U.S. patent application number 13/943150 was filed with the patent office on 2013-11-14 for antimicrobial therapy for bacterial infections.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to George Y. LIU, Victor NIZET.
Application Number | 20130303437 13/943150 |
Document ID | / |
Family ID | 38218400 |
Filed Date | 2013-11-14 |
United States Patent
Application |
20130303437 |
Kind Code |
A1 |
NIZET; Victor ; et
al. |
November 14, 2013 |
ANTIMICROBIAL THERAPY FOR BACTERIAL INFECTIONS
Abstract
The disclosure provides a molecular genetic approach of targeted
mutagenesis and heterologous expression, coupled with in vitro and
in vivo models of bacterial pathogenesis, to demonstrate that the
S. aureus pigment is a virulence factor and potential novel target
for antimicrobial therapy.
Inventors: |
NIZET; Victor; (San Diego,
CA) ; LIU; George Y.; (Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA |
Oakland |
CA |
US |
|
|
Family ID: |
38218400 |
Appl. No.: |
13/943150 |
Filed: |
July 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11918584 |
Oct 15, 2007 |
8507551 |
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PCT/US06/14486 |
Apr 17, 2006 |
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13943150 |
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60672359 |
Apr 18, 2005 |
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Current U.S.
Class: |
514/2.3 ;
514/154; 514/200; 514/23; 514/254.11; 514/464; 514/534;
514/646 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/222 20130101; Y02A 50/30 20180101; A61K 31/216 20130101;
A61K 31/357 20130101; A61K 31/135 20130101; A61K 31/522 20130101;
Y02A 50/475 20180101; A61K 31/138 20130101; A61P 31/04 20180101;
Y02A 50/481 20180101; Y02A 50/473 20180101; A61K 31/426 20130101;
A61K 31/195 20130101 |
Class at
Publication: |
514/2.3 ;
514/534; 514/646; 514/464; 514/200; 514/23; 514/154;
514/254.11 |
International
Class: |
A61K 31/216 20060101
A61K031/216; A61K 31/357 20060101 A61K031/357; A61K 45/06 20060101
A61K045/06; A61K 31/135 20060101 A61K031/135 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] This invention was made with government support under Grant
No. AI048694 awarded by the National Institutes of Health. The
government has certain rights in this invention.
Claims
1. A method for treating, improving on an effective treatment or
preventing a microbial infection in a subject; or for preventing,
treating or improving on an effective treatment for an antibiotic
resistant microbial infection in a subject, comprising: (a)
providing pharmaceutical composition comprising: a small molecule
carotenogenesis inhibitor that interacts with a microbial
carotenoid synthase enzyme and inhibits the production and/or
activity of a carotenoid in a microbe or a bacteria, wherein
inhibition of the production and/or activity of the carotenoid in
the microbe or the bacteria renders the microbe or bacteria more
susceptible to an oxidative damage, wherein optionally the
oxidative damage to the microbe or the bacteria is by a neutrophil
or a human neutrophil in vivo, and optionally targeting or
inhibition of the microbial carotenoid by the small molecule
carotenogenesis inhibitor comprises inhibiting the synthesis,
increasing the degradation or removal, or neutralizing the function
of the microbial carotenoid; and (b) (i) administering to a subject
inflicted with a microbial infection or susceptible to a microbial
invention a therapeutically effective dose of the pharmaceutical
composition, or (ii) coating or applying the pharmaceutical
composition to a device or a catheter, on a device or a catheter,
or with a device or a catheter; thereby treating, improving on an
effective treatment, or preventing the microbial infection, wherein
optionally the subject is a human or a non-human animal.
2. The method of claim 1, wherein the microbial infection
comprises: (a) a bacterial infection; (b) a gram positive or a gram
negative bacterial infection; (c) a Staphylococcus infection, or
the bacterial infection is a Staphylococcus sp. infection, or a
Staphylococcus aureus or a Staphylococcus epidermidis infection;
(d) an Escherichia coli (E. coli), a P. aeruginosa or a Salmonella
typhimurium infection; (e) a Streptococcus pyogenes (group A), a
Streptococcus sp. (viridans group), a Streptococcus agalactiae
(group B), a S. bovis, a Streptococcus (anaerobic species), a
Streptococcus pneumoniae, an Enterococcus sp., a gram-negative
cocci, a Neisseria gonorrhoeae, a Neisseria meningitidis, a
Branhamella catarrhalis, a Bacillus anthracis, a Bacillus subtilis,
a P. acne Corynebacterium diphtheria, a Corynebacterium species, a
diptheroid (aerobic or anaerobic), a Listeria monocytogenes, a
Clostridium tetani, a Clostridium difficile, an Enterobacter
species, a Proteus mirablis, a Pseudomonas aeruginosa, a Klebsiella
pneumoniae, a Salmonella, a Shigella, a Serratia or a Campylobacter
jejuni infection; or (f) a fungal infection, wherein optionally the
fungal infection is an Aspergillus fumigatus, a Burkholderia
cepacia, a Serratia marcesens, a Microsporum canis, a Microsporum
sp., a Trichophyton sp., a T. rubrum, a T. mentagrophyte, a yeast,
a Candida albicans, a C. Tropicalis, a Candida species, a
Saccharomyces cerevisiae, a Torulopsis glabrata, a Epidermophyton
floccosum, a Malassezia furfur, a Pityropsporon orbiculare, a P.
