inhibitor against the gene or protein encoded by the same can be used as an excellent combined antifungal agent which can reduce a conventional amount of an antifungal agent used and increase an efficiency.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of Korean Patent Applications No. 10-2009-0001947, filed on Jan. 9, 2009, and No. 10-2009-0127206, filed on Dec. 18, 2009, and all the benefits accruing therefrom under 35 U.S.C. .sctn.119 which is hereby incorporated by reference as if fully set forth herein.

BACKGROUND

[0002] 1. Field

[0003] The present invention relates to uses of genes for HOG, Ras and cAMP signal transduction pathways to treat fungal infection.

[0004] 2. Description of the Related Art

[0005] The existence and proliferation of an organism in a specific environment is usually determined by an ability to react and adapt to various environmental stresses and maintain cell homeosis. Cells regulate a key process by performing a series of combined signal networks. Among these, a p38/Hog1 mitogen-activated protein kinase (MAPK) dependent signal pathway plays an important role to regulate a wide range of stress reactions in eukaryotes, for example, from yeasts to humans.

[0006] A stress-activated p38 MAPK in a mammal induces various stress-related signals limiting change in osmosis, UV radiation, programmed apoptosis, and adaptation to an immune response by generation of cytokine and control of inflammation. Similar stress-sensitive signal transduction systems have been discovered in other species. Fungi have p38-like MAPKs regulating various stress-related responses. In the budding yeast, Saccharomyces cerevisiae (S. cerevisiae), HogI MAPK regulates a stress-related response to osmotic shock, oxidative damage and heavy metal damage. The fission yeast, Schizosaccharomyces pombe (S. pombe), also has a homolog of HogI, Sty1 (also known as Spc1 and Phh1), which is associated with adaptation to various stresses including osmotic shock, heat shock, oxidative damage and heavy metal damage, carbon deficiency and UV radiation. Interestingly, Sty1 is also associated with growth control, reproduction and differentiation. Hog1 MAPK orthologs are also found in other ascomycete pathogenic fungi including Candida albicans (Hog1) and Aspergillus fumigatus (SakA), and known to mediate reactions induced by various environmental causes including osmotic shock, UV radiation, oxidative damage and high temperature.

[0007] A common molecular mechanism of the p38/HogI MAPK signal transduction network is highly conserved in many eukaryotic cells. While the p38/Hog1 MAPK is non-phosphorylated under normal growth conditions, it is activated by double phosphorylation of Thr and Tyr residues at a TGY motif using a MAPK kinase (MAPKK) activated through phosphorylation by a MAPKK kinase (MAPKKK) in a higher signal system in response to a specific environmental stress. Subsequently, the phosphorylated p38/Hog1 MAPKs are transferred to a nucleus after a dimer is formed to trigger activation of a transcription regulatory factor and induce overproduction of stress-preventing genes resistant to external stress conditions.

[0008] In spite of the conserved regulatory mechanism of the p38/Hog1 MAPK, fungi and mammals have developed a distinctive set of a higher regulatory systems. Particularly, fungi use a two-component-like phosphorelay system, which is not present in mammals, but found only in bacteria, fungi and plants. The fungal phosphorelay system is composed of three components including a hybrid sensor kinase, histidine-containing phosphotransfer protein (HPt), and a response regulator. The three components have not been observed in mammals, and thus are considered a good target for an antifungal agent.

[0009] Basidiomycetous, Cryptococcus neoformans (C. neoformans), also uses a stress-activated Hog1 MAPK system to adapt to various environmental stresses including osmotic shock, UV radiation, heat shock, oxidative damage, toxic metabolites and antifungal agents. C. neoformans is a human pathogenic fungus found everywhere in the world, causing cryptococcal disease in the skin and lungs and cryptococcal encephalomeningitis in immunocompromised patients. While C. neoformans var. grubii (antigen-type A) is the most frequently found (>90% of environmental and clinical strains), C. neoformans var. neoformans (antigen-type D) is common only in a specific region in Europe, but not frequently found (<10%). However, it has been confirmed that C. gattii, known as C. neoformans of antigen-types B and C, are primary pathogens attacking normal people who have no immune problems.

[0010] However, it is inferred that, compared with other fungal Hog1 MAPK systems, the Hog1 MAPK pathway in C. neoformans is not characteristically developed only to correspond to various environmental stresses, but also to regulate production of two pathogenic factors such as an antiphagocytic capsule and an antioxidant melanine and sexual differentiation, and thus plays a critical role as an important signal transduction regulator in C. neoformans cross-talking to another signal transduction pathway. Recently, the inventors found that most Hog1 MAPKs in many C. neoformans strains are always phosphorylated under non-stress conditions, and rapidly dephosphorylated to activate the Hog1 MAPKs in response to the osmotic shock and treatment of an antifungal agent, fluodioxonyl, which clearly contrasts with Hog1 MAPK systems in other fungi. Double phosphorylation at the TGY motif of Hog1 needs Pbs2 MAPKK. A fungus-specific phosphorelay system which is in a higher level of a Pbs2-Hog1 pathway is also found only in C. neoformans. The C. neoformans phosphorelay system includes 7 different sensor hybrid histidine kinases (TcoI-7), a Ypd1 phosphotransfer protein, and two reaction regulators (Ssk1 and Skn7). The Pbs2-Hog1 pathway is generally regulated by Ssk1, not by Skn7. Among the 7 Tco proteins, Tco1 and Tco2 play distinctive and overlapping roles to activate the Ssk1 and the Pbs2-Hog1 MAPK pathway. However, the Tco1 and Tco2 regulate some Ssk1 and Hog1-related phenotypes, and therefore other higher receptor or sensor proteins should be discovered. More recently, a protein, Ssk2 MAPKKK, serving as a linker between the phosphorelay system and the Pbs2-Hog1 MAPK pathway was identified by comparative analysis of a meiotic map between antigen-type D f1 brother strains, B3510 and B3502, showing different phosphorylation patterns of Hog1. The most noticeable fact is that interchange of Ssk2 alleles between two C. neoformans strains showing different Hog1 phosphorylation patterns changes a phenotype controlled by constitutive Hog1 phosphorylation. Unlike S. cerevisiae and S. pombe, C. neoformans has single MAPKKK and Ssk2 regulating the Hog1 MAPK. While a downstream signal transduction network of the Hog1 MAPK pathway in C. neoformans has yet to be discovered, identification and characterization of the downstream signal transduction network of the Hog1 MAPK are needed to develop a target for a new antifungal agent.

[0011] In the past, fungal infections were mainly local infections such as athlete's foot, jock itch, or oral thrush, and rarely systemic infections. However, recently, systemic infections have become as frequent, coming in fourth in frequency among total infections occurring in hospitals.

[0012] The antifungal agents which have been developed so far may be classified into two major groups: those having an azole structure and those not having an azole structure. The azole-based antifungal agents include ketoconazole, fluconazole, itraconazole and voriconazole, while the non-azole-based antifungal agents include terbinafine, flucytosine, amphotericin B and caspofungin.

[0013] The ketoconazole, fluconazole, itraconazole and voriconazole having an azole structure have similar mechanisms to allylamine-based naftifine and terbinafine. These two different antifungal agents serve to inhibit enzymes required for the conversion of lanosterol into ergosterol, which is a main component of a fungal cell membrane. The azole-based antifungal agents inhibit a microsomal enzyme, and the acrylamine-based antifungal agents inhibit a squalene epoxidase, both having a similar effect to the above-mentioned antifungal agents. Flucytocin (5-FC) is a metabolic antagonist inhibiting the synthesis of a nucleic acid, which has an antifungal reaction by non-competitively antagonizing the cause of miscoding a fungal RNA and DNA synthesis. Amphotericin B having a polyene structure has an antifungal reaction by binding to ergosterol in the fungal cell membrane to induce depolarization of the cell membrane and generating a hole to induce loss of the cell contents. An echinocandin-based antifungal agent, caspofungin, has a reaction reversibly inhibiting the formation of a fungal cell wall, and is different from those acting on the cell membrane described above.

[0014] The azole-based drug may lead to death caused by infection when being used on a patient having hypofunction of the liver, and thus a liver function test should precede administration. It is reported that flutocytosin has a dose-dependent bone marrow inhibiting action, liver toxicity, and can cause enterocolitis. Since such side effects are increased when renal insufficiency occurs, monitoring of a renal function is very important to a patient. In addition, flutocytosin is contraindicated for pregnant woman. A major toxicity of amphotericin B is a glomerulus renal toxicity induced by renal artery vasoconstriction, which is dose dependent. Therefore, when a lifetime cumulative dose is 4 to 5 g or more, a rate of permanent loss of the renal function is increased. Furthermore, the renal toxicities such as excessive loss of potassium, magnesium and bicarbonate due to toxicity of a renal tube and low production of erithropoietins may be generated. Moreover, as acute responses, symptoms such as thrombophlebitis, chills, shivering, and hyperpnea may be shown. Since the conventionally developed antifungal agents show various side effects according to kinds of drugs, development of a new therapy which can reduce such side effects and increase an antifungal effect is demanded.

[0015] Meanwhile, in pathogenic fungi distributed in the world, including Aspergillus fumigatus, Candida albicans (C. albicans) and C. neoformans, Ras- and cAMP-signal transduction pathways are evolutionarily conserved, and significantly functional and structural differences are still being found (Pukkila-Worley & Alspaugh, 2004, Rolland et al., 2002, Wong & Heitman, 1999, Thevelein & de Winde, 1999, Alspaugh et al., 1998, Lengeler et al., 2000, and Bahn et al, 2007). In C. neoformans causing fatal fungal encephalomeningitis, the cAMP-signal transduction pathway is important in producing and differentiating pathogenic factors (Idnurm et al., 2005). Like S. cerevisiae and C. albicans, it was confirmed that two major higher signal transduction regulators of adenylyl cyclase (Cac1), adenylyl cyclase-associated protein 1 (Aca1) and G.alpha. subunit protein (Gpa1) regulate a cAMP-signal transduction pathway of C. neoformans (Bahn et al., 2004 and Alspaugh et al., 1997). The disruption of GPA1 genes leads to multiple phenotypes of cells, which include incomplete production of core pathogenic factors, melanin and a capsule, essential for survival and proliferation of C. neoformans in a host, and a decrease in mating, which is important in distribution of infectious spores (Alspaugh et al., 1997). Aca1 physically interacts with a Cac1 adenylyl cyclase, and does not regulate a basic level of cAMP but dominates most cAMP-dependent phenotypes by regulating the induction of cAMP (Bahn et al., 2004). A deletion mutant of CAC1 produces a phenotype more defected than a deletion mutant of gpal.DELTA. or acal.DELTA., and gpal.DELTA. acal.DELTA. double deletion mutants are equivalent to the cac1.DELTA. deletion mutant in phenotype (Bahn et al., 2004). This indicates that Cal1 is activated by both of Aca1 and Gpa1. In a lower signal system of the Cac1 of C. neoformans, two catalytic subunits of a protein kinase A (PKA), Pka1 and Pka2, and a regulatory subunit, Pkr1, are included. While Pka1 plays a dominant role for cAMP signal transduction in a background of an antigen-type A C. neoformans H99 strain, Pka2 also plays the same role in an antigen-type D C. neoformans JEC21 strain (Hicks et al., 2004). Nevertheless, a pka1.DELTA.-pka2.DELTA. double deletion mutant shows a phenotype the same as the cac1.DELTA. deletion mutant, and the cAMP signal transduction from Cac1 is split into two PKA catalytic subunits (Bahn et al., 2004). Interestingly, the deletion of PDE1, not PDE2, repairs some phenotypes including the depletion of a melanin of the gpal.DELTA. deletion mutant, which indicates that different phosphodiesterases act in various fungi (Hicks et al., 2005).

[0016] It is revealed that two Ras proteins, Ras1 and Ras2, are found in Cryptococcus, and play common and distinctive roles (Alspaugh et al., 2000, D'Souza et al., 2001, and Waugh et al., 2002). Among these proteins, Ras1 is a major C. neoformans Ras protein supporting high-temperature growth and invasive growth essential for survival and growth in a host and stimulating sexual differentiation (Alspaugh et al., 2000). Though the ras2.DELTA. deletion mutant does not have a recognizable phenotype, the overexpression of RAS2 somewhat inhibits most of the ras1 mutation phenotypes (Waugh et al., 2002). Like S. cerevisiae, disruption of the RAS1 and RAS2 genes affects cell viability at every temperature, which indicates that the Ras protein is essential for the growth of cells in general. Among various Ras-related phenotypes, only invasive growth and mating are cAMP-dependent, but high-temperature growth is cAMP-independent and a Ras1-specific phenotype (Alspaugh et al., 2000, Waugh et al., 2003). Interestingly, Cac1 does not bear a Leucine-rich repeat (LRR) domain, which is a binding site to a GTP-binding Ras in S. cerevisiae (Shima et al., 1997). Since an adenylyl cyclase/cyclase-related protein complex can provide a secondary Ras-binding site to activate the protein complex as shown in S. cerevisiae (Shima et al., 2000), Ras1 can still interact with an Aca1/Cac1 complex for activating the Ras1 in C. neoformans. Recently, it has been reported that a GEF protein, Cdc24, is a Ras-effecter protein, and regulates the growth of C. neoformans at high temperature in a lower system of Ras1 and a higher system of Rho-like GTPase Cdc42 (Nichols et al., 2007). Consequently, C. neoformans cAMP-signal transduction pathway is regulated by three different higher signal regulators, Ras1, Gpa1 and Aca1.

[0017] Despite the presence of the common higher signal regulators (Ras1, Aca1 and Gpa1) of Cac1, functional correlation between the components and target gene regulated by each regulator in C. neoformans remains still unclear.

SUMMARY

[0018] The present invention provides to finding a new target gene to develop an antifungal agent by investigating a signal transduction network of HOG, Ras and cAMP pathways.

[0019] In one aspect, a use of an inhibitor against at least one protein or a gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.

[0020] In another aspect, a use of at least one protein or a gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein or gene, and a method of screening an antifungal agent including contacting the protein or gene with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein or gene are provided.

[0021] In still another aspect, a use of an inhibitor against at least one protein or a gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.

[0022] In yet another aspect, a use of at least one protein or a gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein or gene, and a method of screening an antifungal agent including contacting the protein or gene with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein or gene are provided.

[0023] In the present invention, to regulate a HOG pathway of C. neoformans, roles of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 genes are investigated to reveal that a biosynthesis level of ergosterol is increased when these genes are inhibited. Since a large amount of ergosterol is distributed on a fungal cell membrane when the genes are inhibited, an efficiency of an ergosterol-binding antifungal agent can be considerably increased due to many working points of the ergosterol-binding antifungal agents. In addition, in the present invention, to regulate Ras and cAMP pathways of C. neoformans, roles of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 genes were investigated to reveal that sensitivity to a polyene- or azole-based drug is increased when these genes are inhibited. Thus, an antifungal pharmaceutical composition including an inhibitor against the gene or protein encoded by the same can be used as an excellent combined antibacterial drug which can reduce an amount of a conventional antifungal agent used and increase efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other aspects of the inventive concept will become more readily apparent by describing in further detail exemplary embodiments thereof which reference to the accompanying drawings, in which:

[0025] FIG. 1 shows identification of genes whose expression is regulated by Hog, Ssk1 and Skn7 of C. neoformans under normal conditions with no stress on the genome level (fold change is expressed by color);

[0026] FIG. 2 shows analysis results for induction of ergosterol biosynthesis genes by disturbance of a HOG signal transduction pathway and an ergosterol content in a cell;

[0027] FIG. 3 shows analysis results showing that the inhibition of the HOG pathway gives an elevated antifungal effect with amphotericin B in C. neoformans;

[0028] FIG. 4 shows analysis results showing that the inhibition of the HOG pathway gives an antagonistic antifungal effect with respect to some azole drugs in C. neoformans;

[0029] FIG. 5 shows analysis results showing that gene coding for an efflux pump of Na.sup.+ and K.sup.+, ENA1 and NHA1, are lower-system target genes regulated by the HOG pathway, and the inhibition of these genes gives high sensitivity to polyene-based drugs and azole-based drugs;

[0030] FIG. 6 shows analysis results for transcripts of ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka2.DELTA. deletion mutants of C. neoformans (fold change is expressed by color);

[0031] FIG. 7 shows functional categories of genes differently regulated by the ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka2.DELTA. deletion mutants of C. neoformans;

[0032] FIG. 8 shows regulation of a significant ratio of Ras- and cAMP-dependent genes by environmental stress;

[0033] FIG. 9 shows an identification result of a cAMP-signal transduction pathway dependent gene in C. neoformans;

[0034] FIG. 10 shows analysis results showing that the inhibition of the Ras- and cAMP-signal transduction pathways increases a sensitivity to polyene-based or azole-based (itraconazole) antifungal agent, independent of ergosterol biosynthesis; and

[0035] FIG. 11 shows analysis results showing that the expression of HSP12 and HSP122 is up-regulated by the cAMP- and HOG-signal transduction pathways, and increases sensitivity to polyene-based antifungal agents by hsp12.DELTA. and hsp122.DELTA. deletion mutants.

DETAILED DESCRIPTION

[0036] Hereinafter, the present invention will be described in detail.

[0037] An antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent is provided.

[0038] A HOG1 pathway is a cell signal transduction system regulating responses induced by various stresses. Particularly, since fungi use a two-element-like phosphorelay system composed of three elements such as a hybrid sensor kinase, a histidine-containing phosphotransfer protein (HPt) and a response regulator, which are not present in mammals, the inventors carried on an investigation of roles of genes involved in the HOG1 pathway to develop a target for a new antifungal agent. As a result, surprisingly, it was found that, in the regulation of the HOG pathway in C. neoformans, a biosynthesis level of ergosterol is increased when SSK1, TCO2, SSK2, PBS2, and HOG1 genes are inhibited. As will be confirmed in the following embodiments, when the genes are inhibited, a large amount of ergosterol is distributed on a fungal cell membrane and working points of the ergosterol-binding antifungal agent are also increased. Therefore, an efficiency of the ergosterol-binding antifungal agent can be considerably increased. In addition, when ENA1 and NHA1 genes, the expression of which is known to be regulated by the HOG signal transduction pathway, are inhibited, it is confirmed that, regardless of the change in ergosterol level, a sensitivity to polyene-based drugs such as amphotericin B and azole-based drugs are considerably increased. Thus, the antifungal pharmaceutical composition including an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans may be used as an excellent combined antibacterial drug which can reduce an amount of the conventional ergosterol-binding antifungal agent or azole-based antifungal agent used and increase efficiency.

[0039] Accordingly, a use of an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor to a subject are provided.

[0040] In the specification, it is understood that SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 used as a target to interrupt a HOG1 signal transduction system indicates an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein, or an SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene. Thus, it is understood that an SSK1, TCO2, SSK2, PBS2, HOG 1, ENA1 or NHA1 inhibitor includes either an inhibitor against an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein or an inhibitor against an SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene.

[0041] In one exemplary embodiment, the inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans may bind to the protein to inhibit an activity thereof, thereby interrupting signal transduction. In another exemplary embodiment, the inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans may inhibit expression of the gene to interrupt signal transduction. In the specification, the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene may be a DNA coding for the gene or mRNA transcripted therefrom. Thus, the inhibitor against the gene may bind to the gene itself to disturb transcription or bind to the mRNA transcripted from the gene to disturb translation of the mRNA.

[0042] In one exemplary embodiment, the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein may have an amino acid sequence of SEQ ID NOs: 1-7 respectively, and the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene may have a nucleic acid sequence of SEQ ID NOs: 8-14 respectively or a cDNA sequence of SEQ ID NOs:15-21 respectively. However, this is merely an example of a sequence of C. neoformans antigen-type A H99 strain, and the sequence of the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 is not limited thereto.

[0043] In the specification, it is understood that the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein or the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene includes a variant or fragment thereof having substantially the same activity as the protein or gene.

[0044] In one exemplary embodiment, the antifungal pharmaceutical composition may include at least one inhibitor against at least one protein selected from the group consisting of Ssk1, Ena1 and Nha1. SSK1 may be a good target to develop an antifungal agent because it is not only an important upstream reaction regulator of HOG1, but also a gene which is not found in mammals. Therefore, the SSK1 inhibitor may reduce a possibility of generating certain side effects and increase a biosynthesis level of ergosterol in a fungus, thereby improving the efficiency of an ergosterol-binding antifungal agent. Meanwhile, ENA1 and NHA1 are defined as lower-system target genes regulated by a HOG pathway. When these genes are inhibited, sensitivity to an azole-based drug such as fluconazole, ketoconazole and itraconazole is also considerably increased as well as that to a polyene-based drug such as amphotericin B. Therefore, the inhibitors simultaneously or independently inhibiting these genes may exhibit very high antifungal activities when used in combination with the polyene- or azole-based drug.

[0045] The inhibition of the Ssk1, Tco2, Ssk2, Pbs2 or Hog1 protein or gene improves the biosynthesis of ergosterol and increases the distribution of ergosterol on a fungal cell membrane. Thus, since binding targets of the ergosterol-binding antifungal agent disrupting the fungal cell membrane by being bound to ergosterol are increased, an effective amount of the ergosterol-binding antifungal agent may be reduced and a killing ability of the ergosterol-binding antifungal agent may be increased. In addition, the inhibitors against ENA1 and NHA1 considerably increase drug sensitivities to the azole-based antifungal agent as well as the polyene-based antifungal agent, and thus amounts of these drugs used can be reduced and a killing ability may be improved. Such an antifungal activity induced by the inhibition of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 may treat cryptococcal disease and encephalomeningitis by infection of C. neoformans.

[0046] Thus, in one exemplary embodiment, use of an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor to prepare a drug for treating a disease such as cryptococcal disease or encephalomeningitis, a pharmaceutical composition for treating a disease such as cryptococcal disease or encephalomeningitis including the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor, and a method of treating a disease such as cryptococcal disease or encephalomeningitis including injecting an effective amount of the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor to a subject are provided.

[0047] Other than cryptococcal disease or encephalomeningitis exemplified in the specification, diseases induced by fungal infection are well known in the art. In the specification, the inhibition of the Ssk1, Tco2, Ssk2, Pbs2, Hog1 protein or gene is revealed to improve the efficiency of the ergosterol-binding antifungal agent or azole-based antifungal agent, and thus those of ordinary skill in the art may inhibit the protein or gene to prevent or treat a disease induced by fungal infection.

[0048] In the specification, the "inhibitor of the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein" used to interrupt a HOG1 signal transduction system includes all inhibitors binding to the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein to interrupt signal transduction. For example, such an inhibitor may be a peptide or compound binding to the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein. Such an inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a known method in the art. In one exemplary embodiment, the inhibitor may be a polyclonal or monoclonal antibody with respect to at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 of C. neoformans. Such a polyclonal or monoclonal antibody may be prepared using a method of preparing an antibody known in the art.

[0049] In the present invention, the "inhibitor against the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene" used to interrupt a HOG1 signal transduction system includes every inhibitor inhibiting the expression of the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene to interrupt signal transduction. For example, such an inhibitor may be a peptide, nucleic acid or compound binding to the gene. The inhibitor may be selected by a screening method shown below in cell-based screening, and may be designed using a known method in the art. In one exemplary embodiment, the inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA or vector including the same with respect to at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans. The antisense oligonucleotide, siRNA, shRNA, miRNA or vector including the same may be prepared using a known method in the art. In the specification, the "vector" refers to a gene construct including foreign DNA inserted into a genome coding for a polypeptide. The vector used herein is a vector in which a nucleic acid sequence inhibiting the gene is inserted into a genome, and may include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.

[0050] A pharmaceutical antifungal pharmaceutical composition including an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans does not exhibit an antifungal activity alone, but increases a fungal killing ability of an antifungal agent in combination with the ergosterol-binding antifungal agent or azole-based antifungal agent. Therefore, the antifungal pharmaceutical composition may be sequentially or simultaneously injected with the ergosterol-binding antifungal agent or azole-based antifungal agent.

[0051] The ergosterol-binding antifungal agent refers to an antifungal agent binding to ergosterol on a fungal cell membrane to induce depolarization of the cell membrane and forming a hole to induce the loss of contents in a cell, thereby killing fungi. Such an ergosterol-binding antifungal agent is known in the art, and any ergosterol-binding antifungal agent considerably increases the antifungal effect when used with the antifungal pharmaceutical composition. In one exemplary embodiment, the ergosterol-binding antifungal agent may be a polyene-based antifungal agent. In one aspect, the polyene-based antifungal agent may be at least one antifungal agent selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin. In the preferable embodiment, the polyene-based antifungal agent is amphotericin B. Meanwhile, the azole-based antifungal agent may be at least one antifungal agent selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

[0052] In this aspect, an antifungal combined formulation including the antifungal pharmaceutical composition including the inhibitor of the present invention; and a known ergosterol-binding antifungal agent or azole-based antifungal agent are provided.

[0053] An antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent is also provided.

[0054] The inventors performed investigation on, rather than simply the genes involved in Ras and cAMP pathways, the roles of the genes involved in Ras and cAMP pathways, to develop a target for a new antifungal agent. The result newly revealed that, surprisingly, in the Ras and cAMP pathways of C. neoformans, when a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene is inhibited, a sensitivity to one of the polyene- or azole-based drugs, an itraconazole antifungal agent, is increased. As will be confirmed in the following exemplary embodiment, when the genes are inhibited, the sensitivity to the polyene- or itraconazole antifungal agent in a fungus may be considerably increased. Thus, the antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans may be used as an excellent combined antibacterial drug which can reduce an amount of a conventional polyene-based or itraconazole antifungal agent used and improve an efficiency.

[0055] Therefore, use of an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.

[0056] In the present invention, it is construed that RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 used as a target to interrupt the Ras- and cAMP signal transduction systems is a Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein, or a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene. Accordingly, it is construed that a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 inhibitor includes every inhibitor against the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or inhibitor against the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene.

[0057] In one exemplary embodiment, an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans may be an inhibitor that binds to the protein to inhibit an activity, thereby interrupting signal transduction. In another exemplary embodiment, an inhibitor against at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans may be an inhibitor inhibiting expression of the gene, thereby interrupting signal transduction. In the present invention, the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene may be DNA coding for the gene or mRNA transcripted therefrom. Therefore, the inhibitor against the gene may bind to the gene to interrupt transcription or bind to mRNA transcripted from the gene to interrupt translation of the mRNA.

[0058] In one exemplary embodiment of the present invention, the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein may have an amino acid sequence of one of SEQ ID NOs: 16-24 respectively, and the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene may have a nucleic acid or cDNA sequence corresponding to the protein. However, the sequence just shows a sequence of a C. neoformans antigen-type A H99 strain, and thus the sequence of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 is not limited thereto.

[0059] In the present invention, it is construed that the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein, or the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene includes a variant or fragment thereof having substantially the same activity as the protein or gene.

[0060] In one exemplary embodiment, the inhibitor may be an inhibitor against a Cac1 or Pka1 protein or gene.

[0061] Inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or gene coding for the same may increase a sensitivity to a polyene- or azole-based antifungal agent, and thus an effective amount of the polyene- or azole-based antifungal agent may be reduced and a killing ability of the antifungal agent may be increased. An antifungal activity caused by the inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 may treat cryptococcal disease and encephalomeningitis induced by infection of C. neoformans.

[0062] Thus, in one exemplary embodiment of the present invention, use of a Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor to prepare a drug for treating diseases such as cryptococcal disease and encephalomeningitis, a pharmaceutical composition for treating diseases such as cryptococcal disease and encephalomeningitis including the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor and a method of treating diseases such as cryptococcal disease and encephalomeningitis including injecting an effective amount of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor into a subject are provided.

[0063] Other than the cryptococcal disease and encephalomeningitis stated herein, diseases induced by fungal infection are well known in the art. In the present invention, it is revealed that the inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or gene coding for the same increases the sensitivity to a polyene- or azole-based itraconazole antifungal agent, thereby increasing the efficiency of the antifungal agent. Therefore, those of ordinary skill in the art can inhibit the protein or genes to prevent or treat a disease induced by the fungal infection.

[0064] In the present invention, the "inhibitor of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein" used to interrupt the RAS or cAMP signal transduction system includes every inhibitor binding to the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein to interrupt signal transduction. For example, such an inhibitor may be a peptide or compound binding to the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein. The inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a method known in the art. In one exemplary embodiment, the inhibitor may be a polyclonal or monoclonal antibody with respect to at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans. The polyclonal or monoclonal antibody may be constructed using a known method of constructing an antibody in the art.

[0065] In the present invention, the "inhibitor of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene" used to interrupt the RAS or cAMP signal transduction system includes every inhibitor inhibiting the expression of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene to interrupt signal transduction. For example, such an inhibitor may be a peptide, nucleic acid or compound binding to the gene. The inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a method known in the art. In one exemplary embodiment, the inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector including the same with respect to at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans. Such an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector including the same may be constructed using a known method in the art. In the present invention, the "vector" is a gene construct including foreign DNA inserted into a genome coding for a polypeptide. The vector related to the present invention may be a vector formed by inserting a nucleic acid sequence inhibiting the gene into the genome, which may be a DNA vector, plasmid vector, cosmid vector, bacteriophage vector, yeast vector or viral vector.

[0066] The antifungal pharmaceutical composition of the present invention including the inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1 , Hsp12 and Hsp122 of C. neoformans increases a fungal killing ability of the antifungal agent in combination with a polyene- or azole-based antifungal agent. Thus, the antifungal pharmaceutical composition of the present invention is sequentially or simultaneously injected with the polyene- or azole-based antifungal agent. The polyene- or azole-based antifungal agent is known in the art, and any one of the polyene- or azole-based antifungal agent significantly increases the antifungal effect when used with the antifungal pharmaceutical composition of the present invention. In one aspect, the polyene-based antifungal agent may be at least one of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin. In a preferable embodiment, the polyene-based antifungal agent may be amphotericin B. In one aspect, the azole-based antifungal agent may be at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

[0067] In another exemplary embodiment, the antifungal pharmaceutical composition may be sequentially or simultaneously injected along with the inhibitor against at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans. As will be confirmed from the following exemplary embodiment, when expression of HOG1 is inhibited as well as expression of CAC1 or PKA1 of C. neoformans, the sensitivity to amphotericin B is proportionally increased. This is because genes of the cAMP pathway increase the sensitivity to amphotericin B by a mechanism different from that increasing the sensitivity to amphotericin B due to the increase in biosynthesis of ergosterol when the genes of the HOG pathway described above are inhibited.

[0068] According to the aspect, the present invention also provides an antifungal combined formulation including the antifungal pharmaceutical composition including the inhibitor of the present invention; and at least one antifungal agent selected from the group consisting of a polyene-based antifungal agent, an azole-based antifungal agent and an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans. Preferably, the antifungal combined formulation may include the antifungal pharmaceutical composition including the inhibitor of the present invention, a polyene-based antifungal agent, and at least one antifungal agent selected from the group consisting of inhibitors each against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans.

[0069] The antifungal pharmaceutical composition or antifungal combined formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically available adjuvant, wherein the adjuvant may be a solubilizer such as a diluting agent, a dispersing agent, a sweetening agent, a binding agent, a coating agent, a blowing agent, a lubricant, a gliding agent or a flavoring agent.

[0070] The antifungal pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition including at least one pharmaceutically available carrier other than an active component for administration.

[0071] In the composition formulated in a liquid-phase solution, a pharmaceutically available carrier may be suitable for sterilization and living organisms, and may be saline, sterilized water, Ringer's solution, buffered saline, albumine injection, dextrose solution, maltodextrin solution, glycerol, ethanol or a mixture of at least one thereof. When necessary, another conventional additive such as an antioxidant, buffer or bacteriostatic agent may be added. In addition, a diluting agent, a dispersing agent, a surfactant, a binding agent or a lubricant may be added, and thus the composition may be formulated in the form of an injectable formulation such as an aqueous solution, a suspension or an emulsion, a pill, a capsule, a granule or a tablet. Furthermore, the composition may be formulated using a suitable method in the art, which is disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. according to diseases or components.

[0072] Types of a pharmaceutical formulation of the pharmaceutical composition of the present invention may include a granule, an acida, a coated tablet, a tablet, a capsule, a suppository, a syrup, a juice, a suspension, an emulsion, a drop or injectable liquid and a sustained-release formulation of an active compound.

[0073] The pharmaceutical composition of the present invention may be injected by a conventional method via an intravenous, intraarterial, abdominal, sternal, percutaneous, nasal, inhaling, local, rectal, oral, intraocular or intradermal route.

[0074] An effective amount of the active component of the pharmaceutical composition of the present invention indicates an amount required for preventing or treating a disease, or achieving an effect of inducing bone growth. Accordingly, the effective amount may vary depending on various factors such as kinds of a disease, severity of a disease, kinds and contents of the active component and other components contained in the composition, kinds of dosage forms and patient's age, weight, health, sex and dietary habits, injection times and routes, release rates of the composition, duration of treatment, and co-injected drugs. For adults, when the composition is injected one or more times a day, the dosages may be, but not limited to, 0.1 ng/kg to 10 g/kg for a compound, 0.1 ng/kg to 10 g/kg for a polypeptide, protein or antibody, and 0.01 ng/kg to 10 g/kg for an antisense oligonucleotide, siRNA, shRNAi or miRNA, respectively.

[0075] In the present invention, the "subject" may be, but not limited to, a human, orangutan, chimpanzee, mouse, rat, dog, cow, chicken, pig, goat or sheep.

[0076] Furthermore, the present invention provides a use of at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, and Nha1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein, and a method of screening an antifungal agent including contacting the protein with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein.

[0077] The present invention also provides a use of at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the gene, and a method of screening an antifungal agent including contacting the gene with a candidate material and determining whether the candidate material inhibits or stimulates expression of the gene.

[0078] The present invention also provides a method of screening an antifungal agent by a yeast two-hybrid system capable of monitoring physical contact between SSK1 and SSK2, SSK1 and YPD1 or YPD1 and TCO2 proteins of C. neoformans. When this method is used, a large amount of the candidate materials can be screened quickly.

[0079] As described above, when SSK1, TCO2, SSK2, PBS2 or HOG1 of C. neoformans is inhibited, the HOG1 signal transduction system is interrupted, and thus biosynthesis of ergosterol is improved. Therefore, the material screened to inhibit the protein or gene may be used as an antifungal agent improving a fungal killing ability, along with an ergosterol-binding antifungal agent. The material screened to inhibit ENA1 or NHA1 may be used as an antifungal agent improving a fungal killing ability when used with an ergosterol-binding antifungal agent or azole-based antifungal agent.

[0080] The present invention also provides a use of at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1 , Pka1, Hsp12 and Hsp122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein, and a method of screening an antifungal agent including contacting the protein with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein.

[0081] The present invention also provides a use of at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the gene, and a method of screening an antifungal agent including contacting the gene with a candidate material and determining whether the candidate material inhibits or stimulates expression of the gene.

[0082] The present invention also provides a method of screening an antifungal agent by a yeast two-hybrid system capable of monitoring physical contact between Gpa1 and Cac1 , Cac1 and Aca1, Ras1 and Cdc24 or Ras2 and Cdc24 proteins of C. neoformans. When this method is used, a large amount of the candidate materials can be screened quickly.

[0083] As described above, when RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 of C. neoformans is inhibited, the RAS or cAMP signal transduction system is interrupted, thereby increasing a sensitivity to a polyene- or azole-based antifungal agent. Thus, the material screened to inhibit the protein or gene may be used as an antifungal agent improving a fungal killing ability when used with the polyene- or azole-based antifungal agent.

[0084] Confirmation of the reaction between the protein or gene and the candidate material may be performed by a conventional method of confirming the reaction between a protein and a protein, a protein and a compound, DNA and DNA, DNA and RNA, DNA and a protein, DNA and a compound, RNA and a protein, or RNA and a compound. For example, a hybrid test for confirming a bond between the gene and a candidate material in vitro, a method of measuring an expression level of the gene through northern blotting, quantitative PCR or quantitative real time PCR after reaction of mammalian cell and a test material, a method of connecting a reporter gene to the gene to introduce the gene into a cell, reacting the cell with a test material and measuring an expression level of a reporter protein, a method of reacting the protein with a candidate material and measuring an activity, a yeast two-hybrid, searching for a phage-displayed peptide clone binding to an Idbf protein, high throughput screening (HTS) using a natural substance and a chemical library, drug hit HTS, cell-based screening or a screening method using a DNA array may be used.

[0085] The screening composition may include distilled water or a buffer stably maintaining the structure of a nucleic acid or protein, other than the protein or gene. In addition, the screening composition may include a cell expressing the protein or gene, or a cell containing a plasmid expressing the gene in the presence of a promoter regulating a transcription rate for an in vivo test.

[0086] In the screening method of the present invention, a test material may be individually a nucleic acid, a protein, a peptide, a different extract or natural substance or a compound assumed to have possibility as a drug inhibiting signal transduction through a HOG1 signal transduction system according to a conventional screening method or randomly selected.

[0087] The matters related to a genetic engineering technique in the present invention are made more clear by the literatures disclosed by Sambrook et al. [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (2001)] and Frederick M. Ausubel et al. [Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)].

[0088] While exemplary embodiments have been disclosed herein, it should be understood that other variations may be possible. Such variations are not to be regarded as a departure from the spirit and scope of exemplary embodiments of the present application, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

EXAMPLES

Experimental Procedures

Strains and Growth Conditions

[0089] The C. neoformans strains used in this examples are listed in Table 1[Bahn Y S, Geunes-Boyer S, Heitman J (2007) Eukaryot Cell 6: 2278-2289.; Bahn Y S, Kojima K, Cox G M, Heitman J (2005) Mol Biol Cell 16: 2285-2300.; Bahn Y S, Kojima K, Cox G M, Heitman J (2006) Mol Biol Cell 17: 3122-3135.; Perfect J R, Ketabchi N, Cox G M, Ingram C W, Beiser C L (1993) J Clin Microbiol 31: 3305-3309; Kwon-Chung K J, Edman J C, Wickes B L (1992) Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 60: 602-605.].

[0090] The C. neoformans strains were cultured in YPD (yeast extract-peptone-dextrose) medium unless indicated separately.

TABLE-US-00001 TABLE 1 Strain Genotype Parent Serotype A H99 MAT.alpha. KN99 MATa CBN45 MAT.alpha. ras1.DELTA.::NEO H99 CBN64 MAT.alpha. ras1.DELTA.::NEO RAS1::NAT CBN45 MWC12 MAT.alpha. ras2.DELTA.::URA5 H99 CBN32 MAT.alpha. cdc24.DELTA.::NEO H99 CBN33 MAT.alpha. cdc24.DELTA.::NEO CDC24::NAT CBN32 YSB6 MAT.alpha. aca1.DELTA.::NAT-STM#43 H99 YSB51 MAT.alpha. ras1.DELTA.::NAT-STM#150 H99 YSB53 MAT.alpha. ras1.DELTA.::NAT-STM#150 H99 YSB64 MAT.alpha. hog1.DELTA.::NAT-STM#177 H99 YSB123 MAT.alpha. pbs2.DELTA.::NAT-STM#213 H99 YSB261 MAT.alpha. ssk1.DELTA.::NAT-STM#205 H99 YSB264 MAT.alpha. ssk2.DELTA.::NAT-STM#210 H99 YSB349 MAT.alpha. skn7.DELTA.::NAT-STM#201 H99 YSB278 MAT.alpha. tco1.DELTA.::NAT-STM#102 H99 YSB281 MAT.alpha. tco2.DELTA.::NAT-STM#116 H99 YSB324 MAT.alpha. tco1.DELTA.::NAT-STM#102 tco2D::NEO YSB278 YSB284 MAT.alpha. tco3.DELTA.::NAT-STM#119 H99 YSB417 MAT.alpha. tco4.DELTA.::NAT-STM#123 H99 YSB286 MAT.alpha. tco5.DELTA.::NAT-STM#125 H99 YSB348 MAT.alpha. tco7.DELTA.::NAT-STM#209 H99 YSB73 MAT.alpha. ras1.DELTA.::NEO H99 YSB42 MAT.alpha. cac1.DELTA.::NAT-STM#159 H99 YSB83 MAT.alpha. gpa1.DELTA.::NAT H99 YSB188 MAT.alpha. pka1.DELTA.::NAT H99 YSB194 MAT.alpha. pka2.DELTA.::NAT-STM#205 H99 YSB200 MAT.alpha. pka1.DELTA.::NAT pka2.DELTA.::NEO YSB188 YSB174 MAT.alpha. aca1.DELTA.::NAT-STM#43 ras1::NEO YSB278 YSB182 MAT.alpha. cac1.DELTA.::NAT-STM#159 ras1::NEO H99 YSB156 MAT.alpha. hog1.DELTA.::NAT-STM#177 cac1::NEO H99 YSB112 MAT.alpha. ura5 pka1::URA5 hog1::NATSTM#177 H99 YSB58 MATa aca1.DELTA.::NEO KN99 YSB79 MATa cac1.DELTA.::NEO KN99 YSB81 MATa hog1.DELTA.::NEO KN99 YSB175 MAT.alpha. aca1.DELTA.::NEO ras1.DELTA.::NATSTM#150 YSB58 YSB187 MAT.alpha. cac1.DELTA.::NEO ras1.DELTA.::NATSTM#150 YSB79 YSB606 MAT.alpha. gre2.DELTA.::NAT-STM#224 H99 YSB607 MAT.alpha. gre2.DELTA.::NAT-STM#224 H99 YSB609 MAT.alpha. pkp1.DELTA.::NAT-STM#224 H99 YSB610 MAT.alpha. pkp1.DELTA.::NAT-STM#224 H99 YSB599 MAT.alpha. hsp12.DELTA.::NAT-STM#224 H99 YSB600 MAT.alpha. hsp12.DELTA.::NAT-STM#224 H99 YSB603 MAT.alpha. hsp122.DELTA.::NAT-STM#224 H99 YSB604 MAT.alpha. hsp122.DELTA.::NAT-STM#224 H99 YSB590 MAT.alpha. ena1.DELTA.::NAT nha1::NEO AI167 YSB591 MAT.alpha. ena1.DELTA.::NAT nha1::NEO AI167 YSB586 MAT.alpha. nha1.DELTA.::NEO H99 YSB587 MAT.alpha. nha1.DELTA.::NEO H99 YSB588 MAT.alpha. nha1.DELTA.::NEO H99 Serotype D JEC21 MAT.alpha. B-3501 MAT.alpha. YSB267 MAT.alpha. pbs2.DELTA.::NAT-STM#213 JEC21 YSB139 MAT.alpha. hog1.DELTA.::NAT-STM#177 JEC21 YSB338 MAT.alpha. ssk2.DELTA.::NAT-STM#210 JEC21 YSB340 MAT.alpha. ssk2.DELTA.::NAT-STM#210 B-3501 Each NAT-STM# indicates the Nat.sup.r marker with a unique signature tag.

DNA Microarray Array Analysis

[0091] For total RNA isolation used in DNA microarray, the wild-type H99, hog1.DELTA. (YSB64), ssk1.DELTA. (YSB261), and skn7.DELTA. (YSB349), ras1.DELTA. (YSB51), aca1.DELTA. (YSB6), gpa1.DELTA. (YSB83), cac1.DELTA. (YSB42) and pka1.DELTA. pka2.DELTA. (YSB200) mutant strains were grown in 50 ml YPD medium at 30.degree. C. for 16 hr. Then 5 ml of the overnight culture was inoculated into a 100 ml of fresh YPD medium and further incubated for 4-5 hr at 30.degree. C. until it approximately reaches to the 1.0 of optical density (OD) at 600 nm (OD600 nm=1.0). For zero-time samples, 50 ml out of the 100 ml culture was sampled and rapidly frozen in liquid nitrogen. To the remaining 50 ml culture, 50 ml of YPD containing 2 M NaCl, 40 .mu.g/ml fludioxonil (PESTANAL, Sigma, 100 mg/ml stock solution in dimethylsulfoxide), or 5 mM H2O2 was added (final concentration of 1 M NaCl, 20 .mu.g/ml fludioxonil, or 2.5 mM H2O2, respectively). During incubation in each stress-inducing medium, 50 ml of the culture was sampled at 30 and 60 min, pelleted in a tabletop centrifuge, frozen in liquid nitrogen, and lyophilized overnight. The lyophilized cells were subsequently used for total RNA isolation. As biological replicates for DNA microarray, 3 independent cultures for each strain and growth condition were prepared for total RNA isolation.

Total RNA Preparation

[0092] For total RNA isolation, the lyophilized cell pellets were added with 3 ml volume of sterile 3 mm glass bead (SIGMUND LINDER), homogenized by shaking, added with 4 ml of TRizol reagent (Tri reagent, Molecular Research Center), and allowed to incubate at room temperature for 5 min. Then 800 .mu.l of chloroform was added, incubated for 3 min at room temperature, transferred to the 15 ml of the round-bottom tube (SPL), and centrifuged by 10,000 rpm at 4.degree. C. for 15 min (Sorvall SS-34 rotor). Two milliliter of the supernatant was transferred to the new round-bottom tube, added with 2 ml isopropanol, inverted several times, and allowed to incubate for 10 min at room temperature. Then the mixture was re-centrifuged by 10,000 rpm at 4.degree. C. for 10 min, and its pellet was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC) treated water and centrifuged by 8,000 rpm at 4.degree. C. for 5 min. The pellet was dried at room temperature and resuspended with 500 .mu.l DEPC-treated water. Concentration and purity of total RNA sample were calculated by measuring OD260 nm and gel electrophoresis, respectively. For control total RNA (for Cy3 labeling), all of total RNAs prepared from wild-type, hog1.DELTA., ssk1.DELTA., skn7.DELTA., ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA. and pka1.DELTA. pka2.DELTA. mutant cells grown in conditions described above were pooled (pooled reference RNAs).

cDNA Synthesis and Cy3/Cy5 Labeling

[0093] For cDNA synthesis, total RNA concentration was adjusted to 1 .mu.g/.mu.l with DEPC-treated water, and 15 .mu.l of the total RNA (15 .mu.g) was added with 1 .mu.l of 5 .mu.g/.mu.l oligo dT (5'-TTTTTTTTTTTTTTTTTTTTV-3')/pdN6 (Amersham)(1:1 mixture of 10 .mu.g/.mu.l, respectively), incubated at 70.degree. C. for 10 min, and place on ice for 10 min. Then 15 .mu.l of cDNA synthesis mixture {3 .mu.l 0.1 M DTT, 0.5 .mu.l RNasin [Promega], 0.6 .mu.l aa-dUTP (5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate)/dNTPs [a mixture of 6 .mu.l dTTP (100 mM), 4 .mu.l aa-dUTP (100 mM), 10 .mu.l dATP (100 mM), 10 .mu.l dCTP (100 mM), 10 .mu.l dGTP (100 mM)], 1.5 .mu.l AffinityScript reverse transcriptase (Stratagene), 3 .mu.l AffinityScript buffer, 7 .mu.l water] was added and incubated at 42.degree. C. for 2 hrs. Then 10 .mu.l of 1 N NaOH and 10 .mu.l of 0.5 M EDTA (pH 8.0) were added and incubated at 65.degree. C. for 15 min. After incubation, 25 .mu.l of 1 M HEPES buffer (pH 8.0) and 450 .mu.l of DEPE-treated water were added, and the whole mixture was concentrated through Microcon30 filter (Milipore) and vacuum-dried for 1 hr.

[0094] For Cy3 and Cy5 (Amersham) labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 .mu.l DMSO and 1.25 .mu.l of each dye was aliquoted into separate tubes. The cDNAs prepared as described above were added with 9 .mu.l of 0.05 M Na-bicarbonate (pH 8.0) and incubate at room temperature for 15 min. The cDNAs prepared from pooled reference RNAs were mixed with Cy3 as a control and the cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy5. Each mixture was further incubated at room temperature for 1 hr in the dark and purified by QIAquick PCR purification kit (QIAGEN).

Microarray Hybridization and Washing

[0095] C. neoformans serotype D 70-mer microarray slide containing 7,936 spots (Duke University) was pre-hybridized at 42.degree. C. in 60 ml of pre-hybridization buffer [42.4 ml sterile distilled water, 2 ml 30% BSA (Sigma), 600 .mu.l 10% SDS, 15 ml 20.times.SSC], washed with distilled water and isopropanol, and dried by brief centrifugation (110.times.g, 2 min). The Cy3- and Cy5-labeled cDNA samples were combined, concentrated through Microcon30 filter, and vaccum-dried. The dried cDNA samples were resuspended with 24 .mu.l of 1.times. hybridization buffer [250 .mu.l 50% formamide, 125 .mu.l 20.times.SSC, 5 .mu.l 10% SDS, 120 .mu.l dH2O, total 500 .mu.l], added with 1 .mu.l polyA tail DNA (Sigma), further incubated at 100.degree. C. for 3 min and allowed to cool for 5 min at room temperature. The microarray slides were aligned into the hybridization chamber (DieTech), removed of any dusts, and covered by Lifterslips (Erie Scientific). The Cy3/Cy5-labeled cDNA samples were applied in between Lifterslips and slides. To prevent slides from being dried, 10 .mu.l of 3.times. SSC buffer was applied onto the slides, which were subsequently incubated for 16 hr at 42.degree. C. After incubation, the microarray slides were washed with three different washing buffers [wash buffer 1 (10 ml 20.times.SSC, 600 .mu.l 10% SDS, 189.4 ml dH2O, preheated at 42.degree. C.), wash buffer 2 (3.5 ml 20.times.SSC, 346.5 ml dH2O), wash buffer 3 (0.88 ml 20.times.SSC, 349.12 ml dH2O)] for 2, 5, and 5 min, respectively, on the orbital shaker.

[0096] For each total RNA sample, 3 independent DNA microarray with 3 independent biological replicates were performed, including one-dye swap experiment.

Microarray Slide Scanning and Data Analysis

[0097] After hybridization and washing, the microarray slides were scanned by GenePix 4000B scanner (Axon Instrument) and the signals were analyzed with GenePix Pro (Ver. 4.0) and gal file (http://genome.wustl.edu/activity/ma/cneoformans). Since we used total RNAs isolated from serotype A C. neoformans strains, 70-mer oligonucleotide sequence printed on the serotype D C. neoformans slides was queried against serotype A C. neoformans genome database by blastp search (e-value cut-off: e-4) to find the corresponding serotype A gene ID. Using the serotype A gene sequence, each S. cerevisiae gene name or ID listed in the Tables was identified by blastp search (e-value cut-off: e-4).

[0098] For further hierarchical and statistical analysis, data transported from GenePix software were analyzed with GeneSpring (Agilent) by employing LOWESS normalization, reliable gene filtering, clustering (standard correlation and average linkage) and zero-transformation, and ANOVA analysis (P<0.01).

Ergosterol Assay

[0099] Ergosterol contents were measured as previously described in "Arthington-Skaggs B A, Jradi H, Desai T, Morrison C J (1999) Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J Clin Microbial 37: 3332-3337", but with slight modification. Briefly, each C. neoformans strain was grown in 100 ml YPD medium for 24 h at 30.degree. C. The 100 ml culture was splitted into two 50 ml cultures for duplicate measurement, pelleted in a tabletop centrifuge, and washed with sterile water. The cell pellet was frozen in liquid nitrogen and lyophilized overnight. The dried cell pellet was weighed for normalization of ergosterol contents, added with 5 ml of 25% alcoholic potassium hydroxide, and transferred to a sterile borosilicated glass screw-cap tube. Subsequently, the cells were incubated at 80.degree. C. for 1 h and allowed to cool to room temperature. Then 1 ml of sterile water and 3 ml of heptane were added and vortexed for 3 min. Then 200 .mu.l of the heptane layer is sampled and mixed with 800 .mu.l of 100% ethanol, and its optical density (OD) was measured at both 281.5 nm and 230 nm. Ergosterol contents were calculated as the following: % ergosterol=[(OD281.5 nm/290).times.F]/pellet weight-[(OD230 nm/518).times.F]/pellet weight, where F is the ethanol dilution factor and 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24(28)dehydroergosterol, respectively.

Stress Sensitivity Test

[0100] Each strain was incubated overnight at 30.degree. C. in YPD medium, washed, serially diluted (1 to 10.sup.4 dilutions) in dH.sub.2O, and spotted (3 .mu.l) onto solid YPD medium containing indicated concentrations of stress-inducing agents or antifungal drugs as previously described in "Bahn Y S, Kojima K, Cox G M, Heitman J (2005) Mol Biol Cell 16: 2285-2300." and "Bahn Y S, Kojima K, Cox G M, Heitman J (2006) Mol Biol Cell 17: 3122-3135". To examine antifungal drug sensitivity, the cells were spotted on agar-solid YPD media containing amphotericin B(Sigma), fluconazole(Sigma), itraconazole(Sigma), ketoconazole (Sigma) and fludioxonil. Then spotted cells were incubated at 30.degree. C. for 2-4 days and photographed.

Disruption of CAMP-Signaling Dependent Genes

[0101] For gene disruption, information of genomic DNA structure (exon and intron) for each gene was obtained from serotype A C. neoformans genome database (http://www.broadinstitute.org/annotation/genome/cryptococcus_ne- oformans/MultiHome.ht ml). The GRE2 (CNAG.sub.--02182.2), HSP12 (CNAG.sub.--03143.2), HSP122 (CNAG.sub.--01446.2) and PKP1 (CNAG.sub.--00396.2) genes were deleted by overlap PCR or double joint PCR PCR) with split markers and biolistic transformation in the C. neoformans serotype A H99 strain as previously described (Bahn et al., 2005, Davidson et al., 2002). Primers for generation of the 5' and 3' flanking regions of each gene and dominant selectable nourseothricin resistant marker (NAT, nourseothricin acteryltransferase) were described in the supplemental table 1. Gold microcarriers beads (0.8.about.1.2 .mu.m [Bioworld Inc] or 0.6 -.mu.m [BioRad]) were coated with gel-extracted deletion cassettes produced by overlap PCR and biolistically transformed into the strain H99. Stable transformants selected on YPD medium containing nourseothricin or G418 were subject to the first screening by diagnostic PCR with primers listed in Table 2. Positive mutants were further confirmed by Southern blot analysis using gene-specific probes prepared by primers listed in Table 2.

TABLE-US-00002 TABLE 2 Primer Name Sequence Description B79 TGTGGATGTCTGGCGGAGGATA Screening primer on ACT promtre B1026 GTAAAACGACGGCCAGTGAGC M13 forward (extended) B1027 CAGGAAACAGCTATGACCATG M13 reverse (extended) B1614 TGTTTAGCACCAGCGGAGTC HSP12-5' screening primer B1615 CACGATGAAAGTGCGTTGAAG HSP12-left flanking primer 1 B1616 GCTCACTGGCCGTCGTTTTACACTGTCGGTGAAAGATTGC HSP12-left flanking primer 2 B1617 CATGGTCATAGCTGTTTCCTGAGAACGACAACCAGGAGTC HSP12-right flanking primer 1 B1618 GCTCTGTGCTGACATTATCTGC HSP12-right flanking primer 2 B1707 GAAAGTGCGTTGAAGTGATG HSP12-probe primer 1 B1708 AGTAGAAGCAGCGGACTAAAG HPS12-probe primer 2 B1619 GCGTAGTGGAGATTGGTTTC GRE2-5' screening primer B1620 ATCCCCTCCACTTTACCTCC GRE2-left flanking primer 1 B1621 GCTCACTGGCCGTCGTTTTACAAGTCTCCCTTAGCGATAG GRE2-left flanking primer 2 B1622 CATGGTCATAGCTGTTTCCTGACCACACCCCTGAAGAAAC GRE2-right flanking primer 1 B1623 AACTGTTTCGTCTTGTGTGTC GRE2-right flanking primer 2 B1705 ATAGCAACTTCTTCCGTCG GRE2-probe primer 1 B1706 TGTTGCCTGTGCTCACTTG GRE2-probe primer 2 B1629 CCTCTGACAGCCACATACTG PKP1-5' screening primer B1630 AATGAAGTTCCTGCGACAG PKP1-left flanking primer 1 B1631 GCTCACTGGCCGTCGTTTTACAATGGGATGAGAACGCAC PKP1-left flanking primer 2 B1632 CATGGTCATAGCTGTTTCCTGAGCATTTTCCAGCATCAGC PKP1-right flanking primer 1 B1633 GGTGTGGAACATCTTTTGAG PKP1-right flanking primer 2 B1711 CTGGTTCATCTTGGGTGTC PKP1-probe primer 1 B1712 TCTGAGCATACCACTCCTTTAC PKP1-probe primer 2 B1666 TCTCATTCGCATCCTCTG HSP122-5' screening primer B1667 GTTGGGCAGATAATGTTTGTG HPS122-left flanking primer 1 B1668 GCTCACTGGCCGTCGTTTTACACGGCGTCAGACATTGTG HSP122-left flanking primer 2 B1669 CATGGTCATAGCTGTTTCCTGACAAGAGAAGTCCACTACTCAG HPS122-right flanking primer 1 B1670 GCAAGGTAATGATGAGCG HSP122-right flanking primer 2 B1709 GCGACTGAGATGTAGACCAAC HSP122-probe primer 1 B1710 CTCGGAACGACATAATAAGC HSP122-probe primer 2 B1673 CACACCTGGTAAGAGATAGCG NHA1-left flanking primer 1 B1674 GCTCACTGGCCGTCGTTTTACAGTGGTAGAAGTAGGGCAGC NHA1-left flanking primer 2 B1675 CATGGTCATAGCTGTTTCCTGACAGGGTCCAACAAGGATG NHA1-right flanking primer 1 B1676 TGCTACGATTGTGGTCAGCC NHA1-right flanking primer 2 B1677 GGACGAGACGAGTTATCAAAC NHA1-screening primer B1698 CTTCATCAACTTGCGTGC NHA1-probe primer

Example 1

DNA Microarray Analysis of C. neoformans hog1.DELTA., ssk1.DELTA., and skn7.DELTA. Mutants

[0102] To investigate Hog1 signaling pathway in C. neoformans, we performed comparative transcriptome analysis of serotype A wild-type (WT, H99) strain, hog1.DELTA., ssk1.DELTA., and skn7.DELTA. mutants under both normal growth conditions and stressed conditions, such as in the presence of osmotic shock (1 M NaCl), oxidative stress (2.5 mM H2O2), and antifungal drug fludioxonil (40 .mu.g/ml), by using DNA microarray analysis. We isolated total RNAs from cells growing in each stress condition after zero (non-stress condition), 30, and 60 min incubation. We prepared 3 independent RNA samples for each condition as biological replicates for DNA microarray analysis. As a control RNA for common Cy3 labeling, we used reference RNAs that were pooled from all RNA samples prepared in this study. We used 70-mer serotype D C. neoformans DNA microarray chips containing total 7,936 spots, based on information from the C. neoformans genome database.

[0103] For basic validation of our array quality, we monitored expression levels of HOG1, SSK1, and SKN7 genes, and known Hog1-regulated genes, such as GPP1 (Glycerol-3-phosphatase) and GPD1 (Glycerol-3-phosphate dehydrogenase), in our array data.

[0104] FIG. 1 shows identification of genes whose expression is regulated by Hog, Ssk1 and Skn7 of C. neoformans under normal conditions with no stress on the genome level (fold change is expressed by color). FIG. 1A shows relative expression levels of HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to WT strain. FIG. 1B shows condition tree analysis result in WT, hog1.DELTA., ssk1.DELTA., skn7.DELTA. mutant. FIG. 1C shows clustering analysis result of 950 genes which are exhibited significantly different expression patterns in hog1.DELTA., ssk1.DELTA., or skn7.DELTA. mutants compared to WT (ANOVA test, P<0.01) under normal growth condition(YDP, 30.quadrature.). FIG. 1D shows Venn diagram presenting HOG1, SSK1, and SKN7-dependent genes that include genes up- or down-regulated over 2 folds.

[0105] As expected, relative expression levels of HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to WT strain were very low(FIG. 1A). In addition, expression of GPD1 (glycerol-3-phosphate dehydrogenase, CNAG.sub.--01745) and GPP1 (DL-glycerol-3-phosphatase, CNAG.sub.--01744) homologous genes, which are well-known Hog1-regulated stress defense genes in other fungi, was highly reduced (4.5-fold and 2.5-fold reductions, respectively) in hog1.DELTA. and ssk1.DELTA. mutants, further supporting the quality of our array data.

[0106] We monitored how HOG1, SSK1, and SKN7 mutations affect gene expression patterns in C. neoformans under unperturbed normal conditions. Among 7,936 spots monitored, 3,858 spots were found to be reliable based on Cross-gene error model (cutoff 10). Supporting the previous finding, the transcription profile of the hog1.DELTA. mutant was considerably similar to that of the ssk1.DELTA. mutant, based on the condition tree analysis (FIG. 1B). A total of 950 genes in the reliable genes exhibited significantly different expression patterns in hog1.DELTA., ssk1.DELTA., or skn7.DELTA. mutants compared to WT (ANOVA test, P<0.01) (FIG. 1C), indicating that about 15% of the whole C. neoformans genes could be transcriptionally affected by perturbation of the two-component system and HOG signaling pathways even under unstressed, normal conditions. Among them, 559 genes exhibited more than 2-fold induction in at least one of the mutants (FIG. 1D). Several key findings were made as the following. First, a majority of the genes (555 genes, 99%) were up- or down-regulated by either Ssk1 or Hog1 under unstressed conditions while only 51 genes (9%) were regulated by Skn7. Among the Skn7-dependent genes, only 4 genes were found to be Skn7-specific (FIG. 1D). Thus it appears to be clear that HOG1 and SSK1 mutations alter genome-wide transcription profiles under normal conditions in a greater scale than the SKN7 mutation (FIG. 1D). Second, there exist significantly higher overlaps between Ssk1- and Hog1-dependent genes (422 out of 555 genes, 76%) than between Skn7- and Hog1-dependent genes (45 out of 467 genes, 10%), further corroborating that Ssk1 is the major upstream regulator of the Hog1 MAPK. Third, regardless of the significant overlap in genes regulated by Ssk1 and Hog1, there were a number of Ssk1-specific (90 genes) and Hog1-specific (40 genes) genes, strongly suggesting that Ssk1 and Hog1 are not strictly in the linear pathway and could have other target(s) or upstream regulators, respectively (FIG. 1D). This explains why the ssk1.DELTA. mutant exhibits slightly different phenotypes (i.e. higher sensitivity to hydrogen peroxide) compared to hog1.DELTA. mutants and Hog1 can still be phosphorylated in the absence of Ssk1 response regulator under exposure to NaCl.

[0107] Genes regulated by Hog1 and Ssk1 cover a wide variety of functional categories, including energy production and conversion, amino acid/carbohydrate/lipid transport and metabolism, translational and protein biosynthesis, post-translational modification, signal transduction, stress-defense mechanisms, and others (Supplementary table 2), indicating that active remodeling of various aspects of cellular functions could occur simply by perturbation of the HOG pathway even without external stresses. Furthermore it should be noticed that more than one third of Hog1 and Ssk1-dependent genes do not have any functional orthologs in other organisms, indicating that C. neoformans appears to develop many cryptococcus-specific Hog1/Ssk1-dependent genes.

[0108] Among Ssk1- and Hog1-regulated genes identified by our array analysis, several groups of genes provided novel insights into the potential mechanism of the HOG pathway in controlling virulence factor and sexual reproduction of C. neoformans. First, a group of genes involved in iron transport and regulation were found to be highly induced in the ssk1.DELTA. and hog1.DELTA. mutants compared to the wild-type strain. These genes include SIT1 (CNAG.sub.--00815 and CNAG.sub.--07138) encoding siderophore-transporters, CFO1 (CNAG.sub.--06241) and CFO2 (CNAG.sub.--02958) encoding ferroxidases, and CFT1 (CNAG.sub.--06242) encoding Fe transporter. The C. neoformans Sit1 are homologous to the S. cerevisiae Arn3/Sit1 having high affinity for the hydroxamate siderophore ferrioxamine and C. neoformans Cfo1/Cfo2 and Cft1 are homologous to high-affinity iron permease/multicopper ferroxidase complex (Ftr1-Fet3) in S. cerevisiae. Since iron transport regulation and melanin synthesis seem to be closely related in C. neoformans, increased melanin synthesis observed in both hog1.DELTA. and ssk1.DELTA. mutants could be correlated with increased expression of a group of genes involved in iron transport.

[0109] Second, the GPA2 gene (CNAG.sub.--00179), encoding a G-protein .alpha.-subunit in the pheromone responsive MAPK pathway, is dramatically upregulated upon ssk1.DELTA. or hog1.DELTA. mutation (12.1- and 13.3-fold increases, respectively). This finding suggests that increased pheromone production and sexual reproduction found in ssk1.DELTA. and hog1.DELTA. mutants may result from enhanced expression of Gpa2 that is induced during mating and promotes the mating process of C. neoformans.

[0110] Third, several genes involved in oxidative stress defense were differentially regulated by HOG1 and SSK1 mutation. As expected from the previous finding that the hog1.DELTA. and ssk1.DELTA. mutants exhibit hypersensitivity to hydrogen peroxide, two genes (CNAG.sub.--04981 and CNAG.sub.--00575), which are homologous to the CTA1 gene encoding catalase A that detoxifies H2O2 to H2O, was drastically downregulated in both mutants (Supplementary Table 1). Furthermore, basal expression levels of the SOD2 gene (mitochondrial superoxide dismutase) were decreased in both hog1.DELTA. and ssk1.DELTA. mutants, further corroborating the role of the HOG pathway in oxidative stress response. Interestingly, however, basal expression levels of some genes involved in oxidative stress response [TRR1 (thioredoxin reductase), TSA1 (thioredoxin peroxidase), GRX5 (glutathione-dependent oxidoreductase), CCP1 (mitochondrial cytochrome-c peroxidase)] were more than 2-fold increased (3.8, 3.1, 2.1, and 9.5 fold changes, respectively) in the hog1.DELTA. mutant, but not in the ssk1.DELTA. mutant (Supplementary Table 2). The SRX1 gene (sulfiredoxin) also involved in oxidative stress response was more reduced in the ssk1.DELTA. mutant (4.2-fold reduction) than the hog1.DELTA. mutants (1.3-fold reductions). These results may explain why the hog1.DELTA. mutants are relatively more resistant to H2O2 than the ssk1.DELTA. mutant.

Example 2

Ergosterol Biosynthesis Genes are Transcriptionally Upregulated by Perturbation of the HOG Signaling Pathway

[0111] Among genes upregulated by mutation of HOG1 and SSK1 genes, a gene homologous to ERG28 (CNAG.sub.--07208) was noticeable since it plays a key role in the fungal sterol biosynthesis. Previous microarray analysis performed in S. cerevisiae revealed that expression of ERG28 is tightly correlated with other ergosterol biosynthetic genes. Erg28 is an endoplasmic reticulum (ER) transmembrane scaffold, protein, which is essential for the yeast sterol biosynthesis by interacting strongly with Erg27, Erg25, Erg11, and Erg6 and weakly with Erg26 and Erg1. This finding led us to monitor expression patterns of other sterol biosynthetic genes in our array data.

[0112] FIG. 2A shows the relative expression profiles of ergosterol biosynthesis genes in hog1.DELTA., ssk1.DELTA., and skn7.DELTA. mutants compared to WT strain. The fold change is illustrated by a color (see color bar scale) and exact value for each gene was indicated in the table placed right side of the hierarchical clustering diagram. FIG. 2B shows cellular ergosterol contents in WT (H99), skn7.DELTA. (YSB349), ssk1.DELTA. (YSB261), ssk2A (YSB264), and hog1.DELTA. (YSB64) mutants. Left and right graphs demonstrate % ergosterol in each strain and relative increase of ergosterol contents compared to WT, respectively. Each bar presents the average from four independent experiments and error bar indicates the standard deviation. Asterisks (*): The ssk1.DELTA., ssk2.DELTA., pbs2.DELTA., and hog1.DELTA. mutants contain significantly higher ergosterol levels compared to the WT (P<0.05, as analyzed by using the Bonferroni multiple comparison test).

[0113] Interestingly, a majority of the ergosterol biosynthetic genes were upregulated in hog1.DELTA. and ssk1.DELTA. mutants, but not in the skn7.DELTA. mutant, compared to the wild-type strain (FIG. 2A). Genes, such as ERG11, ERG6, MVD1, ERG5, ERG25, ERG20, and ERG4, were upregulated in both ssk1.DELTA. and hog1.DELTA. mutants while genes, such as ERG27, ERG13, ERG26, ERG10, IDI1, HMG1, and ERG8, were upregulated only in the ssk1.DELTA. mutant (FIG. 2A). In contrast, none of genes were significantly upregulated in the skn7 mutant and indeed some of genes, including ERG13, ERG1, ERG3, ERG7, and ERG2 genes, were downregulated in the skn7.DELTA. mutant (FIG. 2A).

[0114] To verify our microarray data, we examined whether increased expression levels of some of the ergosterol biosynthesis genes indeed affect cellular ergosterol contents in the hog1.DELTA. and ssk1.DELTA. mutants (FIG. 2B). In accordance with our microarray data, cellular ergosterol contents were much higher in the hog1.DELTA. and ssk1.DELTA. mutants than WT and skn7.DELTA. mutants (FIG. 2B), suggesting that increased expression of some of ergosterol biosynthetic genes leads to enhanced production of cellular ergosterol. The ssk2.DELTA. (MAPKKK) and pbs2.DELTA. (MAPKK) mutants in the HOG pathway were also found to contain significantly higher levels of cellular ergosterol than WT and skn7.DELTA. mutants (FIG. 2B), further corroborating our array data.

[0115] This finding prompted us to investigate the susceptibility of the mutants in the two-component system and the HOG pathway to antifungal drugs that are targeted to the ergosterol biosysnthetic genes or ergosterol itself. First we have examined the susceptibility of the ssk1.DELTA., skn7.DELTA., ssk2.DELTA., pbs2.DELTA., and hog1.DELTA. mutants made in the serotype A H99 strain background to the polyen antifungal drug, amphotericin B, which binds to ergosterol in the fungal cell membrane and ultimately causes lethality by disrupting the membrane integrity. We hypothesized that increased ergosterol contents observed in ssk1.DELTA., ssk2.DELTA., pbs2.DELTA., and hog1.DELTA. mutants could render them to be hypersensitive to amphotericin B due to the increased number of drug targets.

[0116] FIG. 3 shows analysis results showing that the inhibition of the HOG pathway confers synergistic antifungal effects with amphotericin B in C. neoformans. FIG. 3A-3B show pictures photographed after incubation at 30.degree. C. for 72 h of each C. neoformans strain spotted on YPD agar containing indicated concentrations of amphotericin B. FIG. 3C shows pictures photographed after incubation at 30.degree. C. for 72 h of C. neoformans serotype A strains and serotype D strains spotted on YPD agar containing indicated concentrations of amphotericin B.

[0117] Confirming our hypothesis, the ssk1.DELTA., ssk2.DELTA., pbs2.DELTA., and hog1.DELTA. mutants exhibited dramatic hypersensitivity to amphotericin B treatment compared to WT (FIG. 3A), which is in good agreement with the finding that ergosterol contents were significantly higher in the HOG pathway mutants than WT (FIG. 2B). In contrast, the skn7.DELTA. mutant showed WT-levels of resistance to amphotericin B (FIG. 3A), which can be also explained by the previous data showing that cellular ergosterol contents in the skn7.DELTA. mutants are similar to those of WT (FIG. 2B).

[0118] We also monitored amphotericin B-susceptibility of C. neoformans strains having mutation hybrid sensor kinases (Tco1, Tco2, Tco3, Tco4, Tco5, and Tco7), which act upstream of the Ssk1 response regulator. Previously we have shown that Tco1 and Tco2 play redundant and distinct roles in controlling a subset of Hog1-dependent phenotypes. Here we found that Tco1 and Tco2 play discrete roles in sensing and responding to amphotericin B. Among Tco proteins, only Tco2, which is double hybrid sensor kinases containing two response regulator domains and two histidine kinase domains in a single polypeptide, showed hypersensitivity to amphotericin B (FIG. 3B), indicating that Tco2 is involved in sensing and responding to amphotericin B for conferring the drug-resistance via the HOG pathway. However, the fact that the degree of hypersensitivity observed in the tco2.DELTA. mutant is lesser than the ssk1.DELTA. mutant suggests other possibilities. One possibility is that other unknown receptor/sensors may exist to respond to the amphotericin B. The other possibility is that constitutively phosphorylated Hog1 may repress ergosterol biosynthetic pathway under normal conditions hypersensitivity regardless of the presence of receptors/sensors since Ssk1, Ssk2, and Pbs2, but not Tco2 proteins, are all involved in constitutive phosphorylation levels of Hog1.

[0119] To test the hypothesis, we have also examined the amphotericin B sensitivity of other C. neoformans strains, such as JEC21 and B3501-A, showing differential Hog1 phosphorylation levels. To support our second hypothesis, the JEC21 strain where Hog1 is not constitutively phosphorylated exhibited hypersensitivity to amphotericin B even more than the ssk2.DELTA. mutant in the H99 strain background (FIG. 3C). In the JEC21 strain background, mutation of SSK2, PBS2, and HOG1 genes did not affect sensitivity to amphotericin B (FIG. 3C). In contrast, the B3501 strain where Hog1 is constitutively phosphorylated, albeit to a lesser extent than in the H99 strain, exhibited higher resistance to amphotericin B than the JEC21 (FIG. 3C). Similar to the H99 strain, mutation of the SSK2 MAPKKK that abolishes the Hog1 phosphorylations increased the amphotericin B sensitivity (FIG. 3C). All these data strongly indicate that constitutively phosphorylated Hog1 represses ergosterol biosynthetic pathway under normal conditions.