ovale, a Cryptococcus neoformans, an Aspergillus fumigatus, an
Aspergillus nidulans, an Aspergillus sp., a Zygomycetes, a
Rhizopus, a Mucor, a Paracoccidioides brasiliensis, a Blastomyces
dermatitides, a Histoplasma capsulatum, a Coccidioides immitis or a
Sporothrix schenckii infection.
3. The method of claim 1, wherein the microbe or bacteria are
antibiotic resistant, or the microbe or bacteria are methicillin-
or vancomycin-resistant strains, or the microbe or bacteria are
Methicillin-Resistant Staphylococcus Aureus (MRSA).
4. The method of claim 1, wherein the carotenoid is a
Staphylococcus sp., or a Staphylococcus aureus carotenoid.
5. The method of claim 1, wherein the carotenogenesis inhibitor is
a mixed function oxidase inhibitor, wherein optionally the mixed
function oxidase inhibitor comprises: a
2-diethylaminoethyl-2,2-diphenyl-valerate; a
2,4-dichloro-.beta.-phenylphenoxyethylamine; a
2,4-dichloro-6-phenylphenoxyethyldiethylamine; a piperonyl
butoxide, or a combination thereof.
6. The method of claim 1, wherein the contacting is in vivo or in
vitro, or the contacting is on a surface suspected of having a
microbe, or on a surface of a device or a catheter, or applied with
a device or catheter.
7. The method of claim 6, wherein the contacting in vivo is by
topical administration and the pharmaceutical composition is
formulated for topical administration.
8. The method of claim 1, wherein the carotenogenesis inhibitor is
administered in combination with at least one antibiotic, and
optionally the carotenogenesis inhibitor and antibiotic are
administered simultaneously or are administered sequentially.
9. The method of claim 8, wherein the at least one antibiotic
comprises: (a) an antibiotic belonging to a class selected from the
group consisting of aminoglycosides, penicillins, cephalosporins,
carbapenems, monobactams, quinolones, tetracyclines, glycopeptides,
chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole,
nitrofurantoin, rifampin and mupirocin; (b) an amikacin,
gentamicin, kanamycin, netilmicin, t-obramycin, streptomycin,
azithromycin, clarithromycin, erythromycin, erythromycin, estolate,
ethylsuccinate, gluceptatellactobionate, stearate, penicillin G,
penicillin V, methicillin, nafcillin, oxacillin, cloxacillin,
dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin,
mezlocillin, azlocillin, piperacillin, cephalothin, cefazolin,
cefaclor, cefamandole, cefoxitin, cefuiroxime, cefonicid,
cefmetazole, cefotetan, cefprozil, loracarbef, cefetamet,
cefoperazone, cefotaxime, ceftizoxime, ceftriaxone, ceftazidime,
cefepime, cefixime, cefpodoxime, cefsulodin, i-mipenem, aztreonam,
fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin,
enoxacin, lomefloxacin, cinoxacin, doxycycline, m-inocycline,
tetracycline, vancomycin or a teicoplanin or any combination
thereof; (c) any combination of at least two or more of (a) and/or
(b).
10. A pharmaceutical composition comprising a small molecule
carotenogenesis inhibitor and a pharmaceutically acceptable
carrier, wherein the small molecule carotenogenesis inhibitor
interacts with a microbial carotenoid synthase enzyme and inhibits
the production and/or activity of a carotenoid in a microbe or a
bacteria, wherein inhibition of the production and/or activity of
the carotenoid in the microbe or the bacteria renders the microbe
or bacteria more susceptible to an oxidative damage, wherein
optionally the oxidative damage to the microbe or the bacteria is
by a neutrophil or a human neutrophil in vivo, and optionally
targeting or inhibition of the microbial carotenoid by the small
molecule carotenogenesis inhibitor comprises inhibiting the
synthesis, increasing the degradation or removal, or neutralizing
the function of the microbial carotenoid.