[0120] To further support this finding, we also examined the susceptibility of the mutants to azole compounds, including triazoles (fluconazole and itraconzaole) and imidazole (ketoconazole), which inhibit the fungal cytochrome P450 enzyme 14.alpha.-demethylase and eventually prevent conversion of lanosterol to ergosterol.

[0121] FIG. 4 shows analysis results showing that the inhibition of the HOG pathway confers antagonistic antifungal effects with some azole drugs in C. neoformans. It shows pictures photographed after incubation at 30.degree. C. for 72 h of each C. neoformans strain spotted on YPD agar containing indicated concentrations of fluconazole, ketoconazole, and itraconazole.

[0122] We had expected that the ssk1.DELTA. and hog1.DELTA. mutants having increased expression of many ergosterol biosynthesis genes, particularly including ERG11, should show higher resistance to azole compounds. The ssk1.DELTA., ssk2.DELTA., pbs2.DELTA., and hog1.DELTA. mutants all exhibited hyper-resistance to fluconazole and ketoconazole, but not to itraconazole (FIG. 4). Interestingly, the skn7.DELTA. mutants also showed higher resistance to fluconazole and ketoconazole than WT (FIG. 4). Among hybrid sensor kinases, only Tco1 and Tco2 display differential sensitivity to azole compounds. Although to a lesser extent than the HOG mutants, the tco2.DELTA. mutant exhibited higher resistance to fluconazole and ketoconazole than WT (FIG. 4). In contrast, the tco1.DELTA. mutant exhibits hypersensitivity to all azole drugs (FIG. 4), indicating that Tco1 may regulate the HOG pathway in C. neoformans in an opposite manner to Tco2. In conclusion, inactivation of the HOG pathway increases ergosterol contents by induction of ergosterol biosynthesis genes and therefore confers synergistic effects with amphotericin B treatment, but antagonistic effects with fluconazole and ketoconazole.

Example 3

Finding and Characterizing the Downstream Target Genes Controlled by the HOG Pathway

[0123] We found ENA1 (serotype A ID: CNAG.sub.--00531.2) and NHA1 (serotype A ID: CNAG_01678.2) genes as the downstream target genes controlled by the HOG pathway and performed an additional experiment. Cells excrete H+(proton) out of cell membrane using H+-ATPase pump such as Pma1, thereby playing a role in maintaining membrane potential essential to cell growth in a normal condition. On the contrary, potassium ion(K+), an ion useful to cell growth, flows into cells using K+ influx pump such as Trk1/Trk2. Na+, unlike K+, is classified as a toxic ion. When high concentration of Na+ is present in a cell, it should be excreted via efflux pump. Since K+ also has toxicity when it presents in high concentration, an efflux pump is needed. These are Ena1 and Nha1 which play a role as an efflux pump for Na+and K+.

[0124] The result showed that the two genes coding for the two efflux pumps are controlled by the HOG pathway. As shown in FIG. 5A, when the WT, skn7.DELTA., ssk1.DELTA. and hog1.DELTA. mutant strains were exposed to osmotic stress, the expression level of ENA1 and NHA1 was dependent on the deletion mutant of the HOG pathway genes.

[0125] Thus, in order to identify a characteristic of two genes, we prepared deletion mutant of each gene and double mutant (ena1.DELTA. nha1.DELTA.) eliminating both two genes. And then, we examined sensitivity of the mutants to the polyene-based antifungal agent such as amphotericin B (AmpB), and the azole-based antifungal agent such as fluconazole, ketoconazole and itraconazole. The ena1.DELTA. and nha1.DELTA. mutants did not show high sensitivity to the AmpB. However, surprisingly, the ena1.DELTA. nha1.DELTA. double mutant showed considerably increased sensitivity to AmpB (FIG. 5B). Although lower sensitivity than hog1.DELTA., high AmpB sensitivity of the ena1.DELTA. nha1.DELTA. suggests that these two efflux pumps play an important role in the polyene-based drug resistance. It is more noteworthy that ena1.DELTA. and ena1.DELTA. nha1.DELTA. mutants also show high sensitivity to the azole-based drugs (FIG. 5C). It is a distinguished from the hog1.DELTA. mutant which has high resistance to the azole-based antifungal agents and verify that the inhibitors simultaneously or independently targeting Ena1 and Nha1 may exhibit very high antifungal activities when used in combination with the polyene- or azole-based antifungal agents.

Example 4

Comparative Transcriptome Analysis of C. neoformans ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka2.DELTA. Mutants

[0126] To compare the downstream signaling network of Ras1-, Aca1-, and Gpa1-dependent signaling pathways, we performed comparative transcriptome analysis of the serotype A wild-type (WT, H99) strain, ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka1.DELTA. mutants by employing DNA microarray analysis as described in Materials and Methods. For basic validation of our array quality, we checked expression levels of the RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 genes in our array data. The relative expression levels of RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 in each corresponding mutant were very low compared to those in the wild type strain (0.08, 0.03, 0.09, 0.06, 0.07, and 0.12, respectively) (FIG. 6A), which supported the quality of our array.

[0127] From total 7,936 genes monitored by this DNA microarray, 565 genes exhibited differential expression patterns in the Ras- and cAMP mutants at statistically significant levels compared to the wild type strain (ANOVA test, P<0.05) (FIG. 6B). The hierarchical clustering analysis of the Ras- or cAMP-dependent genes revealed several important facts. First, the transcriptome patterns governed by the Ras1-signaling pathway were distinct from those controlled by the cAMP/PKA-signaling pathway. The statistical analysis indicated that basal expression levels of total 400 genes changed significantly in the ras1.DELTA. mutant compared to the WT, whereas expression levels of 132 genes changed significantly in the aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka2.DELTA. mutants (FIGS. 6C and 6D). Besides the number of genes regulated, the expression patterns of a majority of the Ras1-dependent genes were also distinguished from those of the cAMP-dependent genes, which supported that the Ras1-signaling pathway is largely independent of the cAMP-signaling pathway in C. neoformans. Second, the aca1.DELTA. and gpa1.DELTA. mutants showed transcriptome patterns similar to those of the cac1.DELTA. and pka1.DELTA. pka2.DELTA. mutants, indicating that Aca1.DELTA. and Gpa1 are the two major signaling modulators of the cAMP-signaling pathway (FIG. 6D). However, there were a small group of genes whose expression is differentially regulated between the aca1.DELTA. and gpa1.DELTA. mutants. This indicates that Aca1 and Gpa1 could have other minor signaling branches (FIG. 6D). As expected, the cac1.DELTA. mutant exhibited transcriptome patterns almost identical to that of the pka1.DELTA. pka2.DELTA. mutant, further suggesting that Pka1 and Pka2 are necessary and sufficient protein kinase downstream of the adenylyl cyclase in C. neoformans (FIG. 6D).

[0128] The genes regulated by the Ras- and cAMP-signaling pathways cover a wide variety of cellular functions (FIG. 7). The cAMP-signaling dependent genes were over-represented for those involved in signal transduction mechanisms (15.2%), carbohydrate transport and metabolism (9.6%), and amino acid transport and metabolism (8.0%). These findings were rather expected results since the cAMP-pathway is one of central signal transduction cascades that regulate growth, differentiation, and virulence of C. neoformans and is known to sense glucose and amino acids (Bahn et al., 2004, Xue et al., 2006). Similarly, genes involved in signal transduction mechanisms were most over-represented in the ras1.DELTA. mutant (12.1%) (FIG. 7). In contrast to the cAMP-pathway, however, genes involved in cell wall/membrane/envelope biogenesis were over-represented (2.9%), which implies that Ras1 may be implicated in maintenance of cell wall integrity.

[0129] Among the Ras- and cAMP-dependent genes, a significant proportion of them were found to be environmental stress-regulated (FIG. 8). Our prior transcriptome analysis discovered a number of ESR (Environmental Stress Regulated) genes in C. neoformans (Ko et al., 2009). A total of 1,959 genes were found to be more than 2-fold up or downregulated in response to either of osmotic stress, oxidative stress, or antifungal drug (fludioxonil) treatment (Ko et al., 2009). Interestingly, our current array analysis revealed that a subset of the ESR genes (a total of 225 ESR genes) exhibited significant changes in expression levels in either the ras1.DELTA. or cAMP mutants compared to the wild-type strain (ANOVA test, P<0.05) (FIG. 8). Among these, eighty-six ESR genes showed more than 2-fold induction or reduction in the mutants (FIG. 8). Furthermore, a total of 55 CSR (Common Stress Response) genes were found to be differentially regulated (ANOVA test, P<0.05) and 31 genes of them exhibited more than 2-fold induction or reduction in the mutants (FIG. 2B). The major proportion of the Ras- or cAMP-pathway-dependent ESR and CSR genes did not have any other homologs with significant homology (Table S6). Nevertheless, these results implied that the Ras-and cAMP-signaling pathways be implicated in diverse stress response of C. neoformans.

Example 5

Identification of the Ras- or cAMP-Dependent Genes in C. neoformans

[0130] Next we further investigated individual Ras1- and cAMP-dependent genes identified by our transcriptome analysis.

[0131] Among the selected 161 Ras-dependent genes (2-fold cutoff, FIG. 6C), a majority of them (101 genes, 63%) do not have any orthologs in other fungi (Table S4), which indicated that C. neoformans contains a unique set of Ras-dependent genes. Among the evolutionary conserved Ras-dependent genes, three genes, PXL1, RDI1, and BEM3, whose orthologs are known to be involved in regulation of Rho-GTPase Cdc42 in S. cerevisiae, were notable since the Ras1-Cdc24 signaling pathway has been reported to be controlled by one of three Cdc42 homologues in C. neoformans (Nichols et al., 2007). RDI1 and BEM3 encode Rho-GDP dissociation inhibitor and Rho-GTPase activating protein, respectively (Price et al., 2008, Zheng et al., 1994). Notably, in a good agreement with the role of Ras1 in genotoxic stress response of C. neoformans (FIG. 4), a number of genes involved in regulation of DNA damage repair were identified as Ras-dependent genes. These include RNR2/RNR3 (Ribonucleotide-diphosphate reductase), RAD3 (DNA helicase, a subunit of nucleotide excision repair factor 3), RAD14 (a subunit of nucleotide excision repair factor 1), MSH6 (a protein required for mismatch repair), MND1 (a protein required for recombination and repair of DNA double strand breaks), and DNA2 (ATP-dependent nuclease). Finally, several genes, CHS1 (Chitin synthase 1), CDA2 (Chitin deacetylase), BGL2 (glucan 1,3-.beta.-glucosidase), and GSC2 (Glucan synthase), involved in governing cell wall integrity were also identified as Ras-dependent genes 6, which further supported the role of Ras1 in maintaining cell wall integrity of C. neoformans.

[0132] The statistical comparison of transcriptome data obtained from the cAMP mutants (aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka2.DELTA.) with that from the WT strain (ANOVA, P<0.05) identified 163 genes (FIG. 6C). Among these, 38 genes exhibited more than 2-fold induction or reduction in the cAMP mutants, except CAC1, ACA1, PKA1, and GPA1 (FIG. 9). A majority of the cAMP-dependent genes (31 genes, 81%) do not have any known function in C. neoformans or orthologs in S. cerevisiae, which indicated that C. neoformans contains a unique set of cAMP-dependent genes similarly to the Ras-dependent genes. This observation further corroborates that C. neoformans cAMP mutants have unique phenotypic characteristics that have not been observed in other fungi. Five cAMP-dependent genes (GRE2, ENA1, HSP12, CAT1, and PKP1) in C. neoformans appear to be evolutionarily conserved in other fungi. Interestingly, the GRE2, ENA1, and HSP12 genes are known to be transcriptionally regulated by environmental stress in S. cerevisiae. In C. neoformans, it has been recently reported that Ena1 not only controls osmotic stress under carbon starvation condition (Ko et al., 2009), but also is required for survival in alkaline pH and in vivo virulence (Idnurm et al., 2009). The GRE2 (genes de respuesta a estres, stress-responsive gene), a homolog of mammalian 3-.beta.-hydroxysteroid dehydrogenase, is strongly induced in response to a variety of stresses, including osmotic and oxidative stress, upon binding of HOG-dependent Sko1 transcription factor to CRE (cAMP response element) in the promoter region in S. cerevisiae (Garay-Arroyo & Covarrubias, 1999, Rep et al., 2001). The heat shock protein HSP12 (03143) is a small hydrophilic protein whose expression is also induced by diverse stresses and regulated by both HOG and cAMP signaling pathways (Varela et al., 1995). Here we named this gene as HSC1 (HSP12-like C. neoformans gene 1, 03143).

EXAMPLE 6

Inhibition of the Ras and cAMP-Signaling Pathway Increased Polyene Sensitivity

[0133] Gre2 is involved in regulation of some of ergosterol biosynthesis genes, including ERG6, ERG10, and ERG19/MVD1 (Warringer & Blomberg, 2006). Furthermore, GRE2 is reported to be one of six genes whose expression increased with resistance to amphotericin B (AmpB) in S. cerevisiae (Anderson et al., 2009). Therefore, we examined whether the C. neoformans Ras- and cAMP-mutants are more susceptible to AmpB treatment than WT.

[0134] As shown in FIG. 10, the ras1.DELTA. mutant showed higher susceptibility to AmpB than WT whereas the aca1.DELTA. mutant exhibited slightly higher AmpB susceptibility (FIG. 10A). The ras1.DELTA. aca1.DELTA. double mutant exhibited higher AmpB-sensitivity than each single mutant (FIG. 10B), indicating that Ras1 and Aca1 redundantly or independently control AmpB sensitivity. Cdc24 appears to work downstream of Ras1 for regulation of the polyene drug resistance (FIG. 10C). Interestingly, the ras2.DELTA. mutant was also slightly more sensitive to AmpB than WT, indicating that both Ras proteins control resistance to polyene drugs in C. neoformans.

[0135] Notably, the gpa1.DELTA. and cac1.DELTA. mutants showed much higher AmpB-sensitivity than WT and even than the ras1.DELTA. or aca1.DELTA. mutant (FIG. 10A). Downstream of the Cac1 adenylyl cyclase, the pka1.DELTA. mutant, but not the pka2.DELTA. mutant, showed increased susceptibility to AmpB (FIG. 10A), strongly indicating that the Gpa1-Cac1-Pka1 signaling cascade is one of signaling circuits to control the polyene drug sensitivity. The ras1.DELTA. cac1.DELTA. double mutant exhibited even higher AmpB susceptibility than each single mutant (FIG. 10B), indicating that the Ras- and Gpa1-Cac1-Pka1 pathways are independently involved in AmpB susceptibility. The ras1.DELTA. and ras1.DELTA. cac1.DELTA. mutants generated in MATa background (KN99 strain) exhibited the same phenotypes (data not shown).

[0136] To address whether the involvement of the Ras- and cAMP-pathways in the polyene sensitivity is related to the levels of ergosterol biosynthesis, we checked expression levels of ergosterol biosynthesis genes in the mutants from our array data. Interestingly, none of ergosterol biosynthesis genes, except ERG3 and ERG25 (<less than 2-fold), exhibited significant expression changes in the ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA., or pka1.DELTA. pka2.DELTA. mutants compared to WT (Table S2 and S3). Northern blot analysis showed that expression levels of the ERG3 and ERG25 genes in the mutants were not significantly different from those of WT (FIG. 10D). We also checked cellular ergosterol contents in the Ras- and cAMP-mutants and found that cellular ergosterol contents were not significantly increased in the Ras- and cAMP-mutants compared to WT whereas the hog1.DELTA. mutant has increased ergosterol contents as previously reported (data not shown) (Ko et al., 2009). Furthermore, expression levels of ERG11 in the ras1.DELTA. and cAMP mutants were not significantly different from those of WT (FIG. 10D). Supporting this finding, the gpa1.DELTA., cac1.DELTA., pka1.DELTA., pka2.DELTA., and pka1.DELTA. pka2.DELTA. mutants were nearly as resistant to fluconazole, which target to the fungal cytochrome P450 enzyme 14.alpha.-demethylase and inhibit conversion of lanosterol to ergosterol, as the WT strain (data not shown). All these data strongly implied that the Ras and cAMP-signaling pathway independently influence the polyene sensitivity without affecting ergosterol biosynthesis.

[0137] We have found in Examples 1 to 3 that the HOG pathway controls ergosterol biosynthesis of C. neoformans under unstressed conditions and the HOG pathways mutants are hyper-sensitive to AmpB, but hyper-resistance to fluconazole because of the increased cellular ergosterol contents in the mutants (Ko et al., 2009). Therefore, it is easily conceivable that the HOG and cAMP pathways influence the polyene sensitivity in different manners. Supporting this, we found that the hog1.DELTA. cac1.DELTA. and hog1.DELTA. pka1.DELTA. double mutants were even more sensitive to AmpB than the hog1.DELTA., cac1.DELTA., or pka1.DELTA. single mutant (FIG. 10E). Unexpectedly, the hog1.DELTA. cac1.DELTA. double mutants also exhibited hypersensitivity to various azole drugs, such as fluconazole, ketoconazole, and itraconazole (FIG. 10F). Interestingly, the ras1.DELTA., aca1.DELTA., gpa1.DELTA., cac1.DELTA., and pka1.DELTA. mutants all showed increased sensitivity to itraconazole (FIG. 10G). Particularly, both Ras1 and Ras2 appear to be involved in itraconazole susceptibility in a manner dependent of Cdc24 (FIG. 10H). Taken together, these date indicate that the HOG pathway and cAMP-signaling pathways independently control polyene and azole drug susceptibility.

[0138] One of key findings made by this study was that the Ras- and cAMP-signaling pathways controlled the polyene- and azole-based drug susceptibility in C. neoformans. Both Ras1 and Ras2 appeared to be involved in polyene susceptibility by using Cdc24 as a downstream effector. Interestingly, the ras1.DELTA. aca1.DELTA. mutant was also hypersensitive to amphotericin B, indicating that the Ras1 and Aca1 may play a minor role in susceptibility to the polyene drugs. It could be possible that perturbed action cytoskeleton regulation and cell wall integrity by ras1 and aca1 mutation makes cell more susceptible to the polyene drugs.

[0139] The cAMP-signaling pathway was even more significantly involved in polyene sensitivity than the Ras-signaling pathway. Mutation of the GPA1, CAC1, and PKA1, rendered C. neoformans cells to be hypersensitive to the polyene drugs, such as amphotericin B (AmpB). We recently reported that perturbation of the HOG pathway also renders C. neoformans cells to be hypersensitive to AmpB (Ko et al., 2009). However, the cAMP and HOG pathways appear to work differently for modulation of the polyene drug susceptibility. Inhibition of the HOG pathway, but not the cAMP pathway, increases ergosterol biosynthesis, which enhances the polyene drug susceptibility and azole drug resistance (Ko et al., 2009). Furthermore, the hog1.DELTA. mutant exhibited higher sensitivity to AmpB than the cAMP mutants.

Example 7

Characterization of the cAMP-Dependent Genes in C. neoformans

[0140] We also addressed the role of the cAMP-dependent genes and in diverse stress response and antifungal drug susceptibility of C. neoformans due to the involvement of the cAMP-pathway in the process that we discovered in this study.

[0141] Hypersensitivity of the cAMP mutants to the polyene drug appeared to be partly contributed by decreased expression of the two heat shock proteins Hsp12 (H99 gene ID: CNAG.sub.--03143.2), C. neoformans homologs of HSP12, and Hsp122(H99 gene ID: CNAG.sub.--01446.2) (FIG. 11).

[0142] Interestingly, however, the hsp12.DELTA. or hsp122.DELTA. mutant exhibited slightly higher susceptibility to AmpB than WT, although the cac1.DELTA. mutant was more sensitive to AmpB than the hsp12.DELTA. or hsp122.DELTA. mutant (FIG. 11A). Therefore, it was conceivable that decreased expression of HSP12 or HSP122 contributes to hypersensitivity of the cAMP mutants to AmpB.

[0143] To further characterize the regulatory mechanism of HSP12 and HSP122, we performed Northern blot analysis to confirm that the cAMP-signaling pathway modulated expression of the HSP12 and HSP122 genes. In S. cerevisiae, HSP12 is not expressed under unstressed, glucose-rich condition, but is induced in response to environmental stresses (Praekelt & Meacock, 1990, Siderius et al., 1997). Unexpectedly, however, the HSP12 and HSP122 genes were found to be highly expressed genes in the WT strain under unstressed, glucose-rich condition (FIG. 11A). In a good agreement with the microarray data, HSP12 and HSP122 expression was significantly downregulated in the cAMP mutants, including gpa1.DELTA., cac1.DELTA., and pka1.DELTA. pka2.DELTA. mutants (FIG. 11A). In the aca1.DELTA. and ras1.DELTA. mutants, expression levels of the HSP12 and HSP122 genes were only slightly affected (FIG. 11A). These data not only confirmed our microarray data, but also indicated that HSP12 and HSP122 were positively regulated by the cAMP-signaling pathway.

[0144] Interestingly our previous array analysis showed that HSP12 and HSP122 may also be under control of the HOG pathway. HSP12 and HSP122 expression levels were considerably low in the hog1.DELTA. and ssk1.DELTA., but not in the skn7.DELTA. mutant (FIG. 11B). To confirm this, we performed Northern blot analysis and found that expression levels of HSP12 and HSP122 were very high in the WT and skn7.DELTA. mutants, but was undetectable in the hog1.DELTA. and ssk1.DELTA. (FIG. 11B). All these data strongly indicated that the HSP12 and HSP122 gene was co-regulated by the cAMP and HOG signaling pathways.

[0145] As discussed in the above, hypersensitivity of the cAMP mutants to the polyene drug appeared to be partly contributed by decreased expression of the heat shock protein Hsp12 and Hsp122. In S. cerevisiae, Hsp12 plays a role in stabilizing the plasma membrane as a cell wall plasticizer and water replacement molecules (Sales et al., 2000, Shamrock & Lindsey, 2008) and therefore is involved in maintaining cell wall integrity under the stressful conditions in S. cerevisiae (Shamrock et al., 2009). Therefore, the hsp2.DELTA. mutant is unable to grow in the presence of a cell wall destabilizer, Congo red (Motshwene et al., 2004). Therefore, perturbation of the cAMP-signaling pathway reduces basal expression levels of Hsp12, which subsequently weakened cell wall integrity and membrane plasticity of C. neoformans. Similarly, hypersensitivity of the HOG pathway mutants to the polyene drug in part results from decreased expression of HSP12. However, since the cac1.DELTA. mutant is much more sensitive to AmpB than the hsp12.DELTA. mutant, other factors, except ergosterol biosynthesis, may affect resistance to the polyene drug. Supporting this, the hog1.DELTA. cac1.DELTA. or hog1.DELTA. pka1.DELTA. double mutant exhibited even higher polyene drug sensitivity than each single mutant, which indicated that the two pathways play an independent role in the polyene drug susceptibility. Notably, the double mutation of the HOG1 and CAC1 genes renders C. neoformans cells to be hypersensitive to most of azole drugs, including fluconazole, ketoconazole, and itraconazole, with unknown reasons.

[0146] In any case, modulation of each Ras-, cAMP/PKA-, and HOG-signaling pathway (or combination of them) may provide a novel antifungal therapeutic approach in combination with polyene and azole drugs. Simultaneous inhibition of the cAMP and HOG pathways when treated with polyene drugs such as amphotericin B could be one of the most powerful combination therapy for treatment of cryptococcosis.

Sequence CWU 1

1

6811309PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(1309)amino acid sequence of SSK1 1Met Trp Gly Ser Asn Ala Ser Ile Ala Ala Ser Glu Ser Thr Asp Ser1 5 10 15Leu Ser Pro Ala Pro Ser Gln Ser Ala Ala Val Glu Phe Pro Leu Pro 20 25 30Val Ser Ser Arg Pro Ser Leu Thr Ser Ala Ala His Pro Ser Gln Met 35 40 45Ser Ala Ser Ser Ser Ser Thr Ser Ser Gln Pro Leu Phe Asp Trp Arg 50 55 60Ile Pro Lys Pro Thr Ser Pro Arg Thr Arg Met Asp Pro Phe Asp Thr65 70 75 80 Phe Asp Pro Val Ser Ser Ser Ser Glu Asp Asp Pro Val Pro Gln Glu 85 90 95Ser Arg Arg Ala Gly His Gln Arg Ser Val Thr Asp Pro Leu Leu Arg 100 105 110Asp Gly Gln Pro Leu Asp Met Glu Phe Thr Thr Ala Gly Pro Pro Ile 115 120 125Gln Ser Tyr Asp Phe Glu Gln Pro Pro Thr Phe Ser Arg Thr Leu Ser 130 135 140Ser Pro Leu Pro Ala Lys Val Gly Ser Leu Arg His Pro Met Pro Phe145 150 155 160 Thr Ile Asp Asp Leu Ser Ser Arg Asn Val Asn Ser Thr His Arg Pro 165 170 175Gln Pro Thr Thr Pro Leu His Ser Ile Ser Val Glu Leu Ala Asp Ser 180 185 190Leu Gln Ser Ala Ile Gln Thr Leu Leu His Leu Ser Pro Pro His Leu 195 200 205Leu Asp Asn Ala Lys Glu Gln Tyr Ser Gly Cys Thr Val Gln Ile Pro 210 215 220Ala Thr Ser Leu Ser Ala Leu Leu Thr Ser Met Arg Gly Leu Asn Phe225 230 235 240 Leu Ser Ala His Ala Glu Glu Leu Val Asp Met Ser Ala Arg Gly Asp 245 250 255Pro Pro Val Leu His Gln Glu Asp Phe Asp Val Gly Glu Leu Leu Gln 260 265 270Asn Val Ala Asp Met Leu Ser Gly Glu Ala Ala Glu Lys Arg Ile Asp 275 280 285Phe Val Leu Phe His Gly Asp Val Ala Met Arg His Val Ser Val Tyr 290 295 300Gly Asp Ser Asp Gly Ile Ser Tyr Thr Leu Ser His Val Ile Arg Gln305 310 315 320 Ile Leu Ala Val Ala Asn Tyr Asp Asp Thr Ile Glu Leu Gly Leu Gln 325 330 335Val Ile Pro Gln Ser Pro Ser Leu Ala Ser Ala Val Gly Leu Pro Leu 340 345 350Thr Ser Ala Asp Val Ser Gly Gly Gly Gly Val Lys Ser Ala Ser Thr 355 360 365Ser Arg Ser Gly Ser Pro Asn Asn Ser Leu Ser Arg Ser Asn Ser Val 370 375 380His Asp Gly Pro Leu Leu Cys Val Phe Glu Ile Val His Asn Ile Tyr385 390 395 400 Gln Pro Pro Pro Ser Ser Ala Ser Ala Thr Pro Lys Ala Glu Leu Asn 405 410 415Pro Phe Thr His Leu Ala Glu Glu Thr Glu Ala Leu Lys Pro Arg Leu 420 425 430Asp Thr Ala Phe Cys Lys Asn Leu Leu His Arg Gln Asn Ala Val Leu 435 440 445Lys Val Asp Val Gln Pro Ser Ser Pro Leu Gly Ser Gly Met Pro Arg 450 455 460Arg Ala Tyr Ala Leu Ser Val Leu Leu Pro Arg Gly Lys Pro Ile Thr465 470 475 480 Glu Pro Ala Ile Leu Ser Lys Glu Glu Gln Glu Val Arg Gln Pro Phe 485 490 495Ser Ser His Val Leu Ala Arg Glu Pro Thr Leu Asn Glu Leu Ser Glu 500 505 510Phe Ala Glu Ser Leu Arg Gly Arg Lys Val Phe Ile His Ala Asn Leu 515 520 525Ser Ser Val Phe Ala Arg His Leu Thr Ser Tyr Leu Ala Ala Trp Gly 530 535 540Met Asp Ile Ser His Leu Pro Thr Asp Gly Asp Glu Ala Asp Lys Leu545 550 555 560 Lys Asp Val Ala Ala Lys His Asp Ser Ala Tyr Thr Gly Ser Met Gly 565 570 575Val Ser Gly Gly Thr Thr Ser Ser Ala Glu Thr Pro Tyr Ser Ile Lys 580 585 590Pro Thr Gly Val Thr Ala Val Gln Pro Gly His Phe Val Ile Ile Asp 595 600 605Asp Asp Val Ala Val Leu Arg Arg Glu Leu Val Arg Ile Arg Ser Glu 610 615 620Leu Leu Pro Ile Leu Phe Lys Pro Arg Leu Ser Lys Arg Pro Thr Met625 630 635 640 Thr Ser Arg Thr Arg Ser Thr Pro Ser Leu Arg Gln Val Pro Pro Arg 645 650 655Ser Ser Ser Gly Ser Val Leu Ile His Phe Thr Ser Leu Ala Asn Tyr 660 665 670Asn Arg Val Arg Asp Ala Ile Ala Ser Phe Val Gly Ala Pro Gly Leu 675 680 685Thr Asn Pro Glu Thr Tyr Val Gln Pro Glu Val Ile Val Ile Pro Lys 690 695 700Pro Val Gly Pro Arg Arg Phe Leu Thr Ala Leu His Thr Ala Val Lys705 710 715 720 Gln Pro Met Val Asp Pro Phe Phe Ser Pro Ile Ala Thr Ser Pro Arg 725 730 735Ser Pro Gly Gly Gly Tyr Phe Gly Gly Leu Arg Thr Pro Thr Glu Arg 740 745 750Glu Ser Gly Phe Phe Asp Ser Val Ala Glu Glu Pro His Glu Glu Ala 755 760 765Asp Ser Arg Pro Asp Tyr Ala Thr Val Gln Lys Ala Arg Ser Pro Leu 770 775 780Gly Glu Phe Pro Pro Ser Ala Ala Gln Ile Val Arg Thr Asn Gln Gly785 790 795 800 Leu His Leu Ser Leu Pro Thr Pro Asn Glu Ile Met Thr Thr Pro Ala 805 810 815Pro Glu Tyr Phe Ser Gly Ser Ser Lys Ser Pro Ser Ser Gly Ala Ser 820 825 830Gly Val Val Met Gln Ser Pro Asp Gly Arg Pro Phe Gly Met Phe Phe 835 840 845Glu Pro Pro Ile Lys Asn Glu Arg Arg Gly Ser Thr His Arg Thr Pro 850 855 860Ser Asp Ser Ile Arg Arg Lys Gln Ala Asn Arg Arg Ala Ser Thr Ser865 870 875 880 Asp Glu Pro Phe Ser Ser Pro Ser Thr Ala Leu Pro Pro Arg Arg Ser 885 890 895Ser Thr Ile Ser Thr Thr Gly Asn Glu Glu His Arg Ser Ser Pro Ile 900 905 910Ala Asn Val Thr Asp Arg Pro Thr His Ser Arg Val Asn Ser Arg Arg 915 920 925Lys Asn Asn Leu Pro Ala Ala Glu Gln Pro Ile Leu Ala Val Gly Arg 930 935 940Ala Lys Gly Arg Glu Arg Ser Glu Thr Val Thr Lys Gly Gly Asp Leu945 950 955 960 Gly Ser Arg Lys Gly Thr Pro Ala Ala Ser Pro Arg Ile Glu Glu Lys 965 970 975Lys Glu Leu Glu Arg Gly Glu Lys Thr Lys Ser Leu Ala Pro Ser Thr 980 985 990Ala Pro Thr Lys Lys Asn Ala Lys Val Asp Val Val Val Pro Pro Ile 995 1000 1005Asn Val Leu Ile Val Glu Asp Asn Pro Ile Asn Gln Asn Ile Leu 1010 1015 1020Ser Met Phe Leu Arg Lys Lys Lys Ile Lys Asn Ser Ser Ala Lys 1025 1030 1035Asp Gly Ala Glu Ala Val Glu Lys Trp Arg Thr Gly Gly Phe His 1040 1045 1050Leu Ile Leu Met Asp Ile Gln Leu Pro Val Met Asp Gly Ile Ala 1055 1060 1065Ala Thr Lys Glu Ile Arg Arg Leu Glu Arg His Asn Asn Ile Gly 1070 1075 1080Val Phe Pro Ser Thr Pro Ala Ala Glu Leu Pro Arg Gly Gln Asn 1085 1090 1095Val Ala Asp Ser Pro Pro Pro Ser Ser Pro Phe Arg Ser Ser Val 1100 1105 1110Ile Ile Val Ala Leu Thr Ala Ser Ser Leu Gln Ser Asp Arg Val 1115 1120 1125Ala Ala Leu Ala Ala Gly Cys Asn Asp Phe Leu Thr Lys Pro Val 1130 1135 1140Ser Leu Lys Trp Leu Asp Lys Lys Ile Val Glu Trp Gly Cys Met 1145 1150 1155Gln Ala Leu Ile Asp Phe Asp Gly Trp Arg Arg Trp Lys Ser Ser 1160 1165 1170Asp Thr Lys Asn Pro Ser Glu Thr Lys Gln Gly Phe Ser Val Gly 1175 1180 1185Pro Gln Gln Ala Ala Arg Ser Leu Ala Ser Arg Leu Arg Ile Glu 1190 1195 1200Arg Lys Gly Ser Arg Ser Pro Ala Ala Pro Val Ser Thr Pro Arg 1205 1210 1215Leu Asn Leu Gln Ser Ala Thr Pro Asp Arg Pro Glu Thr Pro Pro 1220 1225 1230Asp Ser Thr Ser Gln Met Pro Lys Ala Pro Pro Val Ala Ala Ser 1235 1240 1245Asp Pro Pro Leu Ser Pro Lys Ser Leu Asn Lys Thr Val Asn Asp 1250 1255 1260Val Phe Glu Gln Ala Asp Ala Arg Leu Glu Asn Ala Arg Glu Glu 1265 1270 1275Gln Gly Val Ser Ser Gln Lys Glu Asn Thr Ser Leu Thr Asp Ser 1280 1285 1290Thr Asn Thr Thr Ile Thr Pro Ser Lys Thr Tyr Pro Ala Pro Pro 1295 1300 1305Pro21691PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(1691)amino acid sequence of TCO2 2Met Ile Leu Gly Thr Asp Ile Asp Leu Ser Ser Ile Pro Thr Ala Phe1 5 10 15Leu Glu Ala Tyr Pro Phe Pro Ala Val Val Phe Val Ile Asp Ser Pro 20 25 30Pro Ser Pro Arg Pro Arg Leu His Ser Arg Asn Thr Asp Thr Thr Ile 35 40 45Arg Arg Thr Asp Gly Gln Ile Ser Pro Leu Thr Gly Pro Pro Val Gln 50 55 60Gln Phe Ala Ser Ala Pro Val Val Trp Gly Asn Gln Arg Trp His Glu65 70 75 80Leu Ala Gln Gly Lys Thr Ile Ala Glu Cys Val Asp Val Ala Ser Gln 85 90 95Asn Lys Leu Gln Thr Trp Val Glu Asn Asp Thr Gly Asp Lys Ser Glu 100 105 110Ser Leu Ala Leu Asp Leu Lys Val Pro Gln Gly Val Thr Leu His Leu 115 120 125Ala Lys Thr Ile Leu Pro Leu Ser Pro Pro Ser Ser Ser Gln Ser Leu 130 135 140Cys Ile Leu Ile Ser Gln Tyr Ile Asp Lys Pro Glu Ser Phe Ala Pro145 150 155 160Pro Ile Ser Ser Gly Asp Ile Leu Phe Ser Ser Leu Ser Arg Leu Ser 165 170 175Gln Thr Phe Ser Arg Ser Ser Ser Phe Ser Ser Asn Pro Arg Lys Ser 180 185 190Ile Asp Val Pro Ala Ser Leu Ser Glu His Arg Gly Ser Ala Thr Ser 195 200 205Thr Ser Ser Asn Leu Arg Ser Ser Ile Asp Leu Thr Ser Pro Asn Ser 210 215 220Gln Pro Ser Pro Leu Asn Arg Glu Gln Ser Thr Tyr Phe Thr His Gly225 230 235 240Ser Ala Thr Arg Glu Glu Arg Pro Ser Val Arg Arg Arg Arg Ser Pro 245 250 255Pro Ile Ser Met Thr Arg Pro Lys Pro Leu Glu Ser His Ala Gln Glu 260 265 270Cys Trp Asp Leu Val Glu Asn Phe Asp Trp Ser Lys Thr Ala Leu Gly 275 280 285Pro Arg Glu Gln Trp Met Asp Ala Leu Asp Pro Val Leu Ala Ile Thr 290 295 300Phe Glu Ser Arg Thr Ala Asp Cys Ala Trp Leu Gly Pro Asp Leu Glu305 310 315 320Leu Val Tyr Asn Lys Ala Tyr Gln Glu Leu Val Asp His Pro Asn Ala 325 330 335Phe Gly Lys Pro Ala Arg Gln Val Trp Ala Thr Asn Trp Asp Tyr Leu 340 345 350Glu Pro Leu Val Lys Arg Cys Leu Ser Gly Thr Pro Val Tyr Lys Asp 355 360 365Asn Asp Pro Leu Phe Trp Arg Arg Tyr Gly Asn Gly Arg Leu Leu Glu 370 375 380His Tyr His Thr Trp Arg Tyr Val Pro Ile Thr Gly Lys Asp Gly Ser385 390 395 400Val Leu Gly Ile Phe Asn Gln Ser Ile Glu Val Thr Asp Ser Val Leu 405 410 415Leu Glu Arg Arg Met Gly Thr Thr Arg Glu Leu Ser Glu His Met Ser 420 425 430Phe Ile Arg Thr Thr Glu Asp Phe Phe Ser Ser Val Ala Asp Val Phe 435 440 445Ser Gln Asn Pro Thr Asp Ile Pro Phe Ala Leu Cys Tyr Arg Val Arg 450 455 460Gln Val Asp Thr Asp Gly Thr Phe Val His Leu Asp Val Ser Leu Gln465 470 475 480Ser Ser Val Gly Val Pro Glu Gly His Pro Ser Ala Pro Asp Gln Ile 485 490 495Pro Val Ser Phe Leu Asn Gly Asn Pro Tyr Pro Ser Asn Val Glu Arg 500 505 510Ser Phe Ser Pro Ala Phe Ser Ile Val Ser Ile His Ser Ser Ser Ser 515 520 525His Arg Val Cys His Val Ser Glu Asp Thr Thr Gln Trp Pro Ile Ala 530 535 540Lys Ala Leu Gln Arg Arg Gln Cys Val Ile Ile Glu Glu Cys Ser Gln545 550 555 560Leu Ile Glu Gly Tyr Pro Ile Arg Arg Trp Asp Gly Leu Pro Phe Ser 565 570 575Ala Ile Val Val Pro Ile Cys Ser Glu Gly Ser Pro Glu Ile Pro Asp 580 585 590Ala Val Val Ile Leu Gly Leu Asn Val Arg Arg Cys Phe Asp His Glu 595 600 605Tyr Asp Ser Trp Ile His Ser Ile Arg Ser Gln Leu Ser Ser Ala Leu 610 615 620Val Met Val Lys Ala Arg Glu Ala Glu Gln Lys Met Val Glu Glu Ser625 630 635 640Ala Arg Met Glu Lys Ala Lys Val Ala Trp Phe Arg Gly Ala Ala His 645 650 655Asp Leu Arg Ser Pro Leu Thr Leu Val Ala Gly Pro Leu Ala Asp Val 660 665 670Leu Asp Ser Asp Leu Asn Ser Ser Gln Arg Thr Ala Leu Thr Val Ala 675 680 685Gln Arg Asn Leu Asp Arg Leu Val Arg Leu Val Asn Ala Leu Met Asp 690 695 700Phe Ser Arg Val Glu Ala Gly Arg Met Glu Gly Arg Phe Val Pro Thr705 710 715 720Asn Leu Ser Gln Phe Val Thr Gln Leu Ala Ala Leu Phe Lys Pro Ala 725 730 735Ile Glu Arg Leu Gly Leu Glu Tyr Val Leu Asp Val Gln Pro Ser Glu 740 745 750Glu Leu Val Phe Ile Asp Pro Val Leu Phe Glu Thr Val Val Ser Asn 755 760 765Leu Ile Gly Asn Ala Leu Lys Tyr Thr Glu Thr Gly Ser Ile Thr Val 770 775 780Arg Val Gln Tyr Thr Asp Tyr Ala Glu Val Ser Val Ile Asp Thr Gly785 790 795 800Val Gly Ile Pro Lys Asn Glu Leu Ala Leu Val Thr Glu Trp Phe His 805 810 815Arg Ala Ser Thr Ala Ile His Ser Gly Thr Gln Gly Thr Gly Leu Gly 820 825 830Leu Ala Leu Ala Lys Glu Leu Leu Lys Leu His Lys Gly Glu Leu Leu 835 840 845Val Glu Ser Gln Thr Ala Asn Glu Ser Gly Gly Pro His Gly Ser Ile 850 855 860Phe Thr Ala Lys Ile Pro Leu Asp Phe Lys Pro Ser Pro Ser Ala His865 870 875 880Ile Ile Pro Ser Val Glu Ser His Lys Thr Phe Gly Lys Tyr Ser Lys 885 890 895Ala Val Ala Asp Glu Ala Met Arg Trp Val Gly Asp Ser Asp Ala Ala 900 905 910Ser Glu Ala Tyr Asp Met Ser Ser Gly Thr Gly Val Ser Ser Ala Gly 915 920 925Ser Gly Ser Gly Asn Thr Thr Thr Phe Gly Pro Lys Phe Ala Asp Ala 930 935 940Phe Leu Phe Asp Lys Asn Asp Ile Val Leu Ile Val Glu Asp Asn Val945 950 955 960Asp Met Arg Glu Tyr Ile Arg Gln Leu Phe Ala Pro Tyr Cys Thr Val 965 970 975Leu Glu Ala Ser Asn Gly Glu Gln Ala Tyr Asn Met Ala Thr Gln Asn 980 985 990Pro Pro Asn Leu Ile Leu Ser Asp Val Leu Met Pro Lys Leu Ser Gly 995 1000 1005Met Glu Leu Leu Gln Arg Ile Arg Ser His Pro Asp Thr Arg Ile 1010 1015 1020Val Pro Met Val Leu Ile Ser Ala Ile Ala Gly Asp Glu Ser Arg 1025 1030 1035Val Glu Ala Leu Leu Asn Gly Ala Asp Asp Tyr Leu Ala Lys Pro 1040 1045 1050Phe Lys Pro Lys Glu Leu Ile Ala Arg Val His Leu His Met Gln 1055 1060 1065Val Gly Lys Lys Arg Ala Lys Leu Glu Ala Leu Tyr Ala Gln Arg 1070 1075 1080Glu Thr Glu Leu Thr Ala Leu Ser Asp Tyr Cys Pro Ile Gly Ile 1085 1090 1095Phe Arg Gly Asp Lys Tyr Gly His Ile Val Tyr Ala Asn Ala Ala 1100 1105 1110Trp Arg Ala Gln Ser Gly Leu Leu Val Gly Asp Pro Asn Asp Trp 1115

1120 1125Ala Ser Tyr Val His Pro Asp Ser Lys Ala Gln Leu Leu Glu Gln 1130 1135 1140Trp Asn Gln Trp Leu Arg Gly Asp Leu Lys Glu Phe Arg Ala Ala 1145 1150 1155Trp Arg Trp Ser Asn Gly Ile Pro Val Arg Ser Ile Leu Val Arg 1160 1165 1170Leu Asp Asp Val Lys Glu Gly Phe Ser Gly Leu Ile Gly Cys Val 1175 1180 1185Val Asp Val Ser His Glu Glu Arg Arg Leu Ile Glu Ala Glu Glu 1190 1195 1200Arg Arg Lys Glu Ala Glu Glu Ser Lys His Gln Gln Glu Leu Leu 1205 1210 1215Ile Asp Leu Thr Ser His Glu Ile Arg Thr Pro Val Ser Ala Ile 1220 1225 1230Leu Gln Cys Ser Asp Leu Val Lys Glu Asn Leu Val Ala Leu Lys 1235 1240 1245Asp Gln Leu Arg Gly Ala Gly Pro Lys Gly Phe Val Pro Ser Gln 1250 1255 1260Glu Leu Leu Ala Asp Leu Glu Gln Asp Val Glu Ala Leu Glu Ser 1265 1270 1275Ile Tyr Gln Cys Gly Leu Val Gln Glu Arg Ile Ala Gly Asp Val 1280 1285 1290Leu Ser Leu Ala Arg Ile Gln Leu Asp Met Leu Ser Leu His Asp 1295 1300 1305Ile Asp Val Asn Leu Arg Arg Glu Gly Arg Lys Val Ser Ser Ile 1310 1315 1320Phe Ala Ser Glu Ala Lys Met Lys Asp Ile Asp Leu Gln Leu Glu 1325 1330 1335Phe Gly Pro Thr Ile Glu Gln Ser Lys Val Leu Ala Ile Lys Thr 1340 1345 1350Asp Pro Val Arg Leu Gly Gln Val Val Thr Asn Leu Ile Ser Asn 1355 1360 1365Ala Ile Arg Phe Thr Ser Ser Ser Asp Val Arg Lys Ile Thr Ile 1370 1375 1380Gln Tyr Asp Val Ser Phe Val Pro Pro Ala Asp Asp Ser Cys Ala 1385 1390 1395Leu Pro Ser Ser Val Gly Leu Pro Asp Ile Leu Pro Val Lys Glu 1400 1405 1410Asn Thr Pro Leu Trp Leu Phe Val Ser Val Thr Asp Ser Gly Pro 1415 1420 1425Gly Met Thr Glu Gln Glu Leu Ser Val Leu Phe Gln Arg Phe Ala 1430 1435 1440Gln Gly Asn Lys Met Ile His Thr Lys Tyr Gly Gly Ser Gly Leu 1445 1450 1455Gly Leu Phe Ile Cys Arg Lys Ile Thr Glu Leu Leu Gly Gly Arg 1460 1465 1470Ile Glu Val Leu Ser Gln Val Gly His Gly Ser Val Phe Arg Phe 1475 1480 1485Phe Ile Lys Thr Arg Ala Val Ala Pro Pro Ser Ala Ile Ala Ala 1490 1495 1500Leu Val Glu Ser Ser Pro Leu Lys Pro Val Ser Ala Thr Ser Pro 1505 1510 1515Ser Ser Ser Leu Ala Met Ser Arg Ser Ser Ser Arg Ser Thr Asn 1520 1525 1530Val Thr Thr Pro Ile Glu Gly Gly Gly Thr Glu His Val Leu Ile 1535 1540 1545Val Glu Asp Asn Leu Ile Asn Gln Thr Val Leu Lys Arg Gln Leu 1550 1555 1560Val Lys Ala Gly Leu Ser Cys Asn Val Ala Ser Asn Gly Leu Glu 1565 1570 1575Ala Leu Asn Val Ile Arg Glu Val His Arg Gln His Arg Arg Gly 1580 1585 1590Gly Pro Asn Arg Lys Arg Leu Phe Asp Val Val Leu Met Asp Leu 1595 1600 1605Glu Met Pro Val Met Asp Gly Ile Thr Ala Val Arg Glu Ile Arg 1610 1615 1620Gln Ser Glu Ala Ala Gly Thr Leu Gly Arg Asn Met Val Ile Ala 1625 1630 1635Leu Thr Gly Asn Ala Arg Gln Gly Gln Ile Asp His Ala Leu Ala 1640 1645 1650Ser Gly Phe Asp Asp Val Val Ile Lys Pro Tyr Ile Leu Val Asp 1655 1660 1665Leu Leu Asn Lys Ile Lys Ser Met Lys Val Arg Lys Leu Glu Leu 1670 1675 1680Glu Thr Ala Lys Ala Gln Glu Glu 1685 169031417PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(1417)amino acid sequence of SSK2 3Met Ser Asn Pro Thr Ser Pro Ser Asn Pro Ser Asp Thr Gly Pro Ser1 5 10 15Ser Ala Ser Asn Val Thr Ser Ser Ser Ser Lys Thr Gly Arg Arg Ser 20 25 30Val Arg Leu Phe Ala Pro Asp Glu Glu Asp Ser Ser Asp Glu Asp Gly 35 40 45Gly Leu Ile Gly Val Pro Ala Glu Thr Thr Phe Lys Asp Asp Glu Ile 50 55 60Pro Pro Ser Asn Pro Arg Ser Ala Ser Tyr Pro Gly Pro Pro Ala His65 70 75 80Thr Ser Pro Thr Ser Lys Ile Ser Thr Ile Val Ser Ser Ala Ser Ala 85 90 95Ala Gln Pro Lys Leu Ala Arg Ser Ile Thr Tyr Val Ala Pro Asn Ala 100 105 110Val Ser Ser Arg Pro Ala Tyr Pro Leu Asn Pro Ala Gly Ser Glu Thr 115 120 125Leu His Ala Ser Gly Arg Ser Tyr Thr Asp Pro Asp Ile Gly Tyr Phe 130 135 140Ser His Asp Ala Gly Asp Asp Gly Trp Gly Ser Asp Asp Asp Asp Glu145 150 155 160Leu Arg Ser Pro Gly Trp Gly Ile Ser His His Asn Met Asp Ser Gly 165 170 175Gly Lys Thr Asn Gly Ser Pro Gln Leu Pro Ile Lys Pro Ala Asp Val 180 185 190Thr Glu Asp Glu Gly Gln Glu Arg Leu Asp Trp Gln Gly Met Leu Glu 195 200 205Ser Val Leu Asn Ser Asp Val Leu Lys Val Glu Glu Gln Arg Ile Tyr 210 215 220Asn Ser Met Pro Thr Asp Ser Phe Arg Glu Glu Ile Gly Lys Thr Leu225 230 235 240Trp Trp Gln Ile Arg Ala Lys Leu Arg Gly Arg Thr Glu Ala Glu Glu 245 250 255Lys Lys Arg Val Gln Glu Arg Arg Ala Arg Val Val Asp Pro Val Leu 260 265 270Glu Glu Ile Asn Glu Phe Lys Tyr Asp Pro Lys Asn Asn Pro Glu Gly 275 280 285Glu Glu Asp Ser Asp Gly Asp Pro Gln Asp Ala Thr Ser Thr Ala Ala 290 295 300Pro Gln Ser Lys Ala Leu Asn Gln Val Asn Thr Val Leu Ala Lys Leu305 310 315 320His Ala Ile Lys Gly Leu Tyr Pro Asn Leu Ala Ala Met Arg Ala Asp 325 330 335Lys Val Leu Tyr Thr Asp Glu Asn Phe Arg Lys Arg Ala Asp Ala Leu 340 345 350Thr Ser Trp Ser Ile Ile Val Ser Ser Leu Gln Thr Gln Leu Lys Leu 355 360 365Leu Gln Lys Trp Thr Gly Ser Asp Glu Leu Asp Ile Thr Lys Pro Asn 370 375 380Thr Thr His Glu Lys Ala Leu Val Gly Lys Tyr Lys Tyr His Ser Ile385 390 395 400Asp Ser Lys Gly Thr Pro Gly Arg Asp Ala Ala Asp Asp Ser Ser Phe 405 410 415Leu Asp Arg Val Ile Lys Glu Asp Asn Leu Gln Arg Thr Phe Glu Arg 420 425 430Arg Ala Phe Val Asp Met Ile Asn Leu Val Arg Asn Ala Lys Glu Thr 435 440 445Val Ile Ser Tyr Leu Pro Gln Phe Gln Glu Gln Asn Leu Pro Asp Phe 450 455 460Gln Tyr Glu Ile Val Arg Leu Ile Gly Phe Pro Gly Arg Leu Ile Ile465 470 475 480Glu Ala Val Lys Val Arg Leu Asp Ala Ala Ser Arg Leu Leu Asp Pro 485 490 495Asn Pro Met Val Val Glu Asp Phe Ile Glu Asn Leu Arg Leu Ser Ile 500 505 510Ser Leu Ala Val Leu Ile Arg Lys Gln Tyr Asp Glu Ile Met Ala Pro 515 520 525Asp Ala Glu Gly Arg Trp Lys Ile Pro His Cys Leu Pro Thr Glu Tyr 530 535 540Asn Asp Val Leu Leu Asp Ala Leu Arg Thr Phe Phe Lys Leu Leu His545 550 555 560Trp Arg Leu Arg Gly Val Gly Lys Ala Ser Tyr Tyr Lys Glu Thr Glu 565 570 575Val Leu Glu Glu Glu Ala Pro Phe Leu Tyr Glu Ala Ala Glu Ala Ile 580 585 590Val Gly Gly Asp Met Val Val Ala Glu Gln Tyr Cys Ala Leu Ser Asn 595 600 605Lys Leu Leu Ile Arg Ser Ala Asn Tyr Leu Asp Gln Gln Leu Arg Val 610 615 620Pro Ile His Ser Pro Ser Arg Asp Lys Glu Arg Gly Asp Lys Glu Arg625 630 635 640Asp Gly Ser Ser Ser Ser Gln Arg Asn Arg Asp Gly Arg Asp Ser Ser 645 650 655Leu Pro Gly Pro Pro Lys His Met Lys Val Glu Glu Leu Phe Ser Trp 660 665 670Tyr Ser Lys Leu Leu Asp Ser Ala Arg Met Arg His Arg Lys Thr Gln 675 680 685Arg Phe Cys Arg Lys Leu Thr Gln Arg Phe Asp Asn Ser Ala Glu Tyr 690 695 700Ser Ile Glu Glu Thr Glu Met Asp Met Leu Val Glu Thr Leu Gln Asp705 710 715 720Thr Gly His Phe Leu Val Tyr Thr Gly Lys Phe Glu Ala Asn Gly Thr 725 730 735Tyr Ile Val Ala Asp Gly Ser Leu Trp Gly Gln Pro Asp Asp Val Arg 740 745 750His Leu Leu Lys Arg Val Phe Ser Val Thr Ile Pro Gly Ser Arg Val 755 760 765Arg Pro Arg Gln Thr Thr Ser Gln Val Ser Val Gly Gly Ala Ser Pro 770 775 780Ser Asn Gly Gln Val Ala Ala Gln His Asp Pro Ala Asp Pro Tyr Pro785 790 795 800Glu Ala Asp Asp Phe Asp Asp Glu Ala Leu Ala Ala Tyr Ile Leu Leu 805 810 815Ile Ser Pro Arg Gln Ser Phe Val Trp Ser Gly Ala Val Met Thr Leu 820 825 830Asp Val Asp Tyr Ile Asp Tyr Glu Leu Pro Asp Asn Arg Val Arg Leu 835 840 845Ile Ala Asp Gly Pro Thr Lys Arg Leu Ala Leu Cys Lys Leu Tyr Phe 850 855 860Lys Gln Ala Leu Ile His Pro Asp Thr Gly Glu Thr Ile Asp Leu Pro865 870 875 880Cys Val Val Glu Ala Gln Ala His Leu Pro Thr Ile Gln Lys Gln Leu 885 890 895Val Lys Ile Ala Lys Ser Ser Tyr Arg Leu Ser Glu Cys Ile Val Gln 900 905 910Ser Ala Pro Leu Val Arg Asn Ala Phe Arg Gly Lys Pro Gly Ser Gln 915 920 925Glu Leu Val Glu Asn Trp Tyr Ser Phe Ala Thr Glu His Gly Thr Arg 930 935 940Val Leu Ile His Ile Glu Pro Ser Val Trp Glu Arg Phe Asn Arg Leu945 950 955 960Leu Met Arg Leu Ala Ile Ser Trp Ile Ser Phe Ile Ser Gln Glu Cys 965 970 975Asn Pro Thr Asp Arg Lys Thr Phe Arg Trp Thr Val Ala Ala Leu Thr 980 985 990Tyr Ala Phe Asn Met Thr Arg Gly Ser Asn Ile Leu Ala Leu Asp Arg 995 1000 1005Ser Glu Phe Ser Leu Leu Arg Arg Ser Ser Met Glu Ala Lys Lys 1010 1015 1020Glu Ala Asp Arg Ile Glu Ala Met Arg Arg Leu Gln Arg Leu Gln 1025 1030 1035Glu Asn Leu Asp Asp Glu Phe Leu Pro Arg Thr Pro Thr Glu Ser 1040 1045 1050Gly Asp Gln Pro Arg Ile Asp Arg Ser Ile Arg Leu Thr Val Glu 1055 1060 1065Glu Arg Leu Arg Leu Ile Ala Glu Leu Glu Ala Arg Arg Asp Glu 1070 1075 1080Leu Ala Pro Ala Pro Val Gly Gln Val Leu Asp Glu Glu Val Ser 1085 1090 1095Glu Asp Arg Ala Leu Val Phe Leu Ala Ala Ser Lys Ser Asn Ile 1100 1105 1110Ser Met Arg Trp Gln Gln Gly Ala Tyr Ile Gly Gly Gly Ala Ser 1115 1120 1125Gly Ser Val Tyr Leu Gly Tyr Ser Leu Gln Asp Asn Thr Val Phe 1130 1135 1140Ala Val Lys Ile Leu Pro Thr Val Asp Leu Gln Ser Ser Pro Ala 1145 1150 1155Leu Tyr Glu Ser Ile Lys Arg Glu Ser Asp Val Met Ser Leu Leu 1160 1165 1170Ser His Pro Asn Ile Val Gly Phe Leu Gly Leu Glu Val His Arg 1175 1180 1185Asn Arg Val Cys Leu Phe Gln Glu Tyr Cys Glu Gly Gly Ser Leu 1190 1195 1200Ala Gly Met Leu Glu Tyr Gly Lys Ile Asp Asp Glu Glu Val Val 1205 1210 1215Gly Ala Phe Thr Ile Gln Leu Leu Arg Gly Leu Glu Tyr Leu His 1220 1225 1230Thr Asn Arg Ile Glu His Arg Asp Leu Lys Pro Glu Asn Ile Leu 1235 1240 1245Ile Gly Ala Asn Ser Val Leu Lys Leu Ala Asp Phe Gly Thr Ala 1250 1255 1260Lys Ile Ile Lys Ser Asn Lys Thr Leu Ala Arg Thr Arg Gly Gly 1265 1270 1275Ala His Ala Lys Met Glu Gly Leu Glu Gly Thr Pro Met Tyr Met 1280 1285 1290Ala Pro Glu Met Ile Lys Asn Gln Arg Thr Gly Lys Leu Gly Ala 1295 1300 1305Cys Asp Ile Trp Gly Leu Gly Cys Ile Val Leu Gln Met Ile Thr 1310 1315 1320Gly Arg Lys Pro Trp Ser Phe Leu Asp Phe Asp Asn Glu Trp Ala 1325 1330 1335Ile Met Phe His Leu Gly Ala Thr Lys Glu Pro Pro Pro Leu Pro 1340 1345 1350Asp Pro Asn Glu Met Ser Asp Gln Gly Ile Glu Phe Ile Asp Gln 1355 1360 1365Cys Leu Ser Leu Asp Pro Glu Ala Arg Pro Val Ala Ser Glu Leu 1370 1375 1380Leu Gln Asp Glu Trp Leu Val Pro Met Leu Glu Gln Met Val Ser 1385 1390 1395Cys Leu Ser Cys Arg Ala Gly Ala Arg Ile Pro Arg Tyr Ile Gly 1400 1405 1410Asp Gly Pro Lys 14154609PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(609)amino acid sequence of PBS2 4Met Thr Asp Pro Thr Pro Pro Ala Leu Asp Ser Leu Ser Leu Ala Asp1 5 10 15Lys Ala Pro Thr Pro Glu Glu Ser Pro Glu Asp Ala Ala Glu Gln Pro 20 25 30Lys Pro Ala Ala Ser Pro Ser Ala Gly Thr Pro Gly His Asp Ala Gln 35 40 45Ser Ser Ser Thr Ser Pro Pro Gln Arg Pro Gln Ser Met Gln Thr Asn 50 55 60Asp Lys Ala Pro Asp Thr Ser Ala Pro Ala Ser Arg Pro Gln Pro Gln65 70 75 80His Val Pro Ala Ser Ala Pro Ala Leu Pro Ser Thr Asn Pro Val Arg 85 90 95Pro Gln Pro Gly Ala Arg Pro Gly Ala Ala Arg Gly Met Pro Ala Pro 100 105 110Met Gly Met Arg Ala Gln Ala Gly Arg Gly Ala Gly Gly Pro Gln Met 115 120 125Gln Thr Lys Met Leu Pro Ser Leu Gln Ala Lys Met Asp Lys Ile Ala 130 135 140Ala Ser Arg Gln Gly Pro Pro Pro Ser Ser Gly Met His Asp Pro Asn145 150 155 160Ala Thr Ser Met Gly Ala Leu Leu Arg Ser Gln Ala Leu Arg Ala Pro 165 170 175Gly Ala Ser Gln Ala Pro Pro Gly Pro Gly Pro Ala Ser Gly Pro Phe 180 185 190Gly Leu Ala Ala Arg Arg Ala Ala Ala Gly Gly Pro Pro Arg Pro Asn 195 200 205Leu Gly Met Met Gly Met Gly Ala Ser Ala Pro Gly Ala Val Gly Arg 210 215 220Gly Ser Gly Leu Ala Gly Arg Arg Gly Pro Pro Gly Gly Leu Thr Leu225 230 235 240Ser Gly Met Lys Gly Ala Ile Lys Asp Glu Gly Asn Lys Phe Ser Asp 245 250 255Phe Gln Gly Val Met Asp Pro Ser Gly Ser Leu Arg Phe Ser Lys Lys 260 265 270Ala Val Leu His Ala Lys Gly Val Asp Phe Glu Asp Gly Gln Ser Phe 275 280 285Lys Ile Asn Met Asp Glu Ile Glu Val Leu Gly Glu Leu Gly Lys Gly 290 295 300Asn Tyr Gly Ser Val His Lys Val Phe His Arg Pro Thr Gly Val Thr305 310 315 320Met Ala Met Lys Glu Ile Arg Leu Glu Leu Asp Asp Ser Lys Leu Asn 325 330 335Gly Ile Ile Met Glu Leu Asp Ile Leu His Arg Ala Val Ala Pro Glu 340 345 350Ile Val Glu Phe Tyr Gly Ala Phe Thr Ile Glu Ser Cys Val Tyr Tyr 355 360 365Cys Met Glu Tyr Met Asp Ala Gly Ser Leu Asp Ser Leu Thr Gly Gly 370 375 380Gly Val Ala Ala Lys Asp Gln Thr Lys Asp Glu Glu Asn Asp Ala Thr385 390 395 400Lys Arg Val Pro Glu Asp Val Leu Arg Arg Ile Thr Ala Arg Ile Val 405 410 415Lys Gly Leu Arg Phe Leu Lys Asp Glu Leu Gln Ile Ile His Arg Asp 420 425 430Val Lys Pro Thr Asn Val Leu Ile Asn Gly Lys Gly Glu Val Lys Met 435 440

445Cys Asp Phe Gly Val Ser Gly Gln Leu Glu Lys Ser Leu Ala Lys Thr 450 455 460Asn Ile Gly Cys Gln Ser Tyr Met Ala Pro Glu Arg Ile Lys Ser Glu465 470 475 480Thr Ala Asn Gln Asn Pro Thr Tyr Thr Val Ser Ser Asp Val Trp Ser 485 490 495Val Gly Leu Ser Ile Val Glu Leu Ala Lys Gly Cys Tyr Pro Tyr Pro 500 505 510Pro Glu Thr Tyr Ala Asn Val Phe Ala Gln Leu Gln Ala Ile Val His 515 520 525Gly Thr Pro Pro Thr Leu Pro Pro Gly Tyr Ser Asp Asn Ala Asn Asp 530 535 540Phe Val Ala Lys Cys Leu Glu Lys Asp Pro Asn Arg Arg Pro Thr Tyr545 550 555 560Ala Gln Leu Leu Glu His Pro Phe Leu Val Ala Asp Lys Gly Ala Glu 565 570 575Val Asp Met Val Gly Trp Val Glu Gly Ala Leu Lys Arg Lys Ala Glu 580 585 590Arg Gly Ile Ala Ser Leu Asn Pro Ile Gln Pro Pro Val Pro Leu Glu 595 600 605Pro 5365PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(365)amino acid sequence of HOG1 5Met Ala Asp Phe Val Lys Leu Ser Ile Phe Gly Thr Val Phe Glu Val1 5 10 15Thr Thr Arg Tyr Val Asp Leu Gln Pro Val Gly Met Gly Ala Phe Gly 20 25 30Leu Val Cys Ser Ala Lys Asp Gln Leu Ser Gly Thr Ser Val Ala Ile 35 40 45Lys Lys Ile Met Lys Pro Phe Ser Thr Pro Val Leu Ser Lys Arg Thr 50 55 60Tyr Arg Glu Leu Lys Leu Leu Lys His Leu Arg His Glu Asn Ile Ile65 70 75 80Ser Leu Ser Asp Ile Phe Ile Ser Pro Leu Glu Asp Ile Tyr Phe Val 85 90 95Thr Glu Leu Leu Gly Thr Asp Leu His Arg Leu Leu Thr Ser Arg Pro 100 105 110Leu Glu Lys Gln Phe Ile Gln Tyr Phe Leu Tyr Gln Ile Leu Arg Gly 115 120 125Leu Lys Tyr Val His Ser Ala Gly Val Val His Arg Asp Leu Lys Pro 130 135 140Ser Asn Ile Leu Val Asn Glu Asn Cys Asp Leu Lys Ile Cys Asp Phe145 150 155 160Gly Leu Ala Arg Ile Gln Asp Pro Gln Met Thr Gly Tyr Val Ser Thr 165 170 175Arg Tyr Tyr Arg Ala Pro Glu Ile Met Leu Thr Trp Gln Lys Tyr Asp 180 185 190Val Ala Val Asp Ile Trp Ser Thr Gly Cys Ile Phe Ala Glu Met Leu 195 200 205Glu Gly Lys Pro Leu Phe Pro Gly Lys Asp His Val Asn Gln Phe Ser 210 215 220Ile Ile Thr Glu Leu Leu Gly Thr Pro Pro Asp Asp Val Ile Gln Thr225 230 235 240Ile Ala Ser Glu Asn Thr Leu Arg Phe Val Gln Ser Leu Pro Lys Arg 245 250 255Glu Lys Val Pro Phe Ser Thr Lys Phe Pro Asn Ala Asp Pro Val Ser 260 265 270Leu Asp Leu Leu Glu Lys Met Leu Val Phe Asp Pro Arg Thr Arg Ile 275 280 285Ser Ala Ala Glu Gly Leu Ala His Glu Tyr Leu Ala Pro Tyr His Asp 290 295 300Pro Thr Asp Glu Pro Val Ala Ala Glu Val Phe Asp Trp Ser Phe Asn305 310 315 320Asp Ala Asp Leu Pro Val Asp Thr Trp Lys Val Met Met Tyr Ser Glu 325 330 335Ile Leu Asp Phe His Asn Leu Gly Asp Ile Ser Gln Asn Glu Ala Glu 340 345 350Gly Pro Val Thr Gly Glu Val Pro Ala Ala Pro Ala Ser 355 360 36561090PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(1090)Amino acid sequence of ENA1(CNAG_00531.2) 6Met Ser Ser Glu Lys Gly Gln Ser Asn Thr Asn Glu Lys Gln Leu Ile1 5 10 15Asn Arg Ala Asp Thr Gly Lys Thr Ala Val Ser Asp Ser Pro Leu Pro 20 25 30Phe Lys Pro His Thr Ala Leu Ser Gly Lys Ile Leu Glu Ala Leu Gly 35 40 45Ser Asn Val Thr Ser Gly Leu Ser Asp Asp Glu Ala Ser Arg Arg Leu 50 55 60Gln Gln Tyr Gly Pro Asn Arg Leu Lys Pro Pro Glu Arg Pro Ser Ile65 70 75 80Leu Lys Ile Ile Ala Arg Gln Val Gly Asn Ala Met Thr Leu Val Leu 85 90 95Ile Ala Ala Met Ala Thr Ser Leu Gly Thr Met Asp Trp Ile Ser Gly 100 105 110Gly Val Ile Ala Ala Leu Val Ile Leu Asn Val Ser Val Gly Ala Tyr 115 120 125Thr Glu Trp Gln Ala Glu Lys Thr Val Ala Ser Leu Glu Ser Val Gly 130 135 140Ala Pro Gln Ala Thr Val Val Arg Thr Arg Asn Gly Ser Arg Glu Ala145 150 155 160Thr Val Lys Ile Ile Pro Val Glu Glu Val Val Pro Gly Asp Ile Ile 165 170 175Gln Leu Lys Asn Gly Asp Ile Val Pro Ala Asp Gly Arg Ile Leu Asp 180 185 190Gly His Leu Ser Asn Leu Glu Ala Asp Glu Ala Phe Leu Thr Gly Glu 195 200 205Ser Leu Pro Val Ala Lys Gln Thr Glu Pro Ile Asp Glu Glu Asp Cys 210 215 220Pro Val Gly Asp Arg Val Cys Met Val Phe Ser Gly Ser Gln Ile Thr225 230 235 240Lys Gly Arg Ala Arg Ala Val Ile Thr Ser Thr Gly Met Gly Thr Glu 245 250 255Ile Gly Lys Ile Ala Gln Ala Leu Glu Ser Lys Ala Lys Asn Lys Asn 260 265 270Arg Gly Phe Ala Ala Phe Trp Trp Lys Val Lys Val Ile Leu Gly Val 275 280 285Glu Glu Thr Thr Pro Leu Gln Ile Lys Leu Asn Lys Leu Ala Tyr Phe 290 295 300Leu Leu Ala Cys Ala Leu Val Ile Ala Val Ile Val Val Ala Ser Thr305 310 315 320Gly Phe Asn Asp Val Pro Leu Ser Ile Ala Thr Tyr Ala Val Ala Ala 325 330 335Ala Val Ser Ile Leu Pro Ala Ser Leu Ile Ala Val Val Ser Leu Thr 340 345 350Leu Ala Arg Ala Ser Thr Asp Leu Ala Ser Arg His Ala Leu Val Arg 355 360 365Arg Met Asp Ala Ile Glu Ala Leu Ala Gly Val Glu Asn Val Cys Ser 370 375 380Asp Lys Thr Gly Thr Leu Thr Val Gly Arg Met Val Val Arg Lys Val385 390 395 400Trp Val Pro Ala Leu Asp Trp Arg Pro Asn Glu Phe Ala Pro Leu Asp 405 410 415Thr Ser Gly Gly Gln Ala Tyr Ser Phe Glu Thr Gly Ser Asp Pro Phe 420 425 430Tyr Pro Arg Gly Glu Val Leu Ala Asp Ser Gln Lys Ile Thr Gly Thr 435 440 445Ala Glu Thr Leu Asp Leu Lys Gln Pro Arg Asp Gln Ser Asp Ser Ser 450 455 460Ser Ser Asp Ser Asp Pro Asp Glu Arg Asp Val Glu Glu Gln Glu Arg465 470 475 480Val Ile His Val Glu Asp Met Glu Asn Asn Leu Arg Asp Leu Ala Leu 485 490 495Cys Ile Ser Leu Cys Asn Gln Ala Thr Leu Thr Arg Pro Val Asn Gln 500 505 510Asp Gly Gln Trp Glu Ala Asn Gly Asp Pro Thr Glu Thr Ala Leu Gln 515 520 525Val Ala Ala His Lys Leu Gly His Gly Lys Pro Phe Leu Thr His Ala 530 535 540Ala Lys Pro Ser His Arg Ala Asp Ser Ile Arg Ser Gly His Ser Ser545 550 555 560Arg Pro Leu Val Ala Gly Ile Arg Gly His Phe Val Pro Ile Ile Glu 565 570 575His Pro Phe Asp Ser Thr Val Lys Arg Met Ser Ile Ala Tyr Lys Phe 580 585 590Val Ser Glu Asp Pro Gln Asp Ser His Ile Leu Cys Leu Leu Lys Gly 595 600 605Ala Ile Glu Arg Val Phe Glu Arg Cys Thr Lys Ile Gln Gly Gln Pro 610 615 620Ile Thr Glu Glu His Lys Lys Asn Ile Met Val Lys Val Asp Ala Leu625 630 635 640Ala Ala Gln Gly Leu Arg Val Leu Ala Leu Cys Gly Lys Arg Leu Pro 645 650 655Val Ser Met Val Asp Glu Val Lys Ser Thr Pro Arg Asp Ala Phe Glu 660 665 670Ala Asp Phe His Phe Leu Gly Leu Ala Gly Ile Phe Asp Pro Pro Arg 675 680 685Lys Glu Ser Ala Gly Ala Val Ala Asp Cys Phe Arg Ala Gly Ile Thr 690 695 700Pro Arg Met Leu Thr Gly Asp His Pro Ala Thr Ala Thr Ala Ile Ala705 710 715 720Leu Asn Ile Gly Ile Leu Asp Lys Thr Tyr Ser Lys Asp Ser Val Met 725 730 735Thr Gly Gln Gln Phe Asp Ser Leu Ser Glu Asp Glu Ile Asp Gln Leu 740 745 750Pro Glu Leu Pro Leu Val Val Ala Arg Cys Ala Pro Glu Thr Lys Val 755 760 765Arg Met Val Asp Ala Ile His Arg Arg Gly Gln Ser Thr Val Met Thr 770 775 780Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys Arg Ala Asp Val Gly785 790 795 800Val Gly Met Gly Thr Gly Ser Asp Val Ala Lys Gln Ser Ala Arg Ile 805 810 815Val Leu Ser Asp Asp Asn Phe Ser Thr Ile Ile Arg Ala Ile Arg Lys 820 825 830Gly Arg Ser Val Phe Lys Asn Leu Ser Lys Phe Leu Leu Tyr Leu Leu 835 840 845Ser Gly Asn Leu Ala Glu Ile Ile Val Leu Met Ile Gly Leu Ala Phe 850 855 860Lys Asp Asp Asn Gly Gln Ala Val Phe Pro Leu Ser Pro Val Ala Ala865 870 875 880Leu Trp Ile Asn Thr Leu Ala Ala Gly Pro Pro Ala Leu Ala Leu Gly 885 890 895Leu Glu Pro Thr Ala Ile Asp Ala Met Glu Gln Gly Pro Glu Val Tyr 900 905 910His Arg Ile Phe Thr Leu Glu Phe Tyr Val Asp Leu Ile Phe Tyr Gly 915 920 925Phe Leu Met Gly Ser Ile Ser Leu Val Asn Phe Val Ile Val Leu Trp 930 935 940Gly Tyr Tyr Pro Gly Asp Leu Gly Arg Leu Cys Asn Glu Asp Asp Pro945 950 955 960Ser Ile Cys Asp Pro Val Tyr Gln Ala Arg Ala Ala Cys Phe Ala Thr 965 970 975Leu Val Ile Val Leu Met Ile His Ala Leu Glu Cys Lys His Leu Ser 980 985 990Lys Gly Leu Ala Gln Ile Asn Leu Arg Asp Asn Lys Val Leu Leu Trp 995 1000 1005Cys Val Val Ala Leu Ser Leu Ser Thr Phe Pro Val Val Tyr Ile 1010 1015 1020Pro Val Ile Asn Asn Lys Val Phe Leu Leu Asn Gly Pro Arg Trp 1025 1030 1035Glu Trp Gly Ile Ile Phe Gly Met Ile Leu Val Tyr Leu Ser Ala 1040 1045 1050Thr Glu Leu Tyr Lys Trp Ile Lys Arg Ile Trp Ile Arg Arg His 1055 1060 1065Ala Pro Pro Ser Lys Gly Pro Ser Asp Lys Thr Leu Arg Met Glu 1070 1075 1080Ser Thr Ile Ala Pro Pro Val 1085 10907916PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(916)Amino acid sequence of NHA1 (CNAG_01678.2) 7Met Thr Ala Phe His Pro Phe Glu Val Asn Ala Pro His Leu Ala Tyr1 5 10 15Thr Phe Leu Gly Gly Phe Val Val Ile Phe Gly Met Ile Ser Leu Phe 20 25 30Ile Lys Glu Lys Leu Tyr Val Gly Glu Ala Pro Ile Ala Thr Val Val 35 40 45Gly Ile Ile Ile Gly Pro His Cys Leu Asn Phe Phe Asn Pro Ala Gly 50 55 60Trp Gly Gly Gly Glu Glu Glu Val Ala Ser Asp Val Thr Leu Glu Phe65 70 75 80Thr Arg Val Val Ile Ala Ile Ser Val Phe Ala Val Gly Val Glu Leu 85 90 95Pro Lys Ala Tyr Met Lys Arg His Trp Arg Ser Leu Phe Phe Leu Leu 100 105 110Gly Pro Cys Met Val Trp Gly Trp Met Ile Ser Ala Leu Leu Ile Trp 115 120 125Gly Leu Ile Pro Asp Leu Thr Phe Leu Ala Ser Leu Val Val Ala Ala 130 135 140Gly Val Thr Pro Thr Asp Pro Ile Leu Ala Gln Ala Val Ile Gly Gly145 150 155 160Lys Phe Ala Asp Lys His Val Pro Ala His Ile Arg His Leu Leu Ser 165 170 175Ala Glu Ser Gly Ser Asn Asp Gly Ala Ala Phe Pro Phe Leu Tyr Ile 180 185 190Ala Leu Tyr Leu Leu Leu Asp Ala Ser Pro Gly His Ala Val Gly Glu 195 200 205Trp Phe Tyr Met Thr Trp Val Tyr Glu Ile Ile Leu Gly Val Ile Ile 210 215 220Gly Ala Ile Leu Gly Phe Cys Ala Arg Lys Leu Met Lys Leu Ala Glu225 230 235 240Arg Lys Arg Leu Ile Asp Arg Gln Ser Tyr Val Ala Gln Tyr Val Ser 245 250 255Leu Ala Val Leu Ser Ile Gly Val Thr Ser Leu Leu Gly Ser Asp Asp 260 265 270Leu Leu Ser Ala Phe Ala Cys Gly Cys Ala Phe Ala Trp Asp Gly Phe 275 280 285Phe Asn Lys Ala Thr Glu Asp Ala Val Phe Ser Asn Val Ile Asp Leu 290 295 300Leu Phe Asn Cys Ala Ala Phe Ile Tyr Ile Gly Ala Ile Ile Pro Phe305 310 315 320Asn His Phe Asn Asp Leu Pro Asp Leu Arg Val Trp Arg Leu Val Val 325 330 335Leu Ala Ile Leu Ile Leu Leu Val Arg Arg Leu Pro Ser Ile Ile Ala 340 345 350Cys Tyr Lys Phe Val Pro Asp Ile Lys Thr Phe Arg Glu Ala Leu Phe 355 360 365Thr Gly Trp Phe Gly Pro Met Gly Val Gly Ala Val Phe Ile Ser Thr 370 375 380Leu Ala Arg Ser Ser Leu Pro Glu Gly Glu Pro Glu Gln Asn Thr Glu385 390 395 400Ala Val Asp Arg Leu Lys Asp Val Ile Met Pro Val Thr Leu Phe Leu 405 410 415Val Leu Ser Ser Ile Val Thr His Gly Met Ser Ile Pro Phe Phe Ser 420 425 430Leu Gly Arg Arg Val His Ser Ile Thr Tyr Thr Arg Ser Arg Asn Leu 435 440 445Ser Met Asp Thr Arg Gly Asp Glu Pro Ala Trp Thr Thr His Ala Arg 450 455 460Arg Ile Ile Pro Gly Gln Glu Ile Ile Val Asn Arg Asp Asp Asp Asp465 470 475 480Glu Glu Gly Asp Leu Gly Val Arg Arg Met Asp Thr Leu Thr Ser Asp 485 490 495Ser Asn Gly Arg Ile Arg Glu Lys Ile Glu Glu Glu Asp Ser Gly Glu 500 505 510Ser Ser Ser Ser Arg Thr Arg Gln Gly Glu Met Ile Glu Met Thr Glu 515 520 525Lys Arg Gly Pro Ala Arg His Gly Ser Gln Ala Ser Gln Gly Glu Ala 530 535 540Ala Glu Glu Gly Glu Arg Trp Arg Ser Ser Gly Glu Glu Ser Ser Asp545 550 555 560Leu Ala Asn Asp Pro Glu Thr Gln Arg Glu Val Glu Glu Gly Met Glu 565 570 575Glu Val Glu Asp Lys Glu Gly Gly Gly Arg Arg Thr Pro Pro Leu Ala 580 585 590Lys Tyr Arg Glu Gly Asn His Leu Ile Val Glu Arg Lys Val Lys Asp 595 600 605Ser Asp Glu Val Glu Val Glu Val Ile Arg Asn His Phe Ser Asp Asn 610 615 620Lys Lys Thr Glu Ser Asp Arg Phe Thr His Pro His Arg Leu Lys Ser625 630 635 640Arg Glu Leu Asp Asp Leu Leu His His Leu Pro Lys Ser Leu Glu His 645 650 655Ala Thr Ser Arg Val Gln Asn Gly Gly Lys Asp Ala Val Asp Arg Leu 660 665 670Gly Leu Gly Leu Met Ala Ile Asn Thr Pro Glu Pro Ser Pro Ser Ile 675 680 685Glu Ser His Gly Gly Pro Arg His Asp Tyr Val Asp Gly Leu Glu Arg 690 695 700Thr Gln Ser Pro Glu Gly Leu Ala Asp Glu Asp Arg Asp Ser Glu Gly705 710 715 720Arg Gly Asp Val Ser His Gly Gly Asp Tyr Glu Glu Asn Glu Ala Asp 725 730 735Tyr Glu Asp Val Pro Asn Glu Thr Arg Arg Gln Arg Arg Lys Lys Met 740 745 750Lys Pro Pro Ala Ile Val Val Ser Arg Gln Asn Ser Ala Gly Leu Pro 755 760 765Arg Arg Ser Ile Arg Ser Arg Leu Phe Gly Arg Arg Gln His Ser Ser 770 775 780Asn Ser Pro Ser Arg Ala Glu Glu Gly Leu Ala Pro Pro Asn Pro Ser785 790 795 800Leu Leu Val Pro Ser Ser Ser Pro Ser Arg Pro Gln