11. The pharmaceutical composition of claim 10, wherein the
composition is a lotion, cream, gel, ointment or spray, or the
pharmaceutical composition is formulated for parenteral or for
topical administration, or the pharmaceutical composition is
formulated administration intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity, by inhalation, or
transdermally.
12. A device comprising a small molecule carotenogenesis inhibitor,
wherein the small molecule carotenogenesis inhibitor interacts with
a microbial carotenoid synthase enzyme and inhibits the production
and/or activity of a carotenoid in a microbe or a bacteria, wherein
inhibition of the production and/or activity of the carotenoid in
the microbe or the bacteria renders the microbe or bacteria more
susceptible to an oxidative damage, wherein optionally the
oxidative damage to the microbe or the bacteria is by a neutrophil
or a human neutrophil in vivo, and optionally targeting or
inhibition of the microbial carotenoid by the small molecule
carotenogenesis inhibitor comprises inhibiting the synthesis,
increasing the degradation or removal, or neutralizing the function
of the microbial carotenoid.
13. A catheter comprising a small molecule carotenogenesis
inhibitor, wherein the small molecule carotenogenesis inhibitor
interacts with a microbial carotenoid synthase enzyme and inhibits
the production and/or activity of a carotenoid in a microbe or a
bacteria, wherein inhibition of the production and/or activity of
the carotenoid in the microbe or the bacteria renders the microbe
or bacteria more susceptible to an oxidative damage, wherein
optionally the oxidative damage to the microbe or the bacteria is
by a neutrophil or a human neutrophil in vivo, and optionally
targeting or inhibition of the microbial carotenoid by the small
molecule carotenogenesis inhibitor comprises inhibiting the
synthesis, increasing the degradation or removal, or neutralizing
the function of the microbial carotenoid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application is a continuation of U.S. patent
application Ser. No. 11/918,584, filed Oct. 15, 2007 (now pending),
which claims priority to international patent (PCT) application
PCT/US2006/014486, filed Apr. 17, 2006, which claims priority under
35 USC .sctn.119 to U.S. Provisional Application Ser. No.
60/672,359, filed Apr. 18, 2005. Each of the aforementioned
applications are expressly incorporated herein by reference in
their entirety and for all purposes.
BACKGROUND
[0003] Ogston (1881) coined the genus Staphylococcus to describe
grapelike clusters of bacteria (staphylo=grape, Gr.) recovered in
pus from surgical abscesses. Shortly thereafter, Rosenbach (1884)
isolated this pathogen in pure culture, and proposed the species
name S. aureus (golden, Lat.) for its characteristic surface
pigmentation in comparison to less virulent staphylococci that
normally colonize the skin surface.
[0004] Entering its seventh decade, the era of antimicrobial
therapy has greatly reduced morbidity and mortality from infectious
diseases. However, the emergence of resistant microorganisms has
now reached epidemic proportions and poses great challenges to the
medical community. Worrisome trends are particularly evident in the
pre-eminent Gram-positive bacterial pathogen S. aureus, which has
become increasingly unresponsive to first-line antibiotic
therapies. S. aureus is probably the most common cause of
life-threatening acute bacterial infections in the world, and is
capable of causing a diverse array of diseases, ranging in severity
from a simple boil or impetigo to fulminant sepsis or toxic shock
syndrome. S. aureus is the single leading cause of bacteremia,
hospital-related (nosocomial) infections, skin and soft tissue
infections, wound infections, and bone and joint infections. It is
one of the most common agents of endocarditis and food
poisoning.
[0005] National prospective surveillance of over 24,000 invasive
bacterial isolates show disease-associated S. aureus strains with
methicillin resistance (MRSA) have increased from 22% in 1995 to
57% currently. MRSA are now frequently identified in
community-acquired infections as well as in hospital settings. An
urgent need exists for discovery of novel classes of antibiotics to
address this genuine public health crisis. A half-century of
synthesizing analogs based on <10 antibacterial scaffolds has
resulted in the development and marketing of >100 antibacterial
agents but, with the exception of the oxazolidinone core, no new
scaffolds have emerged in the past 30 years to address the emerging
resistance problems.
[0006] Developing new classes of antibiotics that target bacterial
virulence factors such as the S. aureus carotenoid pigment is an
approach that has not been utilized to anywhere near its potential.