Asn Ile Ala Ala 805 810 815Glu Pro Glu Ser Ile Leu Ala Glu Asp Ser Arg Gly Ser Ser Ser Pro 820 825 830Ser Gln Ser Gln Asn Leu Ala Ile Pro Leu Thr Arg Thr Leu Ser Ala 835 840 845Ser Arg Ser Ser Pro Ala Val Arg Phe Ala Asp Asp Ala Ser Pro Ser 850 855 860Ser Asp Thr Ala Pro Gly Gln Ser Asn Tyr Gly Thr Asn Ala Pro Gly865 870 875 880Phe Lys Lys Asn Pro Ala Leu Ala Met Tyr Arg Ser Ala Ser Val Gln 885 890 895Ser Thr Gly Ser Asn Lys Asp Gly Pro Ser Val Ser Phe Lys Glu Pro 900 905 910Glu Ile Lys Arg 91584121DNACryptococcus neoformans serotype A H99 straingene(1)..(4121)genomic DNA sequence of SSK1 8atgtggggct caaatgcttc catcgccgcc tcggagtcga ccgactccct ttcccccgcc 60ccctcccagt ctgccgccgt agagttcccc ctgcccgtaa gctcccgccc gtctctcact 120tccgccgctc acccctccca gatgtccgct tcctcctcct ccacctcctc ccagcctctc 180tttgattggc gcattcccaa gcccacctca ccccgcacac gcatggaccc attcgacact 240tttgatcctg tatcctcaag ctcagaggat gatccggtcc ctcaagagag ccgccgtgct 300ggtcatcagc gctccgtaac agatcctctt ttacgagatg gccaacccct cgatatggag 360ttcactactg ccgggccgcc tatacagagc tacgactttg aacaaccgcc cacgtttagc 420agaacgttat cttcccctct tccagctaaa gtcggctcgc ttagacaccc tatgccattc 480accattgacg atttaagctc tcgcaatgtg aattcaaccc atcgtccgca gccgactaca 540cctctgcatt ccatatccgt tgagttagcc gattccttgc aatcggccat tcaaacatta 600ctgcatctgt ccccacctca cctactcgat aacgcaaaag aacagtactc tggatgcacg 660gtacagatac ctgctacctc gctttcggcc cttttgacct ctatgagagg cctcaacttc 720ttgtcggcgc atgctgaaga actggtcgac atgagtgcac gtggagatcc acctgtactc 780catcaagaag acttcgatgt aggagaactt ttgcaaaacg ttgcggatat gttgagtgga 840gaagcagcag aaaaacggat tgatttcgtt ttgttccatg gcgacgtagc gatgaggcat 900gtcagtgtgt atggagattc tgatggaatt agctatacct tgagtcatgt aagtcctaca 960tgcacctgct gcatactgac aaacaggtta ttcgacaaat actggcagta gccaattacg 1020atgataccat agaactcggc cttcaagtca ttcctcaaag tccatcttta gcttccgccg 1080tcggacttcc tctaacctct gccgatgtta gtggaggagg tggtgtcaag tcagcgtcca 1140catctcggtc aggctccccc aataacagtc tctctcgatc taattctgtc catgacgggc 1200cccttctctg tgtgttcgaa atagtacata acatctatca gccaccacca agctcggcat 1260ccgccactcc taaagccgag ctgaaccctt tcactcatct tgctgaagaa accgaagcct 1320tgaaaccaag attggataca gcattttgca aaaacctgct tcatcggcaa aatgctgtcc 1380tcaaagttga tgtgcagcct tcatctcctt taggatccgg gatgccccgt agagcttacg 1440cgttatcagt gctcctacca agaggtaaac ccatcactga gcctgcaata ctttctaaag 1500aggaacaaga agttcgtcaa ccattttcat cccacgtact tgcacgagaa cccaccctca 1560atgagctctc ggaatttgct gaatcattac gaggaagaaa ggtgtttatc catgccaatt 1620tgagtagtgt tttcgcgaga cacctcacga gctacctagc tgcatgggga atggatatat 1680cgcatctacc gacagatggc gatgaggctg ataaattgaa ggatgtcgcg gccaaacatg 1740actcggctta tactggatct atgggtgtgt caggcggcac tacttccagc gcagaaacgc 1800cctattcaat taaaccgacc ggcgtgactg ctgttcaacc tggacacttt gtcattatcg 1860acgatgatgt tgcggtcttg cgccgtgaac tcgtgcgcat ccgttcagaa ttacttccca 1920ttctctttaa acctagactg tcaaagcgtc ccactatgac ttctcgaacc cgttccaccc 1980cttcattgcg acaggtcccc ccaaggtcat catcgggttc tgtacttata cactttacct 2040ctcttgccaa ttataaccga gttcgagacg cgattgcgag ctttgtgggg gcgccagggt 2100taaccaatcc ggaaacttat gttcagccgg aggtgatagt gatacccaag cctgttggac 2160cacgaagatt tttgactgct ctgcataccg ctgtgaaaca gcctatggtt gacccatttt 2220tctcccctat cgccacatct cctagatcac caggcggagg ttactttggt ggtttgcgga 2280ctccgacgga gagagaatca ggattctttg attctgttgc agaagaacca catgaagagg 2340cggattcgcg accagattat gccacggtgc agaaagccag atctccttta ggagaatttc 2400cgccttctgc ggcacagatc gttcgtacca accaaggctt gcatctttcg cttcccactc 2460caaatgaaat tatgacaacg cctgctccag aatatttttc tgggtcttcc aagtctccta 2520gctctggtgc gtccggagtc gtgatgcaga gccctgatgg tcgtcctttc ggaatgtttt 2580tcgaaccgcc cataaaaaat gagcgccgcg gatctactca caggacgcct tccgattcca 2640tcaggaggaa acaagcgaac cgccgtgcgt ctacaagtga tgaacccttt tcttcacctt 2700ctaccgccct acctccccgt cgctcgtcca caatttctac gactggcaat gaggaacacc 2760gcagttcacc tatcgctaac gtcacagacc gtcctaccca ttcaagggta aattcaagaa 2820ggaagaacaa tcttccggcg gcggagcaac ctattttggc tgtgggcagg gcaaaaggca 2880gggaaagatc ggagactgtc acgaagggag gggacctcgg gtcgagaaaa ggtacaccag 2940cggcaagccc acgtatagag gagaagaagg aattggaaag aggcgagaag actaaaagcc 3000tggctccttc aactgctcct acgaagaaga atgctaaagt cgatgttgtg gtgccgccca 3060tcaacgtgct gattgttgaa ggtaaatctt ccattcaaat gatttgttca aacaccgact 3120gacagataac tagacaaccc catcaatcaa aacattttga gtatgttcct gagaaaaaag 3180aagataaaga attcctcggc caaggatggc gcagaagctg ttgaaaagtg gaggactgga 3240ggcttccatc tgattctggt aggctatgat ctctttcttt gattcgtgca gtacttattg 3300gacccttcgc agatggatat ccaattgccc gtcatggatg gcatagctgc taccaaagag 3360attcgtcgac ttgaacgtca caataacatt ggcgtttttc catcgactcc agcggccgaa 3420cttcctcggg gtcaaaatgt tgcggattct ccaccaccat cttctccatt tcgctcgtca 3480gttatcattg ttgccctgac agcctcgtcc ttgcaaagcg acagagtagc tgctttggct 3540gctggctgta atgacttctt gaccaagcct gtgtctttga aatggttgga caaaaagatt 3600gtggaatggg gttgtatgca ggttagtgat ctcttctttt tttttgatta tagctgatct 3660aaatataggc attgattgat tttgacggct ggcgacgatg gaagagctcc gataccaaga 3720atcctagcga aactaagcag ggcttctcag tgggccctca acaggctgct aggtcgcttg 3780ctagcagact acgtattgaa cgcaaaggat ctcgatctcc ggcagctcca gtatcaaccc 3840cgcgactcaa tttgcagtcg gcaaccccag ataggccaga aaccccccca gattccacgt 3900cacaaatgcc aaaggccccg cccgttgcag cctctgaccc cccgttatct cccaagtcgc 3960tgaacaagac agttaatgat gtcttcgagc aagcagacgc tagactcgaa aatgcgcggg 4020aggaacaagg agtatcaagt caaaaggaaa acacaagctt aacagattca acaaacacca 4080ccattacgcc ctcaaagacc tatccggctc ctcccccatg a 412195666DNACryptococcus neoformans serotype A H99 straingene(1)..(5666)genomic DNA sequence of TCO2 9atgatcttag gaaccgacat cgacctgtcg tctataccaa cggcgtttct cgaggtgtgt 60atcgctgtcc ctttcgagtt gcaccatgaa gctgaccatg agaacgcatt cgccgccaac 120ttccaaatct gttctgggct tgtcttttgg acaaccatct aggcttatcc cttcccagcg 180gttgtgttcg tgatcgattc tccccctagc ccaagaccgc ggctccattc cagaaataca 240gacacgacca ttcggcgaac ggatggccaa atatcgcctc ttacaggtcc tccagtgcaa 300cagttcgcgt cagcgcccgt ggtatggggc aatcaacgat ggcacgagct ggctcagggg 360aaaacaattg cagagtgcgt ggatgtggcg tcacagaaca agctgcaaac ttgggtggaa 420aatgacaccg gtgacaagtc ggagagtttg gccctggacc tgaaggtgcc gcaaggcgtg 480actcttcatc tggcaaagac catattgcca ttaagtccac cgtcctcctc tcagtcactt 540tgcatcctta tatcgcaata tatcgataag ccggaaagtt tcgcgccacc aatctcatct 600ggagatattc ttttctcttc tctatcgcga ctctcccaga ctttttctcg gtcatcctct 660ttttcatcca accctagaaa atcgattgat gtccctgcgt cactatccga acaccggggt 720tctgctacat cgacaagtag caatctgcgc tcttcgatcg atttgacttc ccctaattct 780caaccctctc cactaaaccg tgaacaaagc acgtacttca cccatggctc cgcgaccaga 840gaagagcgac cctcagtaag gcgtagacgg tcaccgccaa tctcaatgac gaggcccaag 900cctcttgaga gccatgctca agaatgctgg gacttggtag agaatttcga ctggtcaaaa 960acagcattag ggccgagaga acagtggatg gatgcgttag atcctgttct ggcaatcaca 1020tttgaatcca gaacggcaga ttgtgcctgg ttagggcctg atctagagct agtttagtga 1080gatactatcc gtcttgcaaa aataatggcg actaactttt atgatcagca ataaggcgta 1140tcaagagctg gttgaccatc ccaatgcttt tggaaaacct gcaagacaag tttgggctac 1200caattgggac tacttggaac ccctggtcaa gcgatgtctc agtgggaccc cggtctacaa 1260ggacaacgac ccgcttttct ggcgtcgata cggcaatggt cgacttctgg aacattacca 1320cacttggcga tatgtcccga taacgggcaa agatggctca gtgcttggca tcttcaacca 1380gtcaattgag gtcaccgact cagtactgct agagaggcga atgggcacga ccagggaact 1440ttcggaacac atgtcgttta ttcgtacaac tgaggacttt tttagctcgg ttgccgacgt 1500ctttagtcag aaccctactg acataccgtt cgcactttgt taccgggtcc gacaagttga 1560caccgatggg acatttgtcc atttggacgt ctcgcttcag tcgtccgtcg gtgtacccga 1620aggccatccg tctgctccag atcaaattcc cgtcagcttc ttaaatggta acccttaccc 1680tagcaatgtc gagcgatcat tttctcctgc tttctcaatc gtttcaatcc actcttcgag 1740cagtcatcga gtctgtcacg tctctgaaga cactacacaa tggcccatcg ccaaagccct 1800acaaaggcgg caatgtgtca tcatcgaaga atgttcgcaa ttaatagaag gatatcctat 1860ccgtcgctgg gatgggcttc cattctcagc cattgtcgtg cccatatgct ctgaagggtc 1920tcccgaaatc cctgacgccg ttgttattct tggtctcaat gtgcgacgtt gttttgacca 1980tgaatacgat tcctggattc actctattcg gtcacaacta tcttcggccc tcgtgatggt 2040caaggcgcgt gaagctgaac aaaagatggt tgaggaaagc gcacgtatgg agaaagcaaa 2100agtcgcttgg ttcagaggag ccgcgcacga ccttcgtagt ccattaaccc tcgtcgctgg 2160accgcttgcc gatgtgcttg attcggattt gaactcgagt cagcgcacgg ctttgaccgt 2220tgcgcaacgc aatcttgatc gtttagtgcg cttggtcaac gccctcatgg atttctcgag 2280ggtggaagct ggacgaatgg aaggacgatt tgttccgacg aacttgagtc aattcgtgac 2340acagttggca gctcttttca agcctgcaat agaaagattg gggttagaat acgtactaga 2400tgtccagcca agcgaggagc ttgttttcat cgatcctgtt ctgtttgaga ccgtggtatc 2460aaaccttatt ggcaatgcgc tcaaatacac tgaaacgggt tctatcactg ttcgggtgca 2520atacacggat tacgcagagg tctcggtcat cgataccggt gtgggtatac cgaaaaatga 2580gctggcactg gtgaccgaat ggttccacag ggcaagtact gccattcact cgggaaccca 2640gggaacagga ttgggactgg ctttggccaa ggaattgctc aagttgcata aaggagaatt 2700gcttgtcgag tctcaaaccg ccaatgagtc aggaggtcct catgggtcca tttttacagc 2760gaaaattcct cttgatttca agccctctcc atcggctcat atcattccgt ccgtcgaatc 2820tcacaagacg tttggcaaat acagtaaagc cgtcgcagac gaagccatgc gctgggttgg 2880ggactcagat gccgctagtg aggcgtacga catgtcgagc ggtaccggag tctcaagcgc 2940tggtagtggc tctggaaaca cgaccacctt cggacccaag tttgcagatg cctttttgtt 3000tgataagaac gacattgtgc ttattgtgga agacaatgtc gacatgcgtg aatacatacg 3060acagcttttc gccccttatt gtaccgtact cgaagcttcc aatggtgaac aggcttacaa 3120tatggctacc caaaaccctc ccaacctcat tttgtcggac gtgctcatgc ccaaattatc 3180tggtatggag ctactacaaa ggatcagatc ccatcctgac actcgcattg tgcctatggt 3240ccttatttcg gctattgctg gtgatgagtc tagggttgag gctctgctaa acggcgctga 3300tgactatctt gccaagcctt tcaaacccaa ggaactcatc gcgcgtgttc acctgcacat 3360gcaagttggc aagaaacgtg ccaagctcga agcgctatac gcccaacgcg aaacagaatt 3420gacagctcta tctgactatt gtccgatcgg tatcttccga ggagacaaat atggccatat 3480tgtttatgcg aacgcagctt ggcgtgcgca gagcggcctt ttggtgggtg accctaacga 3540ttgggcatct tatgtgcacc cggattcgaa agcgcagctc ttggaacaat ggaatcagtg 3600gttgaggggg gatttgaagg agttccgagc ggcttggaga tggtctaatg gcatccctgt 3660caggagcatc ttggtccggt tagatgacgt caaggaaggg ttttctgggt taattgggtg 3720cgtagtggat gtgtctcatg aagagagacg attaatcgaa gctgaggaaa gaagaaaaga 3780ggcggaagag agtaaacatc agcaagaact ccttattgac ttgacaagtc atgaaattag 3840gaccccggtg tcagcaatcc tgcagtgctc agatcttgtt aaagagaatc ttgtagctct 3900gaaggaccag ttgagaggag cggggccaaa gggctttgtg ccgagtcaag aattactggc 3960tgatcttgag caggatgtgg aagctttgga aagtaattca ccatcccctt tcatgctaat 4020ttcgaaacta acagtgattt tgtaaggtat ttatcagtgc ggtcttgtgc aggaacgcat 4080tgccggagat gttctttcgc tggctcgtat ccaactcgat atgctgagtt tgcacgacat 4140tgacgtcaac ttgcgccgag aaggcaggaa agtttcgtcc atctttgcat cggaagccaa 4200gatgaaggat atcgacctcc aattggaatt tggacctact atcgaacagt ccaaagtgct 4260ggccatcaag acagatcccg tgagattagg ccaggtggta acaaatctca tttccaacgc 4320cattcggttt acatcttcga gtggtgagtt tcatccaacc cataatgtgc tatagtgata 4380ctgaattgta attcatttag atgtccgaaa gattactatc caatacgacg tatcgtttgt 4440ccctcctgcc gatgactctt gcgccctccc ttcatctgtt ggcttgcccg acatacttcc 4500tgtgaaagag aatactccac tatggctgtt tgtcagtgtt accgattctg gacctggtat 4560gacagagcaa gagttatctg ttttgttcca aaggtttgcc cgtaagtcct aagcccattc 4620tctttgtcga ggttacgttg acgaatcttg atatctagag ggcaataaga tgattcatac 4680taagtatggc ggaagcggtt tgggactgtt catctgtcga agtgagtgat tggaggaggt 4740tttttttttt ttttggtggg gggatacatg atgctgaaac catctacaga gattacagag 4800cttcttggcg gtcgtatcga agtgctcagc caagtcgggc acggtagtgg tgagtgcccg 4860tgtgctctgt tatccattgc atgctgctga atcgagtgcc tttcccaagt tttccgattc 4920ttcattaaaa cgcgcgctgt cgctcctccg tccgccatcg ctgctctcgt agaatcctct 4980cctctcaaac cggtatccgc cacttcacct tcttcctcgt tagccatgag ccgatcgtct 5040tctcggagca caaacgtcac tacacctata gagggtggtg ggaccgagca cgtgttgatc 5100gtggaagata acctaatcaa tcagactgtc ctgaagcgac agctcgtcaa ggcgggttta 5160tcgtgcaacg gtgagttctc catcccatcc tctgtgattc ccacatttat cttgattgct 5220gttgtatagt cgcgagtaat ggccttgaag ctcttaatgt catccgtgaa gtccatcggc 5280aacaccgacg cggtgggccg aaccgtaaaa ggctatttga cgtggtattg atggatctcg 5340agatgccagt gatggatggt atcaccgccg tacgagagat acgacagtcc gaggccgcgg 5400ggacattggg caggaatatg gtgattgccc tgacggggaa cgcgagacaa ggacagattg 5460atcatgcctt ggcttctgga tttgacgatg gtgagttggg aagacccttt tttgggggct 5520cagaggacga tgtggattat gaacttttct gacacttgat tgtagtcgtc atcaaaccgt 5580atatcctggt agatttgctg aacaagatca aatctatgaa agttagaaaa ttggagttgg 5640aaactgcgaa agctcaagaa gagtga 5666104744DNACryptococcus neoformans serotype A H99 straingene(1)..(4744)genomic DNA sequence of SSK2 10atgtccaacc caacctcccc ctcaaacccc tcagacaccg gcccgtcctc ggcgtccaac 60gtcacatcct cgtcctcaaa gaccggacgc agatcagtgc gcctctttgc cccagacgag 120gaggacagct cagacgagga cggcggcctc atcggcgtgc ccgcagagac cacattcaag 180gacgacgaga gtgagtagat acgcaggaac caaatgcagc gcatactcac actccacagt 240ccctccttcc aacccacgtt ccgcctccta ccccgggcca ccggcacaca cctcccccac 300ctctaaaatc tcaaccatcg tttcgtccgc ttctgcagcc cagccaaaac ttgcacgttc 360aataacatac gtcgcaccca atgccgtctc ctcccggccg gcatatcccc tcaatcccgc 420agggtcagaa accttacacg cctcgtacga gacatggcga aaaacgcgat ataccctcga 480gtcattcggc agaggatcca aaccagacgg gaagaatacg gcacaaagag gaaggtcgta 540tactgacccc gatataggat actttagcca cgatgcagga gatgatggtt ggggctcaga 600tgatgacgac gaattgagat cccctggctg gggcatatcc catcataaca tggactctgg 660aggcaagacg aacgggtcac cacagttgcc tataaagccc gccgatgtca ccgaggatga 720aggacaggaa cgtttagatt ggcaaggcat gctggaaagt gtcctcaact cggatgttct 780caaggtggag gaacaacgta tctacaattc catgccgaca gattcattca gagaagagat 840tggaaagacc ctttggtggc aaatccgtgc caaactgcgt gggaggacag aggcggagga 900gaagaaacgg gtgcaagagc gacgagcgag agtggtggac ccggtgctgg aagagataaa 960cgagttcaag tacgacccaa aaaataaccc agaaggcgaa gaagacagtg atggcgatcc 1020gcaagacgcg acttcgactg ctgcacccca atccaaagct ctcaatcaag tcaacaccgt 1080tctcgccaaa cttcatgcaa tcaaaggtct ttatcccaac ctcgcagcca tgcgagccga 1140caaggttctc tataccgatg aaaatttccg caaacgcgcc gacgcattga cctcttggtc 1200catcatcgtt tcatccctcc aaacccagct caaactcttg caaaaatgga caggttccga 1260tgagcttgac atcaccaagc ccaacacgac ccacgagaaa gcattggtcg gcaagtacaa 1320gtatcactct atcgacagca agggtacgcc cggcagggat gcagccgatg actcgagttt 1380cctcgatcgt gtgataaaag aagataacct tcaacggaca ttcgagcgtc gagcgtttgt 1440agacatgatc aacctcgtgc gcaacgccaa ggagacggtc atcagctatc tcccccagtt 1500ccaagaacaa aatcttcccg atttccagta cgaaatcgtt cgtcttattg gtttccccgg 1560tcgacttatc attgaagctg tcaaggttcg tttggatgct gcatcccgac tacttgaccc 1620gaaccctatg gtcgtcgaag actttatcga aaaccttcgt ctatccattt cgctcgccgt 1680gctaatccgg aaacaatacg acgaaatcat ggcacccgat gccgagggga gatggaaaat 1740cccgcattgc ttgccgacag agtacaatga tgttctgctc gatgcgctga ggacattttt 1800caaattgttg cattggagat tacgaggagt ggggaaagcg agttattaca aggaaacaga 1860agtgttggaa gaagaggcgc cgttcttgta tgaagcggcg gaggctattg taggcggtga 1920tatggttgtt gcagagcagt attggtgagt ttgaaatcgt atcatcctgg caaggagctt 1980agtgctaagt atcgatgtaa atagcgcgtt atccaacaag ctccttatac gttcagcaaa 2040ttatcttgac cagcaacttc gggtaccaat acattccccg tctcgcgaca aggaacgtgg 2100tgacaaggag cgcgatggct cttcgtcttc tcaacgtaac cgtgacggcc gtgatagctc 2160gctgcccggc ccaccgaaac acatgaaagt cgaagaactc ttctcatggt actccaaact 2220ccttgattcc gctcgtatgc gacaccgtaa aacccaacgt ttctgtcgta aactcaccca 2280acgattcgat aattccgccg aatattcaat cgaggagacg gagatggaca tgctggtgga 2340gacattgcaa gatactggtc atttcttggt atataccggg aaatttgagg cgaatgggac 2400gtatatcgtt gcggatggga gtctctgggg tcagccggac gatgtgagac atctgttgaa 2460gagggtgttt tcagtgacga ttcctggatc tcgagtccgt ccaaggcaga caacctcgca 2520agtatctgtc ggaggtgcga gcccgtccaa tggtcaagtc gcggcgcaac atgatcctgc 2580agatccgtac cccgaggcag acgattttga cgacgaagcg ctcgcggctt acatcctcct 2640catctcccca cgccaaagtt ttgtatggtc cggagcggtc atgacgctgg atgtggatta 2700catcgactat gaactacctg ataaccgagt cagattgatc gctgacggtc ccaccaagcg 2760gttagcgctg tgcaaacttt atttcaagca agcgctcatt caccctgata cgggcgaaac 2820aatcgacttg ccatgtgtgg ttgaggctca agcgcattta ccgaccattc agaaacaact 2880tgtcaagatt gctaaatcga gttatcgtct ttcagagtgc attgtccagt ctgcaccact 2940cgtccgcaat gcgttcaggg gcaaaccggg atcacaagag ttggtggaga attggtacag 3000ttttgcgaca gagcatggga cgagagtgtt gatccatatt gagcctagtg tatgggagcg 3060attcaatcgg ttgttgatgc gtctggcgat cagttggatt agctttatca gtcaagagtg 3120taaccccaca gaccgcaaga cgttccgatg gactgtggca gctttgacct atgcgttcaa 3180catgacgaga gggagtaaca ttctcgcgct tgatcgatca gaattttcgc ttttgaggag 3240gtatgttggt gtttgtgtgt cactgttggt tagccacttt gatatcctcg gcgcaaggtc 3300gagtatggag gccaaaaagg aggcagacag gattgaggcg atgaggaggt tacaacggct 3360tcaagaaaac ctggacgacg aattcctgcc ccggactccg acagagtctg gcgatcaacc 3420acgtatcgac cgctctataa ggctcacagt cgaagaacgt ctccgtctca ttgccgagct 3480cgaagctcgt cgtgacgagt tggcacccgc acccgtcggt caagtccttg atgaagaagt 3540ctctgaagac cgtgcgttgg tgttccttgc agcttccaaa tccaacattt ctatgcgatg 3600gcagcaaggc gcgtacatcg gtggaggtgc atcgggaagc gtgtacttgg gatactcgtt 3660gcaggataac actgtgtttg ctgtcaagat cttgccaacg gtggatctgc agagtagtcc 3720ggcgttgtac gaaagtatca agcgagaatc ggatgtgatg agcttgttga gtcatccgaa 3780tatcgttggt ttccttgggt tggaagtgca taggaacaga gtttgtcttt tccaagaggt 3840aagtgcttgt tgttgtttcc atttgtgttg ggagggtgtg gtgccaaagc tgatgttcgt 3900gattttagta ctgtgaagga gggtcgctgg caggtatgct cgaatatggc aaaattgacg 3960atgaggaagt cgttggagcg tttacgatcc agctgttacg cggccttgag tatctgcaca 4020ccaaccgcat cgaacaccga gatctcaaac cagaaagtaa gctgacaccc atcttttgat 4080cctttccaac acacacacac taactcgtgt tctccacaga tattctcatc ggcgccaatt 4140ctgtcctcaa gctggccgac tttggtaccg ccaaaatcat caaatccaac aagacgctcg 4200cccgtacacg tggtggcgcg cacgccaaga tggagggtct tgagggtaca ccgatgtaca 4260tggcgccaga gatgatcaag