Classic antibiotic approaches attempt to kill or suppress growth of
bacteria by targeting essential cell functions such as cell wall
biosynthesis, protein synthesis, DNA replication, RNA polymerase,
or metabolic pathways. These conventional therapies run a higher
risk of toxicity since many of these cell functions are also
essential to mammalian cells and require fine molecular distinction
between the microbial target and host cell counterpart(s). Second,
the repetitive use of the same essential targets means that when a
bacterium evolves resistance to a particular antibiotic agent
during therapy, it can become simultaneously cross resistant to
other agents acting on the same target, even though the bacterium
has never been exposed to the other agents. Third, conventional
therapies exert a "life-or-death" challenge upon the bacterium, and
thus a strong selective pressure to evolve resistance to the
antimicrobial agent. Finally, many current antibiotics have very
broad spectrums of activity, with the side effect of eradicating
many components of the normal flora, leading to undesired
complications such as Clostridium difficile colitis or secondary
fungal infections (e.g. Candida).
[0007] The emergence of MRSA has compromised the clinical utility
of methicillin and related antibiotics (oxacillin, dicloxacillin)
and all cephalosporins (e.g. cefazolin, cephalexin) in empiric
therapy of S. aureus infections. MRSA often have significant levels
of resistance to macrolides (e.g. erythromycin), beta-lactamase
inhibitor combinations (e.g. Unasyn, Augmentin) and
fluoroquinolones (e.g. ciprofloxacin), and are occasionally
resistant to clindamycin, trimethoprim/sulfamethoxisol (Bactrim),
and rifampin. In serious S. aureus infection, intravenous
Vancomycin is the last resort, but there have now also been
alarming reports of S. aureus resistance to vancomycin, an
intravenous antibiotic commonly used to treat MRSA.
[0008] New anti-MRSA agents such as linezolid (Zyvox or
quinupristin/dalfopristin (Synercid), both of which utilize the
traditional target of binding to the ribosomal subunits to inhibit
RNA synthesis, are quite expensive.
[0009] Subsequent studies of the S. aureus pigment have unraveled
an elaborate biosynthetic pathway that produces a series of
carotenoids. Similar carotenoids produced in dietary fruits and
vegetables are well recognized as potent antioxidants by virtue of
their free radical scavenging properties and exceptional ability to
quench singlet oxygen.
SUMMARY
[0010] The invention demonstrates that carotenoids serve as
virulence/resistance factors in microbes. In one specific example,
the invention demonstrates that S. aureus carotenoid is a virulence
factor that impairs neutrophil killing and promotes disease
pathogenesis by virtue of its antioxidant properties, and (2)
evidence that pharmacological inhibition of the carotenoid pigment
production renders the organism more susceptible to oxidants and
blood killing.
[0011] The invention provides a method of treating a bacterial
infection, comprising administering to a subject inflicted with the
infection an agent that inhibits the production and/or activity of
a carotenoid in the bacteria. In one embodiment, the bacterial
infection is a Staphylococcus infection. In another embodiment, the
bacteria is a Staphylococcus sp. In yet a further embodiment, the
bacteria is Staphylococcus aureus.
[0012] The invention also provides a method of preventing, treating
or improving the effective treatment of MRSA by targeting a
carotenoid. In one aspect, the carotenoid is a Staphylococcus sp.
carotenoid. In another aspect, the method comprises targeting of
the carotenoid comprises a carotenogenesis inhibitor. In yet a
further embodiment, the carotenogenesis inhibitor is a mixed
function oxidase inhibitor such as, for example,
2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A).
[0013] The invention also provides a method of screening for an
MRSA therapeutic agent useful to treat a bacterial infection
comprising contacting a bacteria that produces a carotenoid with a
test agent suspected of inhibiting the production or activity of a
carotenoid and measuring the effect of the agent in the presence
and absence of an oxidative agent.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1A-E shows a genetic manipulation of Staphylococcus
aureus carotenoid pigment and its antioxidant function. A)
Biochemical pathway for S. aureus carotenogenesis and mutagenesis
of crtM (encoding dehydrosqualene synthase) by allelic replacement.
B) Elimination of S. aureus pigmentation in .DELTA.crtM mutant;
heterologous expression of S. aureus 4'4'-diaponeurosporene pigment
in Streptococcus pyogenes. Increased susceptibility of the S.
aureus .DELTA.CrtM mutant to killing by C) hydrogen peroxide or D)
singlet oxygen, with restoration of WT resistance levels upon
complementation with pCrtMN. E)
[0015] Decreased singlet oxygen susceptibility of S. pyogenes
expressing 4' 4'-diaponeurosporene. Error bars represent standard
deviation of depicted variable; results shown are representative of
at least three experiments.