aaccagagga ctggcaagct gggtgcttgt gatatctggg 4320gtttaggatg tatcgttttg cagatgatca ctggtaggaa gccatggagc ttcttggact 4380ttgataatga atggtacgtc ttttcttgca atgatgtttt ccgcgtaggg agttatgagc 4440tgataatatg attagggcaa tcatgttcca tcttggtgcg acaaaggagc cacctcctct 4500acccgatccc aacgagatgt ccgaccaagg tatcgaattc attgatcaat gtctttcttt 4560ggatccggaa gcgaggccgg tggccagcga gttattgcaa gatgaatggc tggttccaat 4620gttggagcag atggtgagtt gtctttcatg tatgtgtaaa aaatggtcag aagcttatct 4680gctttgcaaa acaggccgag ctggagcaag aataccccga tatattggcg atgggccaaa 4740gtga 4744112202DNACryptococcus neoformans serotype A H99 straingene(1)..(2202)genomic DNA of PBS2 11atgacagacc ctacgccccc cgccctggac agtctctccc tggcagacaa ggcgcctact 60cccgaagaaa gtcccgaaga cgccgctgaa cagcccaagc ccgcggcctc accgtccgca 120ggcacacccg gccatgacgc ccaaagctca tccacctcgc ccccgcaacg ccctcagtcc 180atgcagacaa atgacaaggc gccagataca tctgctccgg cttccaggcc ccaaccgcaa 240catgtccctg catcggcacc tgcgcttccc tctaccaacc ccgtccgtcc acagccgggc 300gcccgtcctg gagcggcgag gggtatgccc gcgcccatgg gtatgcgggc gcaagcaggc 360cgaggcgctg gcggccccca gatgcagacc aagatgctgc ccagtttgca ggctaaaatg 420gacaaggtgt gtatcgctcc atcatttatc ccgctgcata ctcatccaga ggctgtgctg 480acaaaccaca ctatgctatc attagatcgc ggcgtctcgg caagggccac ctccctcctc 540tggcatgcat gatccgaatg ccacatccat gggcgccctc ttacgctccc aagccctccg 600cgcccccggc gcatcgcaag ctcctcccgg ccccggaccg gcttcaggcc ctttcggtct 660cgccgctcgg cgcgcagctg ctgggggccc tccgagaccg aatttgggta tgatgggtat 720gggtgcaagt gcgccgggtg cggttggacg gggatcaggt ctggcgggta gacgggggcc 780ccctggagga ctgacactga gtgggatgaa gggtgcgatc aaggatgagg gaaacaagtt 840ttcagacttt cagggtgtca tgtgggttca gcagactcct tttccatgac tgtgggctga 900tctcaagtac agggacccgt ctggatcgct gagattctca aagaaggctg tcctgcatgc 960aaagggcgtg gactttgagg atgggcaaag tttcaagatc aatatggatg agatcgaggt 1020gcttggagaa ttaggaaagg gcaattacgg ttctgtgcac aaagtcttcc accgtccgac 1080aggcgtcacc atggccatga aggtgatctt attctttctt gcgtcgcttc tggtccagta 1140actaacaaac acgacaggaa atccggttag aacttgacga ttccaagctc aacggcatca 1200ttatggaact cgacatccta caccgggccg ttgctcccga aatagtcgaa ttctacggtg 1260cattcaccat tgaatcatgc gtctactact gtatggagta catggatgcc ggttcactcg 1320actctctcac cggtggcggt gtggcggcca aagatcaaac aaaggatgaa gaaaacgatg 1380cgacaaaacg agtgccggag gatgtattga ggaggattac agcgagaatc gtgaaagggt 1440tgaggttctt gaaggatgaa ttgcagatca tccatcgagg tgagttttcc atgtgcaatg 1500aaaacgggag gaaatgtgct gatatgatgt agacgtcaaa cccacaaatg tgttaatcaa 1560tggcaaggga gaggtcaaga tgtgtgactt tggcgtttca ggtcagctcg aaaagagttt 1620ggccaagacc aatatcggtt gtcaatccta catggctgta cgtctttccc tctcctccat 1680ctcaaagagc ctcccagcta acccgattcc ctctctttct ttagcccgaa cgtatcaagt 1740ctgaaactgc caaccagaat cctacatata ctgtctcttc agacgtctgg tctgtcggtc 1800tgtccattgt cgagcttgcc aaggggtgtt acccctaccc accggagacg tatgcgaatg 1860tgtttgcgca gttgcaggcg attgtgcatg gcactccgcc aacgttgcca cctgggtaca 1920gcgataatgc gaatgatttc gttgccaagt ggtacgtctc tcaccccttt ctcttcgtgt 1980ttgaatttga caatgctgat aatgagcgca atctttagtc ttgagaaaga tcccaaccga 2040cgaccgactt atgctcagct cttagaacat cctttcttgg tagcggacaa gggcgcagaa 2100gttgacatgg ttggatgggt ggaaggggcg ttgaagcgca aggcagagag ggggattgcg 2160agcctgaatc ctatccaacc acctgtccct ttggaaccat aa 2202121620DNACryptococcus neoformans serotype A H99 straingene(1)..(1620)genomic DNA sequence of HOG1 12atggccgatt ttgtcaagct ctccatcttt ggaaccgtat gtttctttta ttgctctttc 60tcttttccca ccaccgtcat gatctgctct tccaaccaac caacctacga acacgcggcg 120tttgtttttt ccgttggcca ctggatcata tcgtgttgat tctgtccata cgccggatgg 180aggagatctg taaaggcaag gccgcggacg ctgatggatg ggctttctcc atggataggt 240ttttgaggtt accacgcgtt atgtcgacct ccaacctgtc ggtatgggcg ctttcggtct 300cgtctggtga gtcttgtttt tctcaagcaa ctatcctttc atctggtttt tcaacccagc 360gtcgaaacag gtcgtccgac ctttgcatgt cgatgtagag atgtgaactg acaaaaccat 420cttgtttgat gcagttccgc caaggatcag ctgtctggaa cttctgtggc tatcaagaag 480attatgaagc ccttttcaac ccctgttctt tccaagagga cttaccgaga gctcaagctt 540cttaagcact tgagacatga gaacattatc tctcttagtg acattttcat ctctcctctc 600gaagatatgt gagttttgct caatagttgc atatcaaaga aggggggagg gggcctgctg 660acatttatcc aatagctact ttgtcaccga gctgctcggt actgaccttc atcgactcct 720tacctctcgc cctcttgaga agcaattcat ccaatacttc ctttatcaaa tcctccgtgg 780tctcaagtat gtccactctg ccggtgtagt ccatcgagac ttgaagcctt caaacattct 840cgtcaacgag aactgtgact tgaagatttg cgatttcggc cttgcgagga tccaagaccc 900tcagatgact ggttatgttt ctacgaggta ctaccgagca cccgagatca tgttgacatg 960gcaaaagtat gatgtcgcgg gtgagtttca agttttacgt ttgggggtgg tcttttaatt 1020ggcgatccat gctgaccacg caaaaaatca gttgacattt ggagtaccgg ctgtatcttt 1080gcggagatgc tggagggcaa gccattattc cccggaaagg accacgtgaa ccaattctca 1140atcatcaccg aattgctcgg tactccgccg gacgatgtca ttcaaactat cgcctctgaa 1200aacactctcc gtttcgtcca gagtctgccc aagcgcgaaa aggtcccatt ctccaccaag 1260ttccccaacg ccgaccctgt gtctcttgat ttgttagaga agatgctcgt gtttgaccct 1320cgtacccgta tatccgccgc tgaaggtctc gcgcacgagt atcttgcgcc ttaccatgat 1380cctaccgatg agcctgttgc cgccgaggtg tttgattgga gttttaacga tgcggatttg 1440ccggtggata cttggaaggt gatgatgtat agtgaaattc ttggtaagtc tctgtgcctt 1500gccttttttt gggtattata ctaacgtcgg actttagact tccacaacct cggagatatt 1560tcacagaacg aagcagaggg acccgttact ggcgaagtcc ccgctgctcc tgccagctaa 1620133792DNACryptococcus neoformans serotype A H99 straingene(1)..(3792)Genomic DNA sequence of ENA1(CNAG_00531.2) 13atgtcttctg agaaaggaca atcaaataca aacgagaaac aacttattaa ccgcgccgac 60actggcaaga ctgcagtgtc agactctcct ctccctttca aacctcatac cgctctctct 120ggcaagatcc tcgaggcttt agggagtaat gttacttctg gtctatcaga tgacgaagca 180tcaaggagac tccaacaata tggtcccaat aggctgaagc cccctgagag acctagtatt 240ctcaagatca tcgctaggca agtgggcaat gctatgactc ttgtcctcag taggtgttct 300ctccctttaa cgtttcttaa gctgatcaat ttcgtagtcg ctgccatggc aacttcattg 360ggtaccatgg actggatcag cggtggcgtt attgcggctc tggttatcct caatgtatca 420gtgggagcct acacagaatg gcaagccgaa aaggtacgtg ttttaagatc tgcggagatc 480ccataaaccc cgaggaattc tgacgtcata aatagaccgt ggccagtctc gagtctgttg 540gagctccgca agctactgta gtccgaactc gcaatggctc tcgcgaggct accgtcaaaa 600ttatccccgt agaggaagtc gtacccggtg acattattca actcaaaaat ggtgatattg 660ttcctgcgga cggaagaatc cttgacgggc acctgagtaa cttggaagct gacgaggctt 720tcctgactgg cgaaagtctg ccggttgcaa aacagactga gcctatcgat gaagaggact 780gtcctgttgg gtaagtagaa aagatttccg cacattcagc cacagcctaa ttatgaggac 840agcgaccgtg tttgtatggt cttttctggt tcccagatca ccaaaggtcg agctcgtgcc 900gtcattacca gcactggtat ggggacagag attggaaaaa ttgctcaagc tcttgaatct 960aaagctaaaa ataagaaccg tggatttgct gctttctggt ggaaagtcaa agttattttg 1020ggtgtcgagg agactactcc tttgcaaatc aagtatgtta tcttgctata gtagtgtaat 1080ggttggatgg tactgacgcc gatgctagac ttaataagct cgcatacttc cttttggcgt 1140gtgccctcgt catagccgtc attgttgtcg cctccaccgg ttttaatgat gtccccctct 1200ctattgccac ctacgctgtc gctgccgccg tctccattct ccccgcctct ttgattgcag 1260ttgttagttt gactttggcg cgtgcgtcaa ctgatttagc atctcgacat gctttggtcc 1320gacgaatgga tgctattgag gctttagctg gtgttgagaa tgtgtgctcg gacaaggtaa 1380gttaccattc aatttggctc gaaacctgtt gatacagttt tagccggtac ccttactgtt 1440ggccgcatgg tagttcgcaa agtctgggtt cctgctcttg actggcgccc caatgaattt 1500gctcccctcg acactagtgg tggtcaagca tatagttttg agaccggatc tgatcctttc 1560tatcctcgtg gtgaagtcct ggccgattcc cagaagatca ctgggactgc ggagaccctc 1620gatctcaagc aacctcgtga ccaatctgac tcttcctctt ccgactctga ccccgatgaa 1680cgagacgtag aggaacaaga acgggtcatc cacgttgaag acatggaaaa caaccttcga 1740gaccttgctc tctgtatttc gctttgtaat caagcgactc tcactcgtcc tgtcaaccaa 1800gacggccaat gggaagcaaa cggtgatcct accgaaacgg cccttcaagt tgctgcacac 1860aaacttggtc atggcaagcc ctttcttact catgctgcca agccaagcca ccgtgcggat 1920tctatccgat ctggtcacag ttctcgtccc cttgttgctg gtattcgtgg gcactttgtt 1980ccgataattg agcatccttt cgattccacc gtcaagcgaa tgtcaatcgc ttataaattt 2040gtgagcgagg atcctcagga ttctcacatc ctctgtctcc ttaagggtgc catcgagcgt 2100gtctttgaac gatgcaccaa gatccaagga cagcccatca ccgaagagca taagaagaat 2160atcatggtca aagttgatgc tctcgccgct caaggtcttc gggtcctcgc tctttgtgga 2220aagcgacttc ctgtcagcat ggtagacgaa gtcaaatcca cccctcgaga cgcattcgaa 2280gccgatttcc atttcctcgg tcttgctggt atcttcgatc cgcccagaaa ggaatctgca 2340ggcgccgttg ctgattgttt cagggctggt atcacccctc gaatgttgac aggcgatcat 2400cctgctaccg ctacagctat cgccctcaac attggtattc tcgataagac gtactcaaag 2460gattcagtca tgacgggtca gcagtttgac tctttgagcg aagacgaaat tgatcaactg 2520cccgagttgc ctcttgtcgt tgctcgctgc gcccccgaaa ccaaagtgag ctgttttaac 2580atatctaatg atgtacttgt gcctgacggt tcccagttcg aatggtcgat gccattcatc 2640gacgaggaca aagcactgta atgactggtg atggtgtcaa cgactctccc gccctcaagc 2700gtgctgatgt gggcgttggc atgggtactg gttccgatgt tgccaagcag tcagcgcgta 2760tcgtcctcag tgatgacaac ttcagcacca tcattcgggc tattaggaaa ggtcgttctg 2820tcttcaagaa cttgtctaaa ttcttgctcg tgagtaattc aatgcatgtg atggaaacga 2880agctgatctg gcttccttag tacttgcttt ccggtaactt ggctgaaatc atcgtcctca 2940tgattggtct cgctttcaag gatgacaatg gtcaggctgt tttccccctg tcacctgttg 3000ccgctctttg gatgtacgtg taactacctg attctttgca aggactttga ctgactccca 3060tttagcaaca ctctcgctgc cggacctcct gcccttgccc taggtcttga acctacagct 3120atcgacgcca tggagcaggg acccgaggta taccatcgaa tcttcactct tgaattttac 3180gtcgatctga tcttctacgg tttcctgatg ggctccatca gtttggtcaa cttcgtcatt 3240gtactatggg gatactatcc tgtaagttca gtttgcatcc ccaagaagca tccctaattt 3300atgaataggg agacttaggt cgtctttgta acgaagatga tcccagcatc tgtgatcccg 3360tctatcaggc tcgagctgcc tgttttgcca ccctcgttat tgtcctcatg attcatgctt 3420tggagtgtaa gcacttgagc aaagggttgg cccaaatcaa tttgcgtgac aacaaggtgt 3480tgctgtggtg tgtcgttgcc ctcagtcttt ccactgtaag ccctttacac catctatctg 3540gcctacgata tcagctaatg atgaacatag ttccctgtcg tgtacattcc tgtgatcaat 3600aacaaggtgt ttttgctcaa cggtcccagg tgggaatggg gtatcatctt cggcatgatc 3660ttggtgtatc tcagtgctac tgagctctac aagtggatca aaagaatttg gatccgacga 3720catgcccccc cttccaaagg accttccgac aagaccctta ggatggagag taccattgct 3780cctcctgttt ga 3792143173DNACryptococcus neoformans serotype A H99 straingene(1)..(3173)Genomic DNA sequence of NHA1 (CNAG_01678.2) 14atgactgctt tccacccctt tgaagtcaat gcccctcatc tcgcatacac gttcctcggc 60ggctttgtgg tcatctttgg catgatcagt ttgtttatca aagagaagct ctatgtcggc 120gaagcaccta tagcaactgt agtcggcatc atcattggtc cccattgcct caattttttc 180aatcctgcag gatggggtgg cggggaggaa gaggtcgcga gtgacgttac attggaattc 240actcgcgttg tcattgctat atccgtattc gccgtcggcg ttgaattgcc caaggtaggt 300ggtaacttgt gattcattgg agtagagaaa gcgctgatct tggtgcttgg gaaaggcata 360catgaagcgg cactggcgat cgctcttctt ccttcttggc ccgtgcatgg tgtggggatg 420gatgatctcc gccctgctga tctggggcct gatacctgac ctaacatttc tcgcctcgct 480cgtagttgcg gcgggcgtca cccccacaga tcctatcttg gcccaggcag ttatcggagg 540caagttcgcc gataaacatg ttcccgccca catccgccac ctcctctccg cagaaagtgg 600aagtaacgat ggggccgcct ttcccttcct ctacatcgcc ctctacctcc tactcgatgc 660gagcccaggc catgccgtcg gagaatggtt ctacatgact tggtgcgtag gcaaacctag 720agcatcctca gttacctttg ttcttgtcgc tcacatgtct ttttagggtc tacgaaatta 780ttcttggtgt tatcatcggg gccatcctgg gattctgcgc acgcaagttg atgaagttag 840cggagcgcaa acgtctcatt gataggcagt cttacgtcgc ccagtatgtc agtctggcag 900tgctgtcgat tggtaagcgt gttgaccggc cttgcgatta ttcaatgagc tgaccacgat 960gtaggcgtta caagtttgct cggcagtgac gatttgcttt ctgctttcgc ttgcggttgt 1020gcttttgcat gggagtacgt agtgcttgtg atttatctct tgactgcgct gacgacatta 1080cagcggtttc ttcaacaaag ctacggagga tgcggtgttc tcgaacgtta ttgatctact 1140tttcaattgc gccgccttca tctatatcgg cgctatcatt cctttcaatc attttaacga 1200tttgcccgat gtacgttcgc atgggctacc acatacacga actaactgga tctcagctcc 1260gagtatggcg attggttgtg ttggctatcc tcattcttct agttcgtcgt ctgccttcta 1320taatagcgtg ttacaaattc gttcccgata tcaagacgtt cagagaagct ctttttacgg 1380gatggttcgg tgagttcctt attgtgagta tcggttgggt catgtctgat gagaggttag 1440ggcctatggg cgtcggtgct gtattcatct ccactcttgc tcggtcgtct ttgccagaag 1500gggagcctga acagaataca gaagcggtgg accgcctaaa agacgtcatc atgcctgtca 1560ccttatttct tgtattgtct tcaatcgtaa ctcgtaagtc tatcccgtcg cactcactac 1620tgactcagtg tagacggcat gtcaattcca tttttctctc ttggtcgccg ggtccattcc 1680attacttata ctcgatcacg aaatctttcc atggacacgc gaggcgatga gcctgcctgg 1740acaactcatg ctcggcgtat tattccaggc caggagatca ttgtcaaccg tgatgacgac 1800gacgaagaag gcgacttggg tgttagacgg atggacacac tcacgagcga ttcaaatggt 1860cgtatcaggg aaaagattga ggaagaagat agcggagaaa gtagctcatc ccgaacaagg 1920cagggagaaa tgattgaaat gacagaaaaa cgtggcccgg ctcgccatgg tagccaggcc 1980agccagggcg aagcggcgga ggaaggagag aggtggagaa gttcgggaga agaaagctct 2040gatcttgcga atgaccctga gacacagaga gaggtggaag agggaatgga agaggtcgaa 2100gataaggaag gaggtggtag aagaacgccc cccctggcca agtacagaga aggaaaccac 2160ctcattgtgg agagaaaagt caaggacagt gacgaggtat gctatgcgga tgtcccaatt 2220gcttatcacg acttgctcat gcgtatggct tttaggttga agtcgaggtc atccgaaacc 2280atttttccga caacaagaaa acggaaagtg accgcttcac tcatccccat cgcctcaagt 2340cacgagagct tgacgatttg cttcatcacc ttcccaaaag cctcgagcat gctacttcac 2400gggttcaaaa tggcggcaaa gatgcagttg atcgtctcgg tcttgggctg atggctatta 2460acactccgga accgtcacca tcgatcgaat cgcacggcgg tccaaggcat gattatgtcg 2520atggcttgga gagaacgcag agcccagagg gtcttgcaga cgaggatagg gatagcgagg 2580gccggggcga tgtgtcccat gggggtgatt atgaagaaaa cgaggccgac tatgaggatg 2640ttccgaacga gactcgtcgg caaaggagga agaaaatgaa accaccagca attgtcgtct 2700ctcggcagaa cagcgccggg ctcccgagac gatccatccg ctccaggctg ttcggccgac 2760gacaacattc ttccaactct ccctcccgtg ccgaagaagg cttagcccct cccaatccat 2820ctcttcttgt tccatcctca tccccttcgc gtcctcaaaa cattgctgca gaacccgagt 2880ccatactggc agaagattcg cgcggatcat cctcaccttc ccaatctcaa aatcttgcga 2940tccctctcac aagaaccctt tcagctagcc gatcgtcgcc tgcggtgcgc ttcgccgacg 3000atgctagtcc ttcatcggac acagcgcctg ggcagtcaaa ttatggtact aacgctccag 3060gtttcaagaa gaatccggct ttagcaatgt atcgatcggc cagtgtacaa agtacagggt 3120ccaacaagga tgggcctagc gtatctttca aagaacctga aatcaagcgt tga 3173153930DNACryptococcus neoformans serotype A H99 strainC_region(1)..(3930)coding region sequence of SSK1 15atgtggggct caaatgcttc catcgccgcc tcggagtcga ccgactccct ttcccccgcc 60ccctcccagt ctgccgccgt agagttcccc ctgcccgtaa gctcccgccc gtctctcact 120tccgccgctc acccctccca gatgtccgct tcctcctcct ccacctcctc ccagcctctc 180tttgattggc gcattcccaa gcccacctca ccccgcacac gcatggaccc attcgacact 240tttgatcctg tatcctcaag ctcagaggat gatccggtcc ctcaagagag ccgccgtgct 300ggtcatcagc gctccgtaac agatcctctt ttacgagatg gccaacccct cgatatggag 360ttcactactg ccgggccgcc tatacagagc tacgactttg aacaaccgcc cacgtttagc 420agaacgttat cttcccctct tccagctaaa gtcggctcgc ttagacaccc tatgccattc 480accattgacg atttaagctc tcgcaatgtg aattcaaccc atcgtccgca gccgactaca 540cctctgcatt ccatatccgt tgagttagcc gattccttgc aatcggccat tcaaacatta 600ctgcatctgt ccccacctca cctactcgat aacgcaaaag aacagtactc tggatgcacg 660gtacagatac ctgctacctc gctttcggcc cttttgacct ctatgagagg cctcaacttc 720ttgtcggcgc atgctgaaga actggtcgac atgagtgcac gtggagatcc acctgtactc 780catcaagaag acttcgatgt aggagaactt ttgcaaaacg ttgcggatat gttgagtgga 840gaagcagcag aaaaacggat tgatttcgtt ttgttccatg gcgacgtagc gatgaggcat 900gtcagtgtgt atggagattc tgatggaatt agctatacct tgagtcatgt tattcgacaa 960atactggcag tagccaatta cgatgatacc atagaactcg gccttcaagt cattcctcaa 1020agtccatctt tagcttccgc cgtcggactt cctctaacct ctgccgatgt tagtggagga 1080ggtggtgtca agtcagcgtc cacatctcgg tcaggctccc ccaataacag tctctctcga 1140tctaattctg tccatgacgg gccccttctc tgtgtgttcg aaatagtaca taacatctat 1200cagccaccac caagctcggc atccgccact cctaaagccg agctgaaccc tttcactcat 1260cttgctgaag aaaccgaagc cttgaaacca agattggata cagcattttg caaaaacctg 1320cttcatcggc aaaatgctgt cctcaaagtt gatgtgcagc cttcatctcc tttaggatcc 1380gggatgcccc gtagagctta cgcgttatca gtgctcctac caagaggtaa acccatcact 1440gagcctgcaa tactttctaa agaggaacaa gaagttcgtc aaccattttc atcccacgta 1500cttgcacgag aacccaccct caatgagctc tcggaatttg ctgaatcatt acgaggaaga 1560aaggtgttta tccatgccaa tttgagtagt gttttcgcga gacacctcac gagctaccta 1620gctgcatggg gaatggatat atcgcatcta ccgacagatg gcgatgaggc tgataaattg 1680aaggatgtcg cggccaaaca tgactcggct tatactggat ctatgggtgt gtcaggcggc 1740actacttcca gcgcagaaac gccctattca attaaaccga ccggcgtgac tgctgttcaa 1800cctggacact ttgtcattat cgacgatgat gttgcggtct tgcgccgtga actcgtgcgc 1860atccgttcag aattacttcc cattctcttt aaacctagac tgtcaaagcg tcccactatg 1920acttctcgaa cccgttccac cccttcattg cgacaggtcc ccccaaggtc atcatcgggt 1980tctgtactta tacactttac ctctcttgcc aattataacc gagttcgaga cgcgattgcg 2040agctttgtgg gggcgccagg gttaaccaat ccggaaactt atgttcagcc ggaggtgata 2100gtgataccca agcctgttgg accacgaaga tttttgactg ctctgcatac cgctgtgaaa 2160cagcctatgg ttgacccatt tttctcccct atcgccacat ctcctagatc accaggcgga 2220ggttactttg gtggtttgcg gactccgacg gagagagaat caggattctt tgattctgtt 2280gcagaagaac cacatgaaga ggcggattcg cgaccagatt atgccacggt gcagaaagcc 2340agatctcctt taggagaatt tccgccttct gcggcacaga tcgttcgtac caaccaaggc 2400ttgcatcttt cgcttcccac tccaaatgaa attatgacaa cgcctgctcc agaatatttt 2460tctgggtctt ccaagtctcc tagctctggt gcgtccggag tcgtgatgca gagccctgat 2520ggtcgtcctt tcggaatgtt tttcgaaccg cccataaaaa atgagcgccg cggatctact 2580cacaggacgc cttccgattc catcaggagg aaacaagcga accgccgtgc gtctacaagt 2640gatgaaccct tttcttcacc ttctaccgcc ctacctcccc gtcgctcgtc cacaatttct 2700acgactggca atgaggaaca ccgcagttca cctatcgcta acgtcacaga ccgtcctacc 2760cattcaaggg taaattcaag aaggaagaac aatcttccgg cggcggagca acctattttg 2820gctgtgggca gggcaaaagg cagggaaaga tcggagactg tcacgaaggg aggggacctc 2880gggtcgagaa aaggtacacc agcggcaagc ccacgtatag aggagaagaa ggaattggaa 2940agaggcgaga agactaaaag cctggctcct tcaactgctc ctacgaagaa gaatgctaaa 3000gtcgatgttg tggtgccgcc catcaacgtg ctgattgttg aagacaaccc catcaatcaa 3060aacattttga gtatgttcct gagaaaaaag aagataaaga attcctcggc caaggatggc 3120gcagaagctg ttgaaaagtg gaggactgga ggcttccatc tgattctgat ggatatccaa 3180ttgcccgtca

tggatggcat agctgctacc aaagagattc gtcgacttga acgtcacaat 3240aacattggcg tttttccatc gactccagcg gccgaacttc ctcggggtca aaatgttgcg 3300gattctccac caccatcttc tccatttcgc tcgtcagtta tcattgttgc cctgacagcc 3360tcgtccttgc aaagcgacag agtagctgct ttggctgctg gctgtaatga cttcttgacc 3420aagcctgtgt ctttgaaatg gttggacaaa aagattgtgg aatggggttg tatgcaggca 3480ttgattgatt ttgacggctg gcgacgatgg aagagctccg ataccaagaa tcctagcgaa 3540actaagcagg gcttctcagt gggccctcaa caggctgcta ggtcgcttgc tagcagacta 3600cgtattgaac gcaaaggatc tcgatctccg gcagctccag tatcaacccc gcgactcaat 3660ttgcagtcgg caaccccaga taggccagaa acccccccag attccacgtc acaaatgcca 3720aaggccccgc ccgttgcagc ctctgacccc ccgttatctc ccaagtcgct gaacaagaca 3780gttaatgatg tcttcgagca agcagacgct agactcgaaa atgcgcggga ggaacaagga 3840gtatcaagtc aaaaggaaaa cacaagctta acagattcaa caaacaccac cattacgccc 3900tcaaagacct atccggctcc tcccccatga 3930165076DNACryptococcus neoformans serotype A H99 strainC_region(1)..(5076)coding region sequence of TCO2 16atgatcttag gaaccgacat cgacctgtcg tctataccaa cggcgtttct cgaggcttat 60cccttcccag cggttgtgtt cgtgatcgat tctcccccta gcccaagacc gcggctccat 120tccagaaata cagacacgac cattcggcga acggatggcc aaatatcgcc tcttacaggt 180cctccagtgc aacagttcgc gtcagcgccc gtggtatggg gcaatcaacg atggcacgag 240ctggctcagg ggaaaacaat tgcagagtgc gtggatgtgg cgtcacagaa caagctgcaa 300acttgggtgg aaaatgacac cggtgacaag tcggagagtt tggccctgga cctgaaggtg 360ccgcaaggcg tgactcttca tctggcaaag accatattgc cattaagtcc accgtcctcc 420tctcagtcac tttgcatcct tatatcgcaa tatatcgata agccggaaag tttcgcgcca 480ccaatctcat ctggagatat tcttttctct tctctatcgc gactctccca gactttttct 540cggtcatcct ctttttcatc caaccctaga aaatcgattg atgtccctgc gtcactatcc 600gaacaccggg gttctgctac atcgacaagt agcaatctgc gctcttcgat cgatttgact 660tcccctaatt ctcaaccctc tccactaaac cgtgaacaaa gcacgtactt cacccatggc 720tccgcgacca gagaagagcg accctcagta aggcgtagac ggtcaccgcc aatctcaatg 780acgaggccca agcctcttga gagccatgct caagaatgct gggacttggt agagaatttc 840gactggtcaa aaacagcatt agggccgaga gaacagtgga tggatgcgtt agatcctgtt 900ctggcaatca catttgaatc cagaacggca gattgtgcct ggttagggcc tgatctagag 960ctagtttaca ataaggcgta tcaagagctg gttgaccatc ccaatgcttt tggaaaacct 1020gcaagacaag tttgggctac caattgggac tacttggaac ccctggtcaa gcgatgtctc 1080agtgggaccc cggtctacaa ggacaacgac ccgcttttct ggcgtcgata cggcaatggt 1140cgacttctgg aacattacca cacttggcga tatgtcccga taacgggcaa agatggctca 1200gtgcttggca tcttcaacca gtcaattgag gtcaccgact cagtactgct agagaggcga 1260atgggcacga ccagggaact ttcggaacac atgtcgttta ttcgtacaac tgaggacttt 1320tttagctcgg ttgccgacgt ctttagtcag aaccctactg acataccgtt cgcactttgt 1380taccgggtcc gacaagttga caccgatggg acatttgtcc atttggacgt ctcgcttcag 1440tcgtccgtcg gtgtacccga aggccatccg tctgctccag atcaaattcc cgtcagcttc 1500ttaaatggta acccttaccc tagcaatgtc gagcgatcat tttctcctgc tttctcaatc 1560gtttcaatcc actcttcgag cagtcatcga gtctgtcacg tctctgaaga cactacacaa 1620tggcccatcg ccaaagccct acaaaggcgg caatgtgtca tcatcgaaga atgttcgcaa 1680ttaatagaag gatatcctat ccgtcgctgg gatgggcttc cattctcagc cattgtcgtg 1740cccatatgct ctgaagggtc tcccgaaatc cctgacgccg ttgttattct tggtctcaat 1800gtgcgacgtt gttttgacca tgaatacgat tcctggattc actctattcg gtcacaacta 1860tcttcggccc tcgtgatggt caaggcgcgt gaagctgaac aaaagatggt tgaggaaagc 1920gcacgtatgg agaaagcaaa agtcgcttgg ttcagaggag ccgcgcacga ccttcgtagt 1980ccattaaccc tcgtcgctgg accgcttgcc gatgtgcttg attcggattt gaactcgagt 2040cagcgcacgg ctttgaccgt tgcgcaacgc aatcttgatc gtttagtgcg cttggtcaac 2100gccctcatgg atttctcgag ggtggaagct ggacgaatgg aaggacgatt tgttccgacg 2160aacttgagtc aattcgtgac acagttggca gctcttttca agcctgcaat agaaagattg 2220gggttagaat acgtactaga tgtccagcca agcgaggagc ttgttttcat cgatcctgtt 2280ctgtttgaga ccgtggtatc aaaccttatt ggcaatgcgc tcaaatacac tgaaacgggt 2340tctatcactg ttcgggtgca atacacggat tacgcagagg tctcggtcat cgataccggt 2400gtgggtatac cgaaaaatga gctggcactg gtgaccgaat ggttccacag ggcaagtact 2460gccattcact cgggaaccca gggaacagga ttgggactgg ctttggccaa ggaattgctc 2520aagttgcata aaggagaatt gcttgtcgag tctcaaaccg ccaatgagtc aggaggtcct 2580catgggtcca tttttacagc gaaaattcct cttgatttca agccctctcc atcggctcat 2640atcattccgt ccgtcgaatc tcacaagacg tttggcaaat acagtaaagc cgtcgcagac 2700gaagccatgc gctgggttgg ggactcagat gccgctagtg aggcgtacga catgtcgagc 2760ggtaccggag tctcaagcgc tggtagtggc tctggaaaca cgaccacctt cggacccaag 2820tttgcagatg cctttttgtt tgataagaac gacattgtgc ttattgtgga agacaatgtc 2880gacatgcgtg aatacatacg acagcttttc gccccttatt gtaccgtact cgaagcttcc 2940aatggtgaac aggcttacaa tatggctacc caaaaccctc ccaacctcat tttgtcggac 3000gtgctcatgc ccaaattatc tggtatggag ctactacaaa ggatcagatc ccatcctgac 3060actcgcattg tgcctatggt ccttatttcg gctattgctg gtgatgagtc tagggttgag 3120gctctgctaa acggcgctga tgactatctt gccaagcctt tcaaacccaa ggaactcatc 3180gcgcgtgttc acctgcacat gcaagttggc aagaaacgtg ccaagctcga agcgctatac 3240gcccaacgcg aaacagaatt gacagctcta tctgactatt gtccgatcgg tatcttccga 3300ggagacaaat atggccatat tgtttatgcg aacgcagctt ggcgtgcgca gagcggcctt 3360ttggtgggtg accctaacga ttgggcatct tatgtgcacc cggattcgaa agcgcagctc 3420ttggaacaat ggaatcagtg gttgaggggg gatttgaagg agttccgagc ggcttggaga 3480tggtctaatg gcatccctgt caggagcatc ttggtccggt tagatgacgt caaggaaggg 3540ttttctgggt taattgggtg cgtagtggat gtgtctcatg aagagagacg attaatcgaa 3600gctgaggaaa gaagaaaaga ggcggaagag agtaaacatc agcaagaact ccttattgac 3660ttgacaagtc atgaaattag gaccccggtg tcagcaatcc tgcagtgctc agatcttgtt 3720aaagagaatc ttgtagctct gaaggaccag ttgagaggag cggggccaaa gggctttgtg 3780ccgagtcaag aattactggc tgatcttgag caggatgtgg aagctttgga aagtatttat 3840cagtgcggtc ttgtgcagga acgcattgcc ggagatgttc tttcgctggc tcgtatccaa 3900ctcgatatgc tgagtttgca cgacattgac gtcaacttgc gccgagaagg caggaaagtt 3960tcgtccatct ttgcatcgga agccaagatg aaggatatcg acctccaatt ggaatttgga 4020cctactatcg aacagtccaa agtgctggcc atcaagacag atcccgtgag attaggccag 4080gtggtaacaa atctcatttc caacgccatt cggtttacat cttcgagtga tgtccgaaag 4140attactatcc aatacgacgt atcgtttgtc cctcctgccg atgactcttg cgccctccct 4200tcatctgttg gcttgcccga catacttcct gtgaaagaga atactccact atggctgttt 4260gtcagtgtta ccgattctgg acctggtatg acagagcaag agttatctgt tttgttccaa 4320aggtttgccc agggcaataa gatgattcat actaagtatg gcggaagcgg tttgggactg 4380ttcatctgtc gaaagattac agagcttctt ggcggtcgta tcgaagtgct cagccaagtc 4440gggcacggta gtgttttccg attcttcatt aaaacgcgcg ctgtcgctcc tccgtccgcc 4500atcgctgctc tcgtagaatc ctctcctctc aaaccggtat ccgccacttc accttcttcc 4560tcgttagcca tgagccgatc gtcttctcgg agcacaaacg tcactacacc tatagagggt 4620ggtgggaccg agcacgtgtt gatcgtggaa gataacctaa tcaatcagac tgtcctgaag 4680cgacagctcg tcaaggcggg tttatcgtgc aacgtcgcga gtaatggcct tgaagctctt 4740aatgtcatcc gtgaagtcca tcggcaacac cgacgcggtg ggccgaaccg taaaaggcta 4800tttgacgtgg tattgatgga tctcgagatg ccagtgatgg atggtatcac cgccgtacga 4860gagatacgac agtccgaggc cgcggggaca ttgggcagga atatggtgat tgccctgacg 4920gggaacgcga gacaaggaca gattgatcat gccttggctt ctggatttga cgatgtcgtc 4980atcaaaccgt atatcctggt agatttgctg aacaagatca aatctatgaa agttagaaaa 5040ttggagttgg aaactgcgaa agctcaagaa gagtga 5076174254DNACryptococcus neoformans serotype A H99 strainC_region(1)..(4254)coding region sequence of SSK2 17atgtccaacc caacctcccc ctcaaacccc tcagacaccg gcccgtcctc ggcgtccaac 60gtcacatcct cgtcctcaaa gaccggacgc agatcagtgc gcctctttgc cccagacgag 120gaggacagct cagacgagga cggcggcctc atcggcgtgc ccgcagagac cacattcaag 180gacgacgaga tccctccttc caacccacgt tccgcctcct accccgggcc accggcacac 240acctccccca cctctaaaat ctcaaccatc gtttcgtccg cttctgcagc ccagccaaaa 300cttgcacgtt caataacata cgtcgcaccc aatgccgtct cctcccggcc ggcatatccc 360ctcaatcccg cagggtcaga aaccttacac gcctcaggaa ggtcgtatac tgaccccgat 420ataggatact ttagccacga tgcaggagat gatggttggg gctcagatga tgacgacgaa 480ttgagatccc ctggctgggg catatcccat cataacatgg actctggagg caagacgaac 540gggtcaccac agttgcctat aaagcccgcc gatgtcaccg aggatgaagg acaggaacgt 600ttagattggc aaggcatgct ggaaagtgtc ctcaactcgg atgttctcaa ggtggaggaa 660caacgtatct acaattccat gccgacagat tcattcagag aagagattgg aaagaccctt 720tggtggcaaa tccgtgccaa actgcgtggg aggacagagg cggaggagaa gaaacgggtg 780caagagcgac gagcgagagt ggtggacccg gtgctggaag agataaacga gttcaagtac 840gacccaaaaa ataacccaga aggcgaagaa gacagtgatg gcgatccgca agacgcgact 900tcgactgctg caccccaatc caaagctctc aatcaagtca acaccgttct cgccaaactt 960catgcaatca aaggtcttta tcccaacctc gcagccatgc gagccgacaa ggttctctat 1020accgatgaaa atttccgcaa acgcgccgac gcattgacct cttggtccat catcgtttca 1080tccctccaaa cccagctcaa actcttgcaa aaatggacag gttccgatga gcttgacatc 1140accaagccca acacgaccca cgagaaagca ttggtcggca agtacaagta tcactctatc 1200gacagcaagg gtacgcccgg cagggatgca gccgatgact cgagtttcct cgatcgtgtg 1260ataaaagaag ataaccttca acggacattc gagcgtcgag cgtttgtaga catgatcaac 1320ctcgtgcgca acgccaagga gacggtcatc agctatctcc cccagttcca agaacaaaat 1380cttcccgatt tccagtacga aatcgttcgt cttattggtt tccccggtcg acttatcatt 1440gaagctgtca aggttcgttt ggatgctgca tcccgactac ttgacccgaa ccctatggtc 1500gtcgaagact ttatcgaaaa ccttcgtcta tccatttcgc tcgccgtgct aatccggaaa 1560caatacgacg aaatcatggc acccgatgcc gaggggagat ggaaaatccc gcattgcttg 1620ccgacagagt acaatgatgt tctgctcgat gcgctgagga catttttcaa attgttgcat 1680tggagattac gaggagtggg gaaagcgagt tattacaagg aaacagaagt gttggaagaa 1740gaggcgccgt tcttgtatga agcggcggag gctattgtag gcggtgatat ggttgttgca 1800gagcagtatt gcgcgttatc caacaagctc cttatacgtt cagcaaatta tcttgaccag 1860caacttcggg taccaataca ttccccgtct cgcgacaagg aacgtggtga caaggagcgc 1920gatggctctt cgtcttctca acgtaaccgt gacggccgtg atagctcgct gcccggccca 1980ccgaaacaca tgaaagtcga agaactcttc tcatggtact ccaaactcct tgattccgct 2040cgtatgcgac accgtaaaac ccaacgtttc tgtcgtaaac tcacccaacg attcgataat 2100tccgccgaat attcaatcga ggagacggag atggacatgc tggtggagac attgcaagat 2160actggtcatt tcttggtata taccgggaaa tttgaggcga atgggacgta tatcgttgcg 2220gatgggagtc tctggggtca gccggacgat gtgagacatc tgttgaagag ggtgttttca 2280gtgacgattc ctggatctcg agtccgtcca aggcagacaa cctcgcaagt atctgtcgga 2340ggtgcgagcc cgtccaatgg tcaagtcgcg gcgcaacatg atcctgcaga tccgtacccc 2400gaggcagacg attttgacga cgaagcgctc gcggcttaca tcctcctcat ctccccacgc 2460caaagttttg tatggtccgg agcggtcatg acgctggatg tggattacat cgactatgaa 2520ctacctgata accgagtcag attgatcgct gacggtccca ccaagcggtt agcgctgtgc 2580aaactttatt tcaagcaagc gctcattcac cctgatacgg gcgaaacaat cgacttgcca 2640tgtgtggttg aggctcaagc gcatttaccg accattcaga aacaacttgt caagattgct 2700aaatcgagtt atcgtctttc agagtgcatt gtccagtctg caccactcgt ccgcaatgcg 2760ttcaggggca aaccgggatc acaagagttg gtggagaatt ggtacagttt tgcgacagag 2820catgggacga gagtgttgat ccatattgag cctagtgtat gggagcgatt caatcggttg 2880ttgatgcgtc tggcgatcag ttggattagc tttatcagtc aagagtgtaa ccccacagac 2940cgcaagacgt tccgatggac tgtggcagct ttgacctatg cgttcaacat gacgagaggg 3000agtaacattc tcgcgcttga tcgatcagaa ttttcgcttt tgaggaggtc gagtatggag 3060gccaaaaagg aggcagacag gattgaggcg atgaggaggt tacaacggct tcaagaaaac 3120ctggacgacg aattcctgcc ccggactccg acagagtctg gcgatcaacc acgtatcgac 3180cgctctataa ggctcacagt cgaagaacgt ctccgtctca ttgccgagct cgaagctcgt 3240cgtgacgagt tggcacccgc acccgtcggt caagtccttg atgaagaagt ctctgaagac 3300cgtgcgttgg tgttccttgc agcttccaaa tccaacattt ctatgcgatg gcagcaaggc 3360gcgtacatcg gtggaggtgc atcgggaagc gtgtacttgg gatactcgtt gcaggataac 3420actgtgtttg ctgtcaagat cttgccaacg gtggatctgc agagtagtcc ggcgttgtac 3480gaaagtatca agcgagaatc ggatgtgatg agcttgttga gtcatccgaa tatcgttggt 3540ttccttgggt tggaagtgca taggaacaga gtttgtcttt tccaagagta ctgtgaagga 3600gggtcgctgg caggtatgct cgaatatggc aaaattgacg atgaggaagt cgttggagcg 3660tttacgatcc agctgttacg cggccttgag tatctgcaca ccaaccgcat cgaacaccga 3720gatctcaaac cagaaaatat tctcatcggc gccaattctg tcctcaagct ggccgacttt 3780ggtaccgcca aaatcatcaa atccaacaag acgctcgccc gtacacgtgg tggcgcgcac 3840gccaagatgg agggtcttga gggtacaccg atgtacatgg cgccagagat gatcaagaac 3900cagaggactg gcaagctggg tgcttgtgat atctggggtt taggatgtat cgttttgcag 3960atgatcactg gtaggaagcc atggagcttc ttggactttg ataatgaatg ggcaatcatg 4020ttccatcttg gtgcgacaaa ggagccacct cctctacccg atcccaacga gatgtccgac 4080caaggtatcg aattcattga tcaatgtctt tctttggatc cggaagcgag gccggtggcc 4140agcgagttat tgcaagatga atggctggtt ccaatgttgg agcagatggt gagttgtctt 4200tcatgccgag ctggagcaag aataccccga tatattggcg atgggccaaa gtga 4254181830DNACryptococcus neoformans serotype A H99 strainC_region(1)..(1830)coding region sequence of PBS2 18atgacagacc ctacgccccc cgccctggac agtctctccc tggcagacaa ggcgcctact 60cccgaagaaa gtcccgaaga cgccgctgaa cagcccaagc ccgcggcctc accgtccgca 120ggcacacccg gccatgacgc ccaaagctca tccacctcgc ccccgcaacg ccctcagtcc 180atgcagacaa atgacaaggc gccagataca tctgctccgg cttccaggcc ccaaccgcaa 240catgtccctg catcggcacc tgcgcttccc tctaccaacc ccgtccgtcc acagccgggc 300gcccgtcctg gagcggcgag gggtatgccc gcgcccatgg gtatgcgggc gcaagcaggc 360cgaggcgctg gcggccccca gatgcagacc aagatgctgc ccagtttgca ggctaaaatg 420gacaagatcg cggcgtctcg gcaagggcca cctccctcct ctggcatgca tgatccgaat 480gccacatcca tgggcgccct cttacgctcc caagccctcc gcgcccccgg cgcatcgcaa 540gctcctcccg gccccggacc ggcttcaggc cctttcggtc tcgccgctcg gcgcgcagct 600gctgggggcc ctccgagacc gaatttgggt atgatgggta tgggtgcaag tgcgccgggt 660gcggttggac ggggatcagg tctggcgggt agacgggggc cccctggagg actgacactg 720agtgggatga agggtgcgat caaggatgag ggaaacaagt tttcagactt tcagggtgtc 780atggacccgt ctggatcgct gagattctca aagaaggctg tcctgcatgc aaagggcgtg 840gactttgagg atgggcaaag tttcaagatc aatatggatg agatcgaggt gcttggagaa 900ttaggaaagg gcaattacgg ttctgtgcac aaagtcttcc accgtccgac aggcgtcacc 960atggccatga aggaaatccg gttagaactt gacgattcca agctcaacgg catcattatg 1020gaactcgaca tcctacaccg ggccgttgct cccgaaatag tcgaattcta cggtgcattc 1080accattgaat catgcgtcta ctactgtatg gagtacatgg atgccggttc actcgactct 1140ctcaccggtg gcggtgtggc ggccaaagat caaacaaagg atgaagaaaa cgatgcgaca 1200aaacgagtgc cggaggatgt attgaggagg attacagcga gaatcgtgaa agggttgagg 1260ttcttgaagg atgaattgca gatcatccat cgagacgtca aacccacaaa tgtgttaatc 1320aatggcaagg gagaggtcaa gatgtgtgac tttggcgttt caggtcagct cgaaaagagt 1380ttggccaaga ccaatatcgg ttgtcaatcc tacatggctc ccgaacgtat caagtctgaa 1440actgccaacc agaatcctac atatactgtc tcttcagacg tctggtctgt cggtctgtcc 1500attgtcgagc ttgccaaggg gtgttacccc tacccaccgg agacgtatgc gaatgtgttt 1560gcgcagttgc aggcgattgt gcatggcact ccgccaacgt tgccacctgg gtacagcgat 1620aatgcgaatg atttcgttgc caagtgtctt gagaaagatc ccaaccgacg accgacttat 1680gctcagctct tagaacatcc tttcttggta gcggacaagg gcgcagaagt tgacatggtt 1740ggatgggtgg aaggggcgtt gaagcgcaag gcagagaggg ggattgcgag cctgaatcct 1800atccaaccac ctgtcccttt ggaaccataa 1830191098DNACryptococcus neoformans serotype A H99 strainC_region(1)..(1098)coding region sequence of HOG1 19atggccgatt ttgtcaagct ctccatcttt ggaaccgttt ttgaggttac cacgcgttat 60gtcgacctcc aacctgtcgg tatgggcgct ttcggtctcg tctgttccgc caaggatcag 120ctgtctggaa cttctgtggc tatcaagaag attatgaagc ccttttcaac ccctgttctt 180tccaagagga cttaccgaga gctcaagctt cttaagcact tgagacatga gaacattatc 240tctcttagtg acattttcat ctctcctctc gaagatatct actttgtcac cgagctgctc 300ggtactgacc ttcatcgact ccttacctct cgccctcttg agaagcaatt catccaatac 360ttcctttatc aaatcctccg tggtctcaag tatgtccact ctgccggtgt agtccatcga 420gacttgaagc cttcaaacat tctcgtcaac gagaactgtg acttgaagat ttgcgatttc 480ggccttgcga ggatccaaga ccctcagatg actggttatg tttctacgag gtactaccga 540gcacccgaga tcatgttgac atggcaaaag tatgatgtcg cggttgacat ttggagtacc 600ggctgtatct ttgcggagat gctggagggc aagccattat tccccggaaa ggaccacgtg 660aaccaattct caatcatcac cgaattgctc ggtactccgc cggacgatgt cattcaaact 720atcgcctctg aaaacactct ccgtttcgtc cagagtctgc ccaagcgcga aaaggtccca 780ttctccacca agttccccaa cgccgaccct gtgtctcttg atttgttaga gaagatgctc 840gtgtttgacc ctcgtacccg tatatccgcc gctgaaggtc tcgcgcacga gtatcttgcg 900ccttaccatg atcctaccga tgagcctgtt gccgccgagg tgtttgattg gagttttaac 960gatgcggatt tgccggtgga tacttggaag gtgatgatgt atagtgaaat tcttgacttc 1020cacaacctcg gagatatttc acagaacgaa gcagagggac ccgttactgg cgaagtcccc 1080gctgctcctg ccagctaa 1098203273DNACryptococcus neoformans serotype A H99 strainC_region(1)..(3273)coding region sequence of ENA1(CNAG_00531.2) 20atgtcttctg agaaaggaca atcaaataca aacgagaaac aacttattaa ccgcgccgac 60actggcaaga ctgcagtgtc agactctcct ctccctttca aacctcatac cgctctctct 120ggcaagatcc tcgaggcttt agggagtaat gttacttctg gtctatcaga tgacgaagca 180tcaaggagac tccaacaata tggtcccaat aggctgaagc cccctgagag acctagtatt 240ctcaagatca tcgctaggca agtgggcaat gctatgactc ttgtcctcat cgctgccatg 300gcaacttcat tgggtaccat ggactggatc agcggtggcg ttattgcggc tctggttatc 360ctcaatgtat cagtgggagc ctacacagaa tggcaagccg aaaagaccgt ggccagtctc 420gagtctgttg gagctccgca agctactgta gtccgaactc gcaatggctc tcgcgaggct 480accgtcaaaa ttatccccgt agaggaagtc gtacccggtg acattattca actcaaaaat 540ggtgatattg ttcctgcgga cggaagaatc cttgacgggc acctgagtaa cttggaagct 600gacgaggctt tcctgactgg cgaaagtctg ccggttgcaa aacagactga gcctatcgat 660gaagaggact gtcctgttgg cgaccgtgtt tgtatggtct tttctggttc ccagatcacc 720aaaggtcgag ctcgtgccgt cattaccagc actggtatgg ggacagagat tggaaaaatt 780gctcaagctc ttgaatctaa agctaaaaat aagaaccgtg gatttgctgc tttctggtgg 840aaagtcaaag ttattttggg tgtcgaggag actactcctt tgcaaatcaa acttaataag 900ctcgcatact tccttttggc gtgtgccctc gtcatagccg tcattgttgt cgcctccacc 960ggttttaatg atgtccccct ctctattgcc acctacgctg tcgctgccgc cgtctccatt 1020ctccccgcct ctttgattgc agttgttagt ttgactttgg cgcgtgcgtc aactgattta 1080gcatctcgac atgctttggt ccgacgaatg gatgctattg aggctttagc tggtgttgag 1140aatgtgtgct cggacaagac cggtaccctt actgttggcc gcatggtagt tcgcaaagtc 1200tgggttcctg ctcttgactg gcgccccaat gaatttgctc ccctcgacac tagtggtggt 1260caagcatata gttttgagac cggatctgat cctttctatc ctcgtggtga agtcctggcc

1320gattcccaga agatcactgg gactgcggag accctcgatc tcaagcaacc tcgtgaccaa 1380tctgactctt cctcttccga ctctgacccc gatgaacgag acgtagagga acaagaacgg 1440gtcatccacg ttgaagacat ggaaaacaac cttcgagacc ttgctctctg tatttcgctt 1500tgtaatcaag cgactctcac tcgtcctgtc aaccaagacg gccaatggga agcaaacggt 1560gatcctaccg aaacggccct tcaagttgct gcacacaaac ttggtcatgg caagcccttt 1620cttactcatg ctgccaagcc aagccaccgt gcggattcta tccgatctgg tcacagttct 1680cgtccccttg ttgctggtat tcgtgggcac tttgttccga taattgagca tcctttcgat 1740tccaccgtca agcgaatgtc aatcgcttat aaatttgtga gcgaggatcc tcaggattct 1800cacatcctct gtctccttaa gggtgccatc gagcgtgtct ttgaacgatg caccaagatc 1860caaggacagc ccatcaccga agagcataag aagaatatca tggtcaaagt tgatgctctc 1920gccgctcaag gtcttcgggt cctcgctctt tgtggaaagc gacttcctgt cagcatggta 1980gacgaagtca aatccacccc tcgagacgca ttcgaagccg atttccattt cctcggtctt 2040gctggtatct tcgatccgcc cagaaaggaa tctgcaggcg ccgttgctga ttgtttcagg 2100gctggtatca cccctcgaat gttgacaggc gatcatcctg ctaccgctac agctatcgcc 2160ctcaacattg gtattctcga taagacgtac tcaaaggatt cagtcatgac gggtcagcag 2220tttgactctt tgagcgaaga cgaaattgat caactgcccg agttgcctct tgtcgttgct 2280cgctgcgccc ccgaaaccaa agttcgaatg gtcgatgcca ttcatcgacg aggacaaagc 2340actgtaatga ctggtgatgg tgtcaacgac tctcccgccc tcaagcgtgc tgatgtgggc 2400gttggcatgg gtactggttc cgatgttgcc aagcagtcag cgcgtatcgt cctcagtgat 2460gacaacttca gcaccatcat tcgggctatt aggaaaggtc gttctgtctt caagaacttg 2520tctaaattct tgctctactt gctttccggt aacttggctg aaatcatcgt cctcatgatt 2580ggtctcgctt tcaaggatga caatggtcag gctgttttcc ccctgtcacc tgttgccgct 2640ctttggatca acactctcgc tgccggacct cctgcccttg ccctaggtct tgaacctaca 2700gctatcgacg ccatggagca gggacccgag gtataccatc gaatcttcac tcttgaattt 2760tacgtcgatc tgatcttcta cggtttcctg atgggctcca tcagtttggt caacttcgtc 2820attgtactat ggggatacta tcctggagac ttaggtcgtc tttgtaacga agatgatccc 2880agcatctgtg atcccgtcta tcaggctcga gctgcctgtt ttgccaccct cgttattgtc 2940ctcatgattc atgctttgga gtgtaagcac ttgagcaaag ggttggccca aatcaatttg 3000cgtgacaaca aggtgttgct gtggtgtgtc gttgccctca gtctttccac tttccctgtc 3060gtgtacattc ctgtgatcaa taacaaggtg tttttgctca acggtcccag gtgggaatgg 3120ggtatcatct tcggcatgat cttggtgtat ctcagtgcta ctgagctcta caagtggatc 3180aaaagaattt ggatccgacg acatgccccc ccttccaaag gaccttccga caagaccctt 3240aggatggaga gtaccattgc tcctcctgtt tga 3273212751DNACryptococcus neoformans serotype A H99 strainC_region(1)..(2751)coding region sequence of NHA1 (CNAG_01678.2) 21atgactgctt tccacccctt tgaagtcaat gcccctcatc tcgcatacac gttcctcggc 60ggctttgtgg tcatctttgg catgatcagt ttgtttatca aagagaagct ctatgtcggc 120gaagcaccta tagcaactgt agtcggcatc atcattggtc cccattgcct caattttttc 180aatcctgcag gatggggtgg cggggaggaa gaggtcgcga gtgacgttac attggaattc 240actcgcgttg tcattgctat atccgtattc gccgtcggcg ttgaattgcc caaggcatac 300atgaagcggc actggcgatc gctcttcttc cttcttggcc cgtgcatggt gtggggatgg 360atgatctccg ccctgctgat ctggggcctg atacctgacc taacatttct cgcctcgctc 420gtagttgcgg cgggcgtcac ccccacagat cctatcttgg cccaggcagt tatcggaggc 480aagttcgccg ataaacatgt tcccgcccac atccgccacc tcctctccgc agaaagtgga 540agtaacgatg gggccgcctt tcccttcctc tacatcgccc tctacctcct actcgatgcg 600agcccaggcc atgccgtcgg agaatggttc tacatgactt gggtctacga aattattctt 660ggtgttatca tcggggccat cctgggattc tgcgcacgca agttgatgaa gttagcggag 720cgcaaacgtc tcattgatag gcagtcttac gtcgcccagt atgtcagtct ggcagtgctg 780tcgattggcg ttacaagttt gctcggcagt gacgatttgc tttctgcttt cgcttgcggt 840tgtgcttttg catgggacgg tttcttcaac aaagctacgg aggatgcggt gttctcgaac 900gttattgatc tacttttcaa ttgcgccgcc ttcatctata tcggcgctat cattcctttc 960aatcatttta acgatttgcc cgatctccga gtatggcgat tggttgtgtt ggctatcctc 1020attcttctag ttcgtcgtct gccttctata atagcgtgtt acaaattcgt tcccgatatc 1080aagacgttca gagaagctct ttttacggga tggttcgggc ctatgggcgt cggtgctgta 1140ttcatctcca ctcttgctcg gtcgtctttg ccagaagggg agcctgaaca gaatacagaa 1200gcggtggacc gcctaaaaga cgtcatcatg cctgtcacct tatttcttgt attgtcttca 1260atcgtaactc acggcatgtc aattccattt ttctctcttg gtcgccgggt ccattccatt 1320acttatactc gatcacgaaa tctttccatg gacacgcgag gcgatgagcc tgcctggaca 1380actcatgctc ggcgtattat tccaggccag gagatcattg tcaaccgtga tgacgacgac 1440gaagaaggcg acttgggtgt tagacggatg gacacactca cgagcgattc aaatggtcgt 1500atcagggaaa agattgagga agaagatagc ggagaaagta gctcatcccg aacaaggcag 1560ggagaaatga ttgaaatgac agaaaaacgt ggcccggctc gccatggtag ccaggccagc 1620cagggcgaag cggcggagga aggagagagg tggagaagtt cgggagaaga aagctctgat 1680cttgcgaatg accctgagac acagagagag gtggaagagg gaatggaaga ggtcgaagat 1740aaggaaggag gtggtagaag aacgcccccc ctggccaagt acagagaagg aaaccacctc 1800attgtggaga gaaaagtcaa ggacagtgac gaggttgaag tcgaggtcat ccgaaaccat 1860ttttccgaca acaagaaaac ggaaagtgac cgcttcactc atccccatcg cctcaagtca 1920cgagagcttg acgatttgct tcatcacctt cccaaaagcc tcgagcatgc tacttcacgg 1980gttcaaaatg gcggcaaaga tgcagttgat cgtctcggtc ttgggctgat ggctattaac 2040actccggaac cgtcaccatc gatcgaatcg cacggcggtc caaggcatga ttatgtcgat 2100ggcttggaga gaacgcagag cccagagggt cttgcagacg aggataggga tagcgagggc 2160cggggcgatg tgtcccatgg gggtgattat gaagaaaacg aggccgacta tgaggatgtt 2220ccgaacgaga ctcgtcggca aaggaggaag aaaatgaaac caccagcaat tgtcgtctct 2280cggcagaaca gcgccgggct cccgagacga tccatccgct ccaggctgtt cggccgacga 2340caacattctt ccaactctcc ctcccgtgcc gaagaaggct tagcccctcc caatccatct 2400cttcttgttc catcctcatc cccttcgcgt cctcaaaaca ttgctgcaga acccgagtcc 2460atactggcag aagattcgcg cggatcatcc tcaccttccc aatctcaaaa tcttgcgatc 2520cctctcacaa gaaccctttc agctagccga tcgtcgcctg cggtgcgctt cgccgacgat 2580gctagtcctt catcggacac agcgcctggg cagtcaaatt atggtactaa cgctccaggt 2640ttcaagaaga atccggcttt agcaatgtat cgatcggcca gtgtacaaag tacagggtcc 2700aacaaggatg ggcctagcgt atctttcaaa gaacctgaaa tcaagcgttg a 275122170PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(170)an amino acid sequence of Ras1(CNAG_01672.2) 22Met Ser Gly Asn Gly His Tyr Arg Arg Asp Gln Arg Leu Val Val Val1 5 10 15Gly Cys Gly Ala Phe Arg Glu Tyr Asn Pro Thr Ile Glu Asp Ser Tyr 20 25 30Arg Lys Gln Val Val Val Asp Asn Glu Ala Thr Thr Leu Glu Ile Leu 35 40 45Asp Thr Ala Gly Gln Glu Glu Tyr Ala Ala Met Ala Asp Gln Trp Tyr 50 55 60Thr Phe Gly Ser Gly Phe Leu Leu Val Tyr Ser Leu Thr Asp Arg Ser65 70 75 80Ser Phe Glu Glu Ile Gln Asn Phe His Arg Glu Ile Leu Arg Val Lys 85 90 95Asp Arg Asp Tyr Val Pro Cys Val Ile Ile Cys Asn Lys Cys Asp Leu 100 105 110Gln Lys Tyr Arg Ser Val Gly Gln Leu Glu Gly Arg Glu Leu Ala Arg 115 120 125Ser Val His Ala Pro Phe Ile Glu Cys Ser Ala Ala Glu Arg Val Asn 130 135 140Val Asp Val Ala Phe Asn Glu Leu Val Lys Leu Val Arg Lys Asp Glu145 150 155 160Arg Val Arg Ile Asn Tyr Asp Ile Ala Phe 165 17023227PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(227)an amino acid sequence of Ras2(CNAG_04762.2) 23Met Leu Phe Lys Ile Thr Val Leu Gly Asp Gly Gly Val Gly Lys Thr1 5 10 15Ala Ile Thr Val Gln Thr Cys Lys Thr Tyr Asp Pro Thr Ile Glu Asp 20 25 30Cys Tyr Arg Lys Gln Trp Val Val Asp Glu Gln Pro Cys Leu Leu Glu 35 40 45Val Leu Asp Thr Ala Gly Gln Glu Glu Tyr Thr Ala Leu Arg Asp Gln 50 55 60Trp Ile Arg Glu Gly Glu Gly Phe Leu Ile Val Tyr Ser Ile Thr Ser65 70 75 80Arg Pro Thr Phe Glu Arg Val Glu Arg Ile Val Glu Arg Val Leu Arg 85 90 95Val Lys Asp Glu Ser Gly Leu Pro Leu Pro Pro Leu Ser Ser Ser Leu 100 105 110Ser Asn Asp Pro Tyr Gly Leu Ala Thr Ser Arg Ser Thr Pro Thr Ser 115 120 125Ala Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Met Trp Ala Ala Arg 130 135 140Val Pro Ile Val Ile Val Gly Asn Lys Lys Asp Met Phe His Ser Arg145 150 155 160Glu Val Ser Thr Asp Glu Gly Ala Ser Leu Ala Arg Arg Leu Gly Cys 165 170 175 Glu Phe Tyr Glu Ala Ser Ala Lys Thr Asn Ser Asn Val Glu Ala Ala 180 185 190Phe Lys Cys Leu Val Lys Lys Ile Lys Leu Ala Lys Gln Gly Gly Val 195 200 205Ala Val Gln Ala Glu Arg Val Gly Gly Arg Lys Lys Lys Gln Lys Cys 210 215 220Val Val Leu225241112PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(1112)an amino acid sequence of Cdc24(CNAG_04243.2) 24Met Ser Val Ser Gly Pro Ile Ser Arg Arg Arg Ile Gly Ser Val Ser1 5 10 15Gln Arg Gly Asn Glu Ser Leu Pro Gln Leu Asp Ile Gln Ser Ile Gln 20 25 30Met Pro Ser Asn Pro Gln Asn Ala Leu Ala Leu Lys Thr Ala Ala Leu 35 40 45Ser Thr Ser Thr Arg Ser Leu His Gln Ile Cys Ser Ile Leu Lys Lys 50 55 60Arg Leu Leu Cys Val Asp Gly Phe Lys Ala Phe Leu Glu Gln Pro Pro65 70 75 80Asn Ala Glu Pro Leu Asp Val Val Ser His Met Cys His Leu Phe Arg 85 90 95Leu Gly Ser Pro Leu Cys His Leu Tyr Asn Leu Leu Ile Pro Ser Phe 100 105 110Val Asp Cys Leu Ser Pro Leu Tyr Ala Asp Leu Pro Ala Pro Ala Lys 115 120 125Ile Glu Tyr Asp Phe Pro Gln Phe Tyr Asp Ser Pro Asn Gly Val Arg 130 135 140Asn Trp Ala Lys Arg Pro Glu Asn Ala Lys Pro Cys Gln Arg Tyr Ile145 150 155 160Ala Ala Phe Cys Met Ala Met Lys Lys Arg Ile Glu Glu Gly Arg Trp 165 170 175Thr Ser Asp Met Trp Ala Leu His Glu Leu Trp Gly Lys Ser Thr Gly 180 185 190Glu Asp Ile Glu Ala Tyr Asp Ser Thr Gly Leu Met Lys Val Leu Ser 195 200 205Thr Val Glu Glu Met Leu Asp Asn Leu Pro Glu Ser Ala Met Ser Pro 210 215 220Ile Ser Pro Gln Thr Pro Phe Thr Ala Ser Gly Ser Ile Ala Gln Arg225 230 235 240Ala Gln Ser Arg Gln Ser Tyr Asp Leu Pro Phe Ser Met Gly Gly Ile 245 250 255Gly Ser Gly Ala Ser Ala Val Ala Asn Met Ala Ala Thr Met Asn Gly 260 265 270Gly Val His Val Glu Thr Gly Pro Ser Glu Asn Ser Pro Thr Ala Ala 275 280 285Glu Met Gln Arg Gly Leu Ser Thr Ser Leu Ala Glu Ala Asn Ala Phe 290 295 300Lys Ser Val Glu Glu Leu Val Ala Ser Glu Lys Ser Tyr Val Gln Glu305 310 315 320Leu Glu Ile Leu Val Arg Cys Ser Gln Glu Met Leu Glu Ala Gln Leu 325 330 335Val Ser Thr Glu Thr Asn His Gln Ile Phe Ser Asn Leu Ser Lys Ile 340 345 350Leu Asp Phe His Arg Lys Phe Leu Ile Lys Leu Glu Thr Glu Tyr Glu 355 360 365Pro Ile Gln Glu Arg Gly Pro Gly Ala Trp Ala Glu Gly Val Trp Gly 370 375 380Arg Pro Phe Ile Leu Ser Glu Ala Glu Phe Asp Cys Tyr Gly Pro Tyr385 390 395 400Cys Ala Asn Tyr Leu Asp Ala Ile Thr Val Val Asn Glu Gln Met Pro 405 410 415Ile Leu Met Arg Gly Gln Glu Leu Ser Pro Gly Glu Arg Pro Cys Leu 420 425 430Asp Pro Gln Arg Glu Leu Gln Ala Phe Met Ile Lys Pro Ile Gln Arg 435 440 445Ile Thr Lys Tyr Gly Leu Leu Leu Asp Ala Ile Leu His Ala Thr Ala 450 455 460Lys His Glu Tyr Pro Phe Arg Pro Glu Leu Glu Glu Ala Ser Ala Ala465 470 475 480Val Lys Arg Ile Ala Ala Gly Ile Asn Glu Val Thr Asp Phe Lys Ala 485 490 495Lys Gln Ala Thr Val Arg Glu Leu Ile Glu Arg Val Asp Asp Trp Lys 500 505 510Gly His Asp Val Asp Lys Phe Gly Pro Leu His Ile Asp Asp His Phe 515 520 525Thr Val Thr Lys Ala Asp Gln Pro Arg Glu Tyr His Val Phe Leu Phe 530 535 540Glu Lys Met Met Leu Cys Cys Lys Glu Ile Thr Pro Glu Lys Lys Lys545 550 555 560Gln Asn Lys Asn Ser Ser Met Leu Arg Lys Asp Arg Gly Thr Ser Lys 565 570 575Ser Gly Pro Leu Asp Lys Lys Lys Leu Ala Leu Lys Gly Arg Ile Phe 580 585 590Val Ser Asn Ile Lys Glu Ala Thr Ile Leu Pro Thr Glu Pro Gly Asp 595 600 605Ala Tyr Gly Val Ala Arg Leu Leu Ile Gly Trp Thr Ile Pro Leu Arg 610 615 620Asn Gln Asp Gly Tyr His Asp Asp Gln Glu Asp Ser Phe Val Met Ile625 630 635 640Gly Lys Ser Glu Glu Gln Met Arg Lys Trp Ser Glu Lys Val Met Glu 645 650 655Leu Ala Asn Asn Glu Arg Lys Ile Gln Glu Asp Met Arg Ala Ala Arg 660 665 670Met Lys Ala Gly Arg Phe Ser Gly Ser Glu Arg Gln Tyr Tyr Gln His 675 680 685Ser Phe Phe Gly Pro Pro Thr Pro Ala Thr Glu His Pro Pro Met Thr 690 695 700Pro Phe Asn Met Pro Pro Leu Pro Asn Gly Ser Ala Thr Pro Tyr Tyr705 710 715 720Ser Glu Asp Glu Asp Pro Glu Gly Leu Arg Ser Gly Arg Thr Thr Pro 725 730 735Ser Ile Leu Gly His His Pro Tyr Ala Tyr Ser Gly Gln Pro Ser Ala 740 745 750Ser Arg Arg Val Gln Ser Gln Gln Ser Met Thr Ser Val Met Pro Thr 755 760 765Glu Leu Arg Ala Arg Ala Met Thr Glu Asp Gln Tyr Gly Pro Ser Met 770 775 780Thr Gln Trp Arg Thr Gln Gln Pro Met Ala Pro Pro Leu Pro Arg Leu785 790 795 800Thr Ser Ala Met Ser Gly Met Ser Val Ala Ser Glu Leu Ser Phe Gly 805 810 815Ser Gly Pro Asn Asn Ile Gly Ile Arg Thr Gly Met Val Arg Gln Met 820 825 830Ser Ser Thr Arg Leu Pro Arg Ala Thr Glu Val Asp Glu Ala Glu Glu 835 840 845Asn Pro Val Asp Thr Arg Asp Ser Tyr Gly Arg Tyr Gly Ser Leu Arg 850 855 860Gly Ile Met Arg Ala Pro Ser His Ala Met Pro Ser Val Pro His Pro865 870 875 880Pro Pro Leu Arg Asn Arg Ser Ala Ser Ser Pro Asn Val Tyr Gln Gln 885 890 895Pro Thr Val Thr Gly Ala Ala Ser Leu Pro Tyr Thr Ala Gly Pro Asn 900 905 910Gly Thr Trp Thr Thr Ser Pro Leu Ala Ser Thr Leu Gln Met Ser Thr 915 920 925His Pro Tyr Val Gln Ser Thr Pro Val Pro Gly Phe Gly Pro Ser Ser 930 935 940Ser Thr Thr Leu Val Gly Gly Thr Ala Tyr Phe Asn Lys Arg Met Ser945 950 955 960Asn Glu Lys Arg Ser Ser Gly Glu Ser His His Ser Thr Thr Thr Thr 965 970 975Asp Thr Ser Asp Gln Thr Ser Pro Ala Thr Pro Tyr Gly Ser Gly Asn 980 985 990Gly Asp Ile Arg Gly Pro Ser Arg Gln Asn Ser Gly Asp Asn Val Ser 995 1000 1005Gly Ser Val Leu Val Lys Leu Arg Phe Gly Asn Asp Gln Phe Ile 1010 1015 1020Leu Gly Val Ser Gln Gly Ile Asp Phe Ile Thr Leu Tyr Gln Lys 1025 1030 1035Ile His Lys Lys Ile Arg Leu Cys Ser Ser Ser Asn Arg Pro Thr 1040 1045 1050Asn Glu His Asp Lys Leu Gln Ile Arg Tyr Val Asp Asn Asp Gly 1055 1060 1065Asp Glu Ile Gln Val Lys Phe Asp Ser Asp Val Glu Leu Met Phe 1070 1075 1080Glu Asp Ala Arg Asp Gln Ala Gly His Ile Asn Leu Ile Ala Arg 1085 1090 1095Trp Ala Glu Asp Arg Arg Gly Thr Pro Gln Gly Glu Ile Tyr 1100 1105 111025432PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(432)an amino acid sequence of Gpa1(CNAG_04505.2) 25Met Gly Gly Cys Met Ser Thr Pro Glu Ala Pro Arg Lys Ala Ala Glu1 5 10 15Thr Lys Gln Val Pro Ser Thr Ser Thr Thr Ser Arg Pro Pro Gln Ala 20 25 30Ser Thr Ser Ala Thr Ala Thr Ala Ala Gly Ala Ser Thr Ser Pro Pro 35 40 45Asn Gly Thr Ala Asn Gly Ile Lys Gly Asp Thr Thr Ala Ala Asn Arg 50 55 60Thr Gly Ala Ser Ala Gly Gln Gly Ile Val Ala Ala Leu Ala Ser Thr65 70 75 80Glu Pro Pro Gly Ala Gln Asp Ser Lys Gly Asn Lys Asp Arg Ser Asn 85 90 95Gln Ile Asp Arg Gln Leu

Glu Asp Asp Gln Lys Lys Phe Arg Lys Glu 100 105 110Cys Lys Ile Leu Leu Leu Gly Ser Gly Glu Ser Gly Lys Ser Thr Ile 115 120 125Val Lys Gln Met Lys Ile Ile His Gln Asn Gly Tyr Ser Lys Asp Glu 130 135 140Leu Leu Ser Phe Arg Gly Val Ile Tyr Lys Asn Val Leu Asp Ser Ala145 150 155 160Gln Ala Leu Ile Met Ala Met Arg Lys Ile Gly Val Asp Pro Glu Asp 165 170 175Ala Asn Asn Arg Ser Tyr Ala Asp Arg Ile Leu Glu Tyr Arg Met Asp 180 185 190Ala Asp Leu Asn Ala Val Ile Pro Ser Glu Ile Leu Tyr Asn Ile Glu 195 200 205Ser Leu Trp His Asp Pro Val Ile Pro Ser Val Met Asp Arg Ser Ser 210 215 220Glu Phe Tyr Ile Met Asp Ser Ala Thr Tyr Phe Phe Ala Asn Ile Arg225 230 235 240Lys Ile Ala Gly Pro Asp Tyr Val Pro Asp Glu Ala Asp Val Leu Arg 245 250 255Ala Arg Thr Lys Thr Thr Gly Ile Ser Glu Thr Arg Phe Asn Met Gly 260 265 270Gln Leu Ser Ile His Met Phe Asp Val Gly Gly Gln Arg Ser Glu Arg 275 280 285Lys Lys Trp Ile His Cys Phe Glu Ala Val Thr Ser Ile Ile Phe Cys 290 295 300Val Ala Leu Ser Glu Tyr Asp Gln Val Leu Leu Glu Glu Ser Gly Gln305 310 315 320Asn Arg Met Gln Glu Ser Leu Val Leu Phe Glu Ser Val Ile Asn Ser 325 330 335Arg Trp Phe Leu Arg Thr Ser Val Ile Leu Phe Leu Asn Lys Ile Asp 340 345 350Leu Phe Lys Gln Lys Leu Pro Lys Val Pro Leu Val Gln Tyr Phe Pro 355 360 365Glu Tyr Thr Gly Gly Ala Asp Ile Asn Lys Ala Ala Lys Tyr Ile Leu 370 375 380Trp Arg Phe Thr Gln Thr Asn Arg Ala Arg Leu Ser Val Tyr Pro His385 390 395 400Leu Thr Gln Ala Thr Asp Thr Ser Asn Ile Arg Leu Val Phe Ala Ala 405 410 415Val Lys Glu Thr Ile Leu Gln Asn Ala Leu Arg Asp Ser Gly Ile Leu 420 425 430262250PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(2250)an amino acid sequence of Cac1(CNAG_03202.2) 26Met Pro Met Phe Arg Arg Ser Ala Ser Ser His Ser Thr Ser Asp Ala1 5 10 15Ala Ala Pro Pro Thr Ile Thr Asn Val Thr Glu Gly Ser Pro Val Ser 20 25 30Ser Gly Ser Ile Thr Arg Gln Lys Arg Ser Arg Ser His Gly Gly Gly 35 40 45Gly Ser Pro Ala Ser Thr Phe Ser Ser Arg Phe Gly Ile Ser Arg His 50 55 60Leu Ser His Gln Phe Gln Gln Pro His Glu Gln Glu Gln Gly Gln Val65 70 75 80Pro Pro Pro Glu Lys Pro Ala Thr Arg Glu Val Pro Val Ser Leu Glu 85 90 95Pro Glu Ile Tyr Gly Ser Glu Ala Gly Glu Asp Ser Asn Phe Gly Gln 100 105 110Glu Pro Ala Gly Glu Gln Leu Ile Pro Glu Thr Arg Ser Ser Ser Arg 115 120 125Gln Ser Arg Arg Ser Ser Arg Glu Leu Ser Val Gln Thr Ile Glu Pro 130 135 140Ser Ser Asp Asp Glu Ile Arg Ser Pro Glu Lys Arg Arg Val Ser Pro145 150 155 160Leu Met Lys Arg Leu Gly Glu Glu Pro Pro Phe Met Leu Pro His Pro 165 170 175Ser Leu Ser Asp Ser Thr Tyr Gln Ala Tyr Pro Gly Val Val Ile Gly 180 185 190Ser Phe Ala Gly Gly Gly Pro Asp Thr Leu Phe Gly Asn Gly Met Gln 195 200 205Leu Glu Gly Thr Val Asp Asp Ile Leu Asp Pro Asn Ala Ala Arg Gln 210 215 220Glu Asp Arg Gly Asn Gln Val Pro Ala Gly Ala Asn Ile Ala Pro Trp225 230 235 240Leu Met Asp Asp Gly Pro Pro Ser Arg Ser Glu Asn Pro Ser Pro Ala 245 250 255Leu Ser Glu Thr Gln Glu Arg Pro Val Lys Gly Pro Ala Ala Ala Leu 260 265 270Arg Glu Lys Asp Pro Arg Lys Thr Ser Thr Val Leu Asn His Phe Ser 275 280 285Ser Val Pro Ser Leu Pro Lys Ile Arg Arg His Gly Arg Ala Ala Thr 290 295 300Thr Thr Pro Asp Gln Thr Pro Arg Gly Ser Thr His Ser Gln Ser Asn305 310 315 320Leu Ala Ser Ser Ser Ser Ser Leu Asn Asn Glu Ser Arg Glu Ser Arg 325 330 335Ala Gly Ser Asp Asp Ser Ile Gln Thr Thr Leu Thr Gln Lys Gly Arg 340 345 350Arg Gln Ser Pro Gly Glu Trp Gly Gln Ala Ser Ala Val Pro Pro Pro 355 360 365Ser Lys Gly Thr Arg Pro Gly Arg Phe Gly Ser Thr Ala Ser Ile Ile 370 375 380Ser Gly Thr Gly Ser Val Gly Glu Lys Lys Lys Ser Leu Phe Gly Gly385 390 395 400Leu Leu Lys Arg Lys Thr Asn Pro Asn Leu Ser Leu Asn Pro Ile Ser 405 410 415His Asp Phe Thr Thr Ser Glu His Arg Gly Ser Ala Gly Ser Ile Pro 420 425 430Leu Ser Ala Ser Ser Ser Lys Leu Ser Ser Cys Ser Leu Ser Ser Leu 435 440 445Pro Ser Lys Ser Pro Pro Phe Thr Ser Pro Pro Glu Ala Phe Ser Arg 450 455 460Gln Phe Leu Pro Ala Asn Tyr Val His Glu Gly Ala Val Ser Pro Leu465 470 475 480Gln Glu Ile Ser Glu Ser Pro Phe His Leu Asp Met Asn Leu Asp Asp 485 490 495Met Glu Gly Ile Ile Asp Pro Ala Lys Ala Gly Leu Pro Ser Thr Val 500 505 510Ala Tyr Arg Pro Ser Ala Ser Ser Glu Val Thr Thr Asp Ser Ser Ala 515 520 525Ser Glu Ser Met Arg Leu Glu Glu Ala Leu Asn Gln Thr Ser Ser Phe 530 535 540Gly Thr Ser Val Ser Gly Ala Ser Arg Gly Ser Asp Gly Thr Lys Met545 550 555 560Pro Gly Arg Ile Val Leu Gly Glu Ala Glu Arg Leu Pro Thr Pro Phe 565 570 575Thr Gly Thr Asp Pro Phe Gln Gln His Arg Glu Ser Ala Ser Thr Thr 580 585 590Gly Ser Asp Gly Lys Pro Phe Ser Pro Pro Ser Pro His Thr Leu Ser 595 600 605Pro Lys His His Leu Pro Ser Ser Ser Ala Asn Gln Pro Arg Arg Pro 610 615 620Ser Ala Leu Arg Asn Val Glu Thr Gly Gln Val Asp Glu Thr Pro Gln625 630 635 640Leu Ser Ala Ser Glu Gly Ser Ile Gln Pro Ile Ser Pro Ser Trp Ala 645 650 655Gly Gly Ser Gly Ile Thr Val Phe Asn Asp Pro Phe Ser Thr Ser Arg 660 665 670Gln Arg Gln Glu Gln Pro Pro Asn Ser Ala Gly Leu Ser Pro Ser Thr 675 680 685Thr Ala Tyr Pro Ser Ala Val Thr Gln Pro Gly Pro Ser Thr Ala Arg 690 695 700Phe Leu Ile Thr Ala Gly Ser Thr Thr Pro Ser Ala Ala Trp Ala Ala705 710 715 720Pro Glu Ser Trp Gly Val Glu Ala Asp Glu Ala Pro Ala Glu Glu Ile 725 730 735Thr Ser Ser Asp Glu Asp Asp Trp Ala Gly Leu Gly Val Glu Glu Val 740 745 750Ala Ser Ala Ser Pro Thr Ser Asp Thr Leu Pro Ser Pro Pro Thr Ser 755 760 765Pro Arg Ala Ser Ser Leu Pro Ser Pro Lys Arg Ala Pro Pro Phe Gly 770 775 780Phe Lys Ser Gln Gln Arg Ala Lys Pro Gly Thr Ser Gly Thr Thr Asp785 790 795 800Ser Thr Thr Ser Ala Ala Gly Arg Arg Lys Gly Lys Arg Val Gly Ser 805 810 815Ser Gly Arg Pro Ala Thr Gly Arg Pro Gly Thr Ser Gly Ser Ala Tyr 820 825 830Asn Pro Ser Ser Leu His Trp Ile Arg Ile Tyr Arg Ala Asp Lys Ser 835 840 845Tyr Met Leu Tyr Asn Leu Pro Leu Asn Thr Ser Thr Gly Glu Leu Leu 850 855 860Ala Leu Leu Ala Ala Gln Ala Glu Gln Gly Met Val Arg Gly Lys Asn865 870 875 880Val Ala Ile Asn Met Lys Leu Tyr Ile Cys Glu Arg Gly Gln Asn Arg 885 890 895Met Leu Leu Pro Ser Glu Lys Pro Leu Thr Ile Gln His Arg Arg Leu 900 905 910Leu Gln Leu Gly His Thr Glu Ala Asp His Leu Asp Glu Leu Gly Lys 915 920 925Asn Asp Met Ala Val Leu Cys Arg Phe Ile Tyr Gln Ala Pro Ile Leu 930 935 940Pro Ile Met Asp Pro Glu Glu Glu Ser Ser Tyr Asp Ser Phe Glu Phe945 950 955 960Ile Asp Ile Ala Ser Arg Asp Leu Gln Thr Ile Pro Ile Phe Leu His 965 970 975Leu His Ala His Asp Ile Ile Ile Leu Asn Ile Ser Lys Asn Pro Met 980 985 990Thr Asp Ile Pro Leu Asp Phe Ile Gln Ala Cys Thr Ser Leu Lys Glu 995 1000 1005Leu Arg Met Ser Asn Met Ala Leu Lys Arg Val Pro Ile Ser Ile 1010 1015 1020Arg Ala Ser Thr Thr Leu Ala Arg Leu Asp Val Ser Cys Asn Arg 1025 1030 1035Ile Ala Asp Leu Glu Ser Val Ala Leu His Glu Val Glu Thr Leu 1040 1045 1050Val Ser Leu Lys Val Gln Asn Asn Lys Leu Thr Ser Met Pro Ser 1055 1060 1065Tyr Phe Ala Gln Met Lys Ser Leu Lys Tyr Leu Asn Ile Ser Asn 1070 1075 1080Asn Lys Phe Glu Thr Phe Pro Ser Val Val Cys Glu Met Ser Asn 1085 1090 1095Leu Val Asp Leu Asp Val Ser Phe Asn Asn Ile Ala Glu Leu Pro 1100 1105 1110Ala Lys Met Ser Asp Leu Lys Ser Leu Glu Lys Leu Gly Leu Tyr 1115 1120 1125Ser Asn Asp Ile Ser Lys Phe Pro Glu Ser Phe Cys Thr Leu Ala 1130 1135 1140Asn Leu Arg Ile Leu Asp Val Arg Arg Asn Lys Ile Thr Asp Leu 1145 1150 1155Ser Ala Val Tyr Ala Leu Pro Asn Leu Ala Thr Leu Gln Ala Asp 1160 1165 1170Asn Asn Asn Ile Val Thr Leu Asp Ala Gln Leu Gly Ala Asn Val 1175 1180 1185Arg Gln Phe Ser Val Pro His Asn Ser Val Thr Arg Phe Thr Leu 1190 1195 1200Ala Pro Pro Pro Asn Met Ala Val Val Thr Tyr Met Leu Thr Asn 1205 1210 1215Leu Asp Leu Ser His Gly Lys Ile Ser Thr Leu Ala Asp Glu Ala 1220 1225 1230Phe Ser Gly Leu Thr Asn Leu Val Thr Leu Asn Leu Asn Phe Asn 1235 1240 1245Gln Phe Thr Lys Leu Pro Ala Thr Leu Gly Arg Leu Thr Ser Leu 1250 1255 1260Glu Val Phe Ser Cys Thr Asp Asn Met Leu Asn Leu Val Pro Ala 1265 1270 1275Gly Phe Gly Lys Leu Gln Arg Leu Arg Met Ile Asn Leu His Asn 1280 1285 1290Asn Asn Leu Lys Ser Leu Pro Glu Asp Leu Trp Ala Cys Gly Ala 1295 1300 1305Leu Glu Val Phe Asn Ala Ser Ser Asn Leu Leu Asp Ser Phe Ile 1310 1315 1320Pro Pro Pro Ala Asp Ile Glu Ser Val Val Gly Arg Val Gly Ser 1325 1330 1335Gly Thr Ser Gln Thr Ser Asn Gly Arg Lys Lys Tyr Ser Val Pro 1340 1345 1350Pro Ile Gly Leu Ser Ile Arg Lys Leu Phe Leu Ala Asp Asn Arg 1355 1360 1365Leu Asn Asp Asp Val Phe His Trp Ile Ser Leu Met Pro Ser Leu 1370 1375 1380Arg Ile Ile Asn Leu Ser Phe Asn Asp Ile Tyr Glu Leu Thr Asn 1385 1390 1395Leu Pro Ser Glu Asp Leu Glu Lys Leu Gln Ser Leu Lys Val Leu 1400 1405 1410His Leu Asn Gly Asn Lys Leu Gln Thr Leu Pro Ser Glu Leu Gly 1415 1420 1425Ala Ile Lys Thr Leu Gln His Leu Asp Val Gly Ser Asn Val Leu 1430 1435 1440Lys Tyr Asn Ile Ala Asn Trp Pro Tyr Asp Trp Asn Trp Asn Trp 1445 1450 1455Asn Thr Ser Leu Arg Tyr Leu Asn Leu Ser Gly Asn Lys Arg Leu 1460 1465 1470Glu Ile Lys Pro Thr Ser Ala His Glu Met Ser His Ala Ser Ser 1475 1480 1485Phe Arg Lys Glu Leu Ser Asp Phe Thr Ala Leu Thr Gln Leu Arg 1490 1495 1500Val Leu Gly Leu Met Asp Val Thr Leu Arg Ile Pro Ser Leu Pro 1505 1510 1515Asp Glu Ser Glu Glu Lys Arg Val Arg Thr Ser Phe Ser Asp Ile 1520 1525 1530Asn Asn Met Ala Tyr Gly Ile Ser Asp Met Leu Gly Ser Ile Asp 1535 1540 1545Asn Leu Ala Met Phe Asp Leu Val Val Pro His Phe Arg Gly Lys 1550 1555 1560Glu Asn Glu Cys Leu Phe Gly Met Phe Gly Arg Val Thr Thr Thr 1565 1570 1575Leu Gln Gly Gly Lys Ile Ala Lys Tyr Val Gln Glu Ile Phe Ala 1580 1585 1590Glu Thr Leu Thr Ala His Leu His Gln Leu Glu Pro Gly Glu Glu 1595 1600 1605Pro Ser Glu Ala Leu Arg Arg Thr Phe Leu Leu Gly Asp Arg Lys 1610 1615 1620Ala Phe Glu Phe Phe Ser Asp Lys Leu Gln Leu Glu Lys Glu Arg 1625 1630 1635Lys Pro Ser Trp Thr Ser Phe Ala Ser Phe Asp Ser Met Phe Arg 1640 1645 1650Gly Trp Thr Pro Gly Val Asn Ser Val Leu Arg Thr Gly Ala Ser 1655 1660 1665Gly Ala Val Val Tyr Leu Val Asp Lys Val Leu His Val Gly Ser 1670 1675 1680Ile Gly Asp Thr Leu Val Val Leu Ser Arg Lys Gly Asp Ala Glu 1685 1690 1695Leu Leu Ser Lys Arg His Asp Pro Thr Asp Arg Glu Glu Ser Ala 1700 1705 1710Arg Ile Arg Lys Ala Glu Ala Trp Val Ser Thr Lys Gly Phe Val 1715 1720 1725Asn Asp Asp Lys Asp Leu Asp Ile Ser Arg Ala Phe Gly Tyr Trp 1730 1735 1740His Glu Cys Pro Ala Val Asn Ala Ala Pro Glu Ile Arg Thr Arg 1745 1750 1755Arg Leu Gln Glu Ser Asp Glu Phe Val Ile Ile Gly Asn His Ala 1760 1765 1770Leu Trp Gln Phe Cys Ser Tyr Gln Thr Ala Val Asp Ile Ala Arg 1775 1780 1785Thr Glu Arg Asp Asp Pro Met Met Ala Ala Gln Lys Leu Arg Asp 1790 1795 1800Phe Ala Ile Ser Tyr Gly Ala Glu Gly Asn Val Met Val Met Val 1805 1810 1815Val Asn Val Ser Asp Leu Phe Leu Ala Lys Gly Gly Arg Ala Arg 1820 1825 1830Gly Pro Ser Lys Gln Thr Ala Thr Asp Ala Asn Ala Asp Val Glu 1835 1840 1845Gly Tyr Ala Val Ala Lys Arg Gln Val Arg Arg Arg Tyr Asp Glu 1850 1855 1860Val Gly Asp Arg Thr Leu Asn Arg Leu Gln Gln Glu Ile Glu Pro 1865 1870 1875Pro Val Gly Gln Val Ala Ile Val Phe Thr Asp Ile Val Asn Ser 1880 1885 1890Thr His Leu Trp Glu Thr Asn Pro Ala Met Pro Thr Ala Ile Lys 1895 1900 1905Met His His Asn Leu Met Arg Arg Gln Leu Arg Leu Asp Gly Gly 1910 1915 1920Tyr Glu Val Lys Thr Glu Gly Asp Ser Phe Met Val Ser Phe Gln 1925 1930 1935Ser Val Ala Ser Ala Leu Leu Trp Ser Phe Asn Cys Gln Ile Gly 1940 1945 1950Leu Leu Gln Gln Glu Trp Pro Arg Glu Leu Leu Glu Ala His Asp 1955 1960 1965Gly Lys Val Val Tyr Asp Ser Asn Gly Thr Ile Val Gln Arg Gly 1970 1975 1980Leu Arg Val Arg Met Gly Val His Trp Gly Ala Pro Glu Cys Glu 1985 1990 1995Lys Asp Pro Ile Thr Arg Arg Met Asp Tyr Tyr Gly Pro Met Val 2000 2005 2010Asn Arg Ala Ala Arg Ile Asn Ala Ser Ala Asp Gly Gly Gln Leu 2015 2020 2025Met Ala Ser Gln Asp Val Leu Asn Glu Ile Ala Pro Leu Met Glu 2030 2035 2040Tyr Leu Asn Ser Ser Asp Glu Gln Val Leu Asn Asp Leu Gln Gly 2045 2050 2055Asp Leu Lys Arg Glu Val Met Glu Leu Arg Arg Ile Gly Leu Glu 2060 2065 2070Val Arg Asp Met Gly Asp Arg Lys Leu Lys Gly Leu Glu Val Pro 2075 2080 2085Glu Arg Leu His Leu Leu Tyr Pro Lys Thr Leu Ala Gly Arg Leu 2090 2095 2100Glu Ile Ser Asn Glu Ile Arg Ala Glu Val

Glu Val Asn Asp Ala 2105 2110 2115Arg Lys Ser Ala Glu Arg Gln Arg Ser Val Asp Ile Asp Gln Val 2120 2125 2130Tyr Gln Leu Ser Asp Ile Ala Leu Arg Leu Glu Ala Val Cys Cys 2135 2140 2145Tyr Asn Pro Thr Pro Ser Ser Pro Gly Asp Thr Pro Thr Ala Gly 2150 2155 2160Val Met Arg Leu His Pro Pro Ala Ser Tyr Leu Gly Pro Ser Ile 2165 2170 2175Arg Glu Asp Met Asn Asp Glu Glu Leu Trp Thr Ile Ile Glu Ser 2180 2185 2190Leu Val Gly Arg Ile Glu Asn Val Met Ser Thr Leu Tyr Leu Lys 2195 2200 2205Asn Phe Gly Glu Phe Ser Ala Val Leu Ala Ala Leu Glu Ser Ala 2210 2215 2220Thr Lys Ile Asp Gln Lys Leu Ile Val His Ala Leu Ala Leu Met 2225 2230 2235Asn Glu Ala Met Gly Lys Asp Glu Glu Asn Ala Ile 2240 2245 225027499PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(499)an amino acid sequence of Aca1(CNAG_05218.2) 27Met Ala Thr Ser Gln Gly Ile His Ser Ile Ser Thr Ile Leu Arg Leu1 5 10 15Glu Asp Ile Ala Val Thr Gln Ala Pro His Gly Ser Ser Val Lys Ser 20 25 30Pro Ala Pro Ala Ser Asp Thr Pro Thr Gly Val Ala Pro Pro Ala Pro 35 40 45Pro Pro Pro Pro Ala Pro Glu Ala Pro Lys Ala Ala Glu Met Thr Gln 50 55 60Pro Ala Gln Ser Pro Ala Ser Lys Val Tyr Gln Asp Glu Ile Ile Asn65 70 75 80Gly Ala Leu Asn Asp Phe Leu Ser Lys Ser Lys Glu Val Gly Gly Leu 85 90 95Val Ala Glu His Ser Ala Leu Ile Gly Pro Leu Cys Glu Ala Gln Leu 100 105 110Ser Phe Leu Gln Phe Ala Ser Asn His Ala Lys Pro Ala Thr Pro Asn 115 120 125Ala Leu Ala Pro Leu Leu Glu Pro Gln Gly Lys Ala Ile Glu Ala Ile 130 135 140Met Glu Thr Lys Asp Lys Leu Ser Arg Ser Lys Glu Gly Arg Glu Trp145 150 155 160Gly Val Cys Phe Asn Val Leu Gly Glu Gly Val Pro Ala Trp Gly Trp 165 170 175Val Gln Val Glu Pro Thr Pro Ala Pro Tyr Val Gly Glu Met Lys Asn 180 185 190Ala Ala Gln Phe Trp Ser Asp Arg Val Ile Lys Gln Tyr Lys Glu Thr 195 200 205Asn Ala Ser Ala Val Ala Trp Ala Lys Ser Phe Ile Ala Leu Ile Ala 210 215 220Ala Leu Glu Ser Tyr Val Lys Gln Trp His Thr Thr Gly Val Val Trp225 230 235 240Asn Pro Lys Gly Ser Pro Ala Pro Pro Ser Met Pro Lys Ala Ser Ala 245 250 255Ser Ala Pro Ser Pro Pro Pro Pro Pro Pro Ser Gly Ser Ala Pro Ala 260 265 270Ala Pro Thr Ser Gly Ser Gly Ala Ala Ala Leu Leu Ala Asp Leu Asn 275 280 285Arg Gly Gly Ala Val Thr Ser Gly Leu Arg Lys Val Asp Ser Ser Gln 290 295 300Met Thr His Lys Asn Pro Ser Leu Arg Ser Ala Gly Thr Val Ser Asp305 310 315 320Asn Ala Lys Lys Gly Pro Pro Leu Lys Pro Lys Pro Gly Ala Lys Pro 325 330 335Ala Lys Lys Pro Ala Lys Ile Glu Leu Glu Asp Gly Asn Lys Trp Ile 340 345 350Ile Glu Asn Gln Glu Asp Asn Lys Ser Ile Lys Ile Asp Asn Thr Glu 355 360 365Leu His His Thr Val His Ile Phe Gly Cys Val Asn Ser Val Val Gln 370 375 380Ile Ser Gly Lys Ile Asn Ala Val Thr Met Ala Gly Cys Lys Lys Thr385 390 395 400Ser Val Val Leu Asp Thr Ala Val Ser Ser Phe Ser Ile Thr Ser Ser 405 410 415Pro Ser Phe Glu Val Gln Ile Ile Gly Ser Ile Pro Thr Ile Gln Ile 420 425 430Asp Thr Thr Asp Ser Gly Gln Val Tyr Leu Ser Lys Asp Cys Met Glu 435 440 445Val Val Glu Ile Val Thr Ser Lys Ser Ser Ser Ile Asn Ile Ser Val 450 455 460Pro Thr Gly Glu Asp Gly Asp Phe Val Glu Arg Pro Val Pro Glu Gln465 470 475 480Met Lys Ser Arg Ile Ile Asp Gly Lys Leu Val Thr Glu Ile Val Glu 485 490 495His Ser Gly 28515PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(515)an amino acid sequence of Pka1(CNAG_00396.2) 28Met Phe Gln Lys Val Ser Asp Lys Phe His Arg Lys Gln Gln Ser Ser1 5 10 15Thr Ser Pro Gly Lys Thr Gln Gln Val Pro Asn Ser Pro Ser Ser Val 20 25 30Leu Ala Lys Ala Asn Ser Gln Ala Gln Gln Ala Tyr Ser Ser Gln Asp 35 40 45His Ser Pro Met Glu Gly Ile Gln Ser Asp Ser Thr His Ile Gln Gln 50 55 60Pro Met Ala Thr Gln Lys Ala Pro Ile Val Gly Pro Ser Thr Ser Thr65 70 75 80Ser Leu Ser Thr Val Pro Val Gln Asp Gly Thr Leu Pro Leu Thr Pro 85 90 95Gly Ala Gln Gly Met Leu Ala Gly Thr Thr Asp Gly His Arg Gln Val 100 105 110Gln Ser Pro Val Ser Arg Ser Ser Ser Ala Gly Glu Asp Lys Met Arg 115 120 125Glu Lys Ala Arg Asp Ala Gln Glu Gln Ala Ala Gln Ala Gln Ala Asn 130 135 140Leu His Arg Val Thr Gln Gln Ala Arg Val Ala Ala Ile Asn Ala Ala145 150 155 160Ala Thr Gln Ala Ala Leu Glu Thr Ala Thr Gln Leu Pro Ala Thr Ala 165 170 175Arg Val Pro Thr Ser Gly Thr Gly Ala Glu Pro Gly Gln Ala Arg Arg 180 185 190Lys Thr Ala Gly Arg Tyr Ala Leu Ser Asp Phe Leu Ile Glu Arg Thr 195 200 205Leu Gly Thr Gly Ser Phe Gly Arg Val His Leu Val Arg Ser Arg His 210 215 220Asn Gly Arg Phe Tyr Ala Val Lys Val Leu Asn Lys Glu Lys Val Ile225 230 235 240Lys Met Lys Gln Val Glu His Thr Asn Ser Glu Arg Glu Met Leu Val 245 250 255Arg Val Arg His Pro Phe Leu Val Asn Leu Trp Gly Thr Phe Gln Asp 260 265 270Val Asn Asn Leu Tyr Met Val Met Asp Phe Val Ala Gly Gly Glu Leu 275 280 285Phe Ser Leu Leu Arg Lys Ser Gln Arg Phe Pro Asn Ser Val Ala Lys 290 295 300Phe Tyr Ala Ala Glu Val Ala Leu Ala Leu Asp Tyr Leu His Ser Leu305 310 315 320Asp Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Gly Ala 325 330 335Asp Gly His Val Lys Val Thr Asp Phe Gly Phe Ala Lys Tyr Val Pro 340 345 350Asp Ile Thr Trp Thr Leu Cys Gly Thr Pro Asp Tyr Leu Ala Pro Glu 355 360 365Val Val Gln Ser Lys Gly Tyr Asn Lys Ser Val Asp Trp Tyr Ala Leu 370 375 380Gly Val Leu Ile Phe Glu Met Leu Ala Gly Tyr Pro Pro Phe Phe Thr385 390 395 400Glu Asp Gly Asn Pro Met Lys Leu Tyr Glu Lys Ile Ile Ala Gly Lys 405 410 415Val Arg Tyr Pro Thr Tyr Phe Asp Val Leu Ala Lys Glu Leu Leu Lys 420 425 430Asn Leu Leu Ile Gly Asp Leu Thr Lys Arg Tyr Gly Asn Leu Arg Ala 435 440 445Gly Ser Ser Asp Ile Phe Ala His Gly Trp Phe Ala Glu Val Asp Trp 450 455 460Asp Lys Leu Tyr Arg Arg Glu Ile Pro Ala Pro Tyr Val Pro Lys Ile465 470 475 480Asp Gly Glu Gly Asp Ala Ser Gln Phe Asp Arg Tyr Gln Glu Ala Asp 485 490 495Val Ser Ala Tyr Gly Lys Val Gly Asn Gly Pro Tyr Asp His Phe Phe 500 505 510Val Glu Phe 5152983PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(83)an amino acid sequence of Hsp12(CNAG_03143.2) 29Met Ser Asp Ala Gly Arg Gln Ser Phe Thr Asp Lys Ala Gly Ala Ala1 5 10 15Met Lys Pro Asp Ser Glu Lys Ser Tyr Leu Glu Gln Ala Lys Asp Thr 20 25 30Ile Gly Gly Lys Ala Asp Ser Ala Ala Ser Thr Gly Gln Pro Gln Ser 35 40 45Gln Lys Ser Tyr Thr Gln Glu Ile Gly Asp Ala Phe Ser Gly Asn Lys 50 55 60Asn Asp Asn Gln Glu Ser Leu Thr Asp Lys Ala Lys Asn Ala Phe Gly65 70 75 80Ala Asn Gln3070PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(70)an amino acid sequence of Hsp122(CNAG_01446.2) 30Met Ser Asp Ala Gly Arg Gln Ser Leu Ala Asp Lys Ala Ser Ser Ser1 5 10 15Met Lys Pro Asp Ser Glu Lys Ser Tyr Val Glu Gln Ala Ser Asp Phe 20 25 30Ile Ser Gly Lys Leu Asp Ser Ala Ala Ser Ala Val Gln Pro Gln Gln 35 40 45Glu Lys Ser Thr Thr Gln Lys Ile Gly Asp Ala Val Ser Gly Asp Asn 50 55 60Arg Asn Arg Asp Val Ala65 703120DNAArtificial SequenceSynthetic oligonucleotide 31tttttttttt tttttttttt 203221DNAArtificial SequenceSynthetic oligonucleotide 32tgtggatgct ggcggaggat a 213321DNAArtificial SequenceSynthetic oligonucleotide 33gtaaaacgac ggccagtgag c 213421DNAArtificial SequenceSynthetic oligonucleotide 34caggaaacag ctatgaccat g 213520DNAArtificial SequenceSynthetic oligonucleotide 35tgtttagcac cagcggagtc 203621DNAArtificial SequenceSynthetic oligonucleotide 36cacgatgaaa gtgcgttgaa g 213740DNAArtificial SequenceSynthetic oligonucleotide 37gctcactggc cgtcgtttta cactgtcggt gaaagattgc 403840DNAArtificial SequenceSynthetic oligonucleotide 38catggtcata gctgtttcct gagaacgaca accaggagtc 403922DNAArtificial SequenceSynthetic oligonucleotide 39gctctgtgct gacattatct gc 224020DNAArtificial SequenceSynthetic oligonucleotide 40gaaagtgcgt tgaagtgatg 204121DNAArtificial SequenceSynthetic oligonucleotide 41agtagaagca gcggactaaa g 214220DNAArtificial SequenceSynthetic oligonucleotide 42gcgtagtgga gattggtttc 204320DNAArtificial SequenceSynthetic oligonucleotide 43atcccctcca ctttacctcc 204440DNAArtificial SequenceSynthetic oligonucleotide 44gctcactggc cgtcgtttta caagtctccc ttagcgatag 404540DNAArtificial SequenceSynthetic oligonucleotide 45catggtcata gctgtttcct gaccacaccc ctgaagaaac 404620DNAArtificial SequenceSynthetic oligonucleotide 46aactgtttcg tcttgtgtgc 204719DNAArtificial SequenceSynthetic oligonucleotide 47atagcaactt cttccgtcg 194819DNAArtificial SequenceSynthetic oligonucleotide 48tgttgcctgt gctcacttg 194920DNAArtificial SequenceSynthetic oligonucleotide 49cctctgacag ccacatactg 205019DNAArtificial SequenceSynthetic oligonucleotide 50aatgaagttc ctgcgacag 195139DNAArtificial SequenceSynthetic oligonucleotide 51gctcactggc cgtcgtttta caatgggatg agaacgcac 395240DNAArtificial SequenceSynthetic oligonucleotide 52catggtcata gctgtttcct gagcattttc cagcatcagc 405320DNAArtificial SequenceSynthetic oligonucleotide 53ggtgtggaac atcttttgag 205419DNAArtificial SequenceSynthetic oligonucleotide 54ctggttcatc ttgggtgtc 195522DNAArtificial SequenceSynthetic oligonucleotide 55tctgagcata ccactccttt ac 225618DNAArtificial SequenceSynthetic oligonucleotide 56tctcattcgc atcctctg 185721DNAArtificial SequenceSynthetic oligonucleotide 57gttgggcaga taatgtttgt g 215839DNAArtificial SequenceSynthetic oligonucleotide 58gctcactggc cgtcgtttta cacggcgtca gacattgtg 395943DNAArtificial SequenceSynthetic oligonucleotide 59catggtcata gctgtttcct gacaagagaa gtccactact cag 436018DNAArtificial SequenceSynthetic oligonucleotide 60gcaaggtaat gatgagcg 186121DNAArtificial SequenceSynthetic oligonucleotide 61gcgactgaga tgtagaccaa c 216220DNAArtificial SequenceSynthetic oligonucleotide 62ctcggaacga cataataagc 206321DNAArtificial SequenceSynthetic oligonucleotide 63cacacctggt aagagatagc g 216441DNAArtificial SequenceSynthetic oligonucleotide 64gctcactggc cgtcgtttta cagtggtaga agtagggcag c 416540DNAArtificial SequenceSynthetic oligonucleotide 65catggtcata gctgtttcct gacagggtcc aacaaggatg 406620DNAArtificial SequenceSynthetic oligonucleotide 66tgctacgatt gtggtcagcc 206721DNAArtificial SequenceSynthetic oligonucleotide 67ggacgagacg agttatcaaa c 216818DNAArtificial SequenceSynthetic oligonucleotide 68cttcatcaac ttgcgtgc 18

Claims

1. A method of treating fungal infection, comprising: injecting an effective amount of an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans into a subject.

2. The method of treating fungal infection of claim 1, wherein the inhibitor against at least one protein selected from the group consisting of Ssk1, Ena1 and Nha1.

3. The method of treating fungal infection of claim 1, wherein an ergosterol-binding antifungal agent or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.

4. The method of treating fungal infection of claim 3, wherein the ergosterol-binding antifungal agent is a polyene-based antifungal agent.

5. The method of treating fungal infection of claim 4, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin.

6. The method of treating fungal infection of claim 5, wherein the polyene-based antifungal agent is amphotericin B.

7. The method of treating fungal infection of claim 3, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

8. A method of treating fungal infection comprising: Injecting an effective amount of an inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans into a subject.

9. The method of treating fungal infection of claim 8, wherein the inhibitor against at least one gene selected from the group consisting of SSK1, ENA1 and NHA1.

10. The method of treating fungal infection of claim 8, wherein an ergosterol-binding antifungal agent or azole-based antifungal agent is sequentially or continuously injected with the inhibitor.

11. The method of treating fungal infection of claim 10, wherein the ergosterol-binding antifungal agent is a polyene-based antifungal agent.

12. The method of treating fungal infection of claim 11, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.

13. The method of treating fungal infection of claim 12, wherein the polyene-based antifungal agent is amphotericin B.

14. The method of treating fungal infection of claim 10, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

15. An antifungal combined formulation, comprising: an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans; and an ergosterol-binding antifungal agent or azole-based antifungal agent.

16. A method of screening an antifungal agent comprising: contacting at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates an activity of the protein.

17. The method of claim 16, wherein the antifungal agent is an inhibitor against an Ssk1, Ena1 or Nha1 protein.

18. A method of screening an antifungal agent comprising: contacting at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates an activity of the gene.

19. The method of claim 18, wherein the antifungal agent is an inhibitor against an SSK1, ENA1 or NHA1 gene.

20. A method of treating fungal infection, comprising: injecting an effective amount of an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans into a subject.

21. The method of treating fungal infection of claim 20, wherein the inhibitor is an inhibitor against a Cac1 or Pka1 protein.

22. The method of treating fungal infection of claim 20, wherein a polyene- or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.

23. The method of treating fungal infection of claim 22, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.

24. The method of treating fungal infection of claim 23, wherein the polyene-based antifungal agent is amphotericin B.

25. The method of treating fungal infection of claim 22, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

26. The method of treating fungal infection of claim 20, wherein an inhibitor against at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans is sequentially or simultaneously injected with the inhibitor.

27. A method of treating fungal infection, comprising: injecting an effective amount of an inhibitor against at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of Cryptococcus neoformans into a subject.

28. The method of treating fungal infection of claim 27, wherein the inhibitor is an against CAC1 or PKA1 gene.

29. The method of treating fungal infection of claim 27, wherein a polyene- or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.

30. The method of treating fungal infection of claim 29, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.

31. The method of treating fungal infection of claim 30, wherein the polyene-based antifungal agent is amphotericin B.

32. The method of treating fungal infection of claim 29, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

33. The method of treating fungal infection of claim 27, wherein an inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans is sequentially or simultaneously injected with the inhibitor.

34. An antifungal combined formulation, comprising: an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans; and at least one antifungal agent selected from the group consisting of a polyene-based antifungal agent, an azole-based antifungal agent, and an inhibitor against at least one protein or gene coding for the same selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans.

35. A method of screening an antifungal agent comprising: contacting at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates an activity of the protein.

36. The method of claim 35, wherein the antifungal agent is an inhibitor against a Cac1 or Pka1 protein.

37. A method of screening an antifungal agent comprising: contacting at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates the activity of the gene.

38. The composition method of claim 37, wherein the antifungal agent is an inhibitor against a CAC1 or PKA1 gene.