[0016] FIG. 2A-F shows Staphylococcus aureus carotenoid pigment
confers resistance to oxidant killing in neutrophils and whole
blood. Survival of WT and .DELTA.CrtM S. aureus in A) coculture
with isolated human neutrophils and B) murine whole blood. Also
shown in (B) is whole blood survival of .DELTA.CrtM complemented
with vector alone or pCrtMN. (C) Effect of plasmid expression of
crtMN on survival of Streptococcus pyogenes in mouse whole blood.
D) Effect of oxidative burst inhibitor DPI on survival of WT and
.DELTA.CrtM mutant S. aureus human neutrophil coculture. Relative
survival of WT and .DELTA.CrtM mutant S. aureus in E) normal and
gp47.sup.phox-/- patient lacking NADPH oxidase function or F) the
blood of wild-type CD1 and C57B1/6 mice and gp91.sup.phox-/- mice.
Results representative of at least three experiments. The assay
using blood from the CGD human patient was performed twice.
[0017] FIG. 3A-C shows Staphylococcus aureus carotenoid contributes
to virulence in a subcutaneous abscess model. Mice were injected
subcutaneously in opposite flanks with the two bacterial strains
under comparison. Line graphs depict sum cumulative skin lesion
size generated by the indicated bacterial strain. Dots on scatter
graphs=ratio of cfu of pigmented vs. nonpigmented strains recovered
from skin lesions in each individual mouse. Photographic image
depicts representative mouse in each treatment group. A) Wild-type
(WT) vs. .DELTA.CrtM mutant S. aureus in CD1 mice, B) WT vs.
.DELTA.CrtM mutant S. aureus in gp91.sup.phox-/- mice, and C)
Streptococcus pyogenes+/-expression of staphylococcal
4'4'-diaponeurosporene.
[0018] FIG. 4A-B shows the inhibition of Staphylococcus aureus
pigment production increases oxidant sensitivity and phagocytic
clearance. Wild-type and .DELTA.CrtM mutant S. aureus were cultured
in the presence or absence of SKF 525-A at the indicated
concentrations. Depicted are the observed effects on A)
pigmentation phenotype, B) singlet oxygen susceptibility, and C)
survival in murine whole blood. Results shown are representative of
at least three experiments.
[0019] FIG. 5A-D demonstrates a lack of pleomorphic effects upon
allelic replacement of the S. aureus crtM gene. A) Similarity of
stationary phase concentrations of viable bacteria in stationary
phase cultures of WT S. aureus vs. .DELTA.crtM mutant and
subsequent growth kinetics of bacteria in fresh THB media. Lack of
measurable difference between WT S. aureus and .DELTA.crtM mutant
in terms of B) buoyant density assessed by migration in Percoll, C)
hydrophobicity as measured by partition into N-hexadecane, and D)
surface charge determined by cytochrome C binding.
[0020] FIG. 6A-F shows a further analyses regarding the
antiphagocytic properties of the Staphylococcus aureus carotenoid
pigment. A) WT and .DELTA.crtM mutant S. aureus are phagocytosed by
human neutrophils at a comparable rate. Deconvolution fluorescence
microscopy of representative study shows intracellular (green) and
extracellular (red) organisms. B) WT and .DELTA.crtM mutant S.
aureus provoke similar degrees of human neutrophil oxidative burst
as measured by nitroblue tetrazolium reduction. C) Effect of
oxidative burst inhibitor diphenyleneiodonium (DPI) on survival of
WT and .DELTA.CrtM mutant S. aureus in normal mouse blood.
Sensitivity of WT and .DELTA.CrtM mutant S. aureus strains to
killing by D) the granule proteases cathepsin G and human
neutrophil elastase or E) the murine cathelicidin mCRAMP. F)
Differences in neutrophil intracellular survival between S. aureus
WT and .DELTA.CrtM mutant S. aureus are similar in experiments
using preopsonization with autologous serum as those without (e.g.
FIG. 6A).
DETAILED DESCRIPTION
[0021] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a protein" includes a plurality of such proteins and reference to
"the cell" includes reference to one or more cells known to those
skilled in the art, and so forth.
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and
materials are described herein.
[0023] The publications discussed above and throughout the text are
provided solely for their disclosure prior to the filing date of
the present application. Nothing herein is to be construed as an
admission that the inventors are not entitled to antedate such
disclosure by virtue of prior disclosure.
[0024] Methods and compositions useful for treatment of microbial
infections, wherein the microbe expresses a carotenoid are
provided. For example, methods and compositions useful for the
treatment of S. aureus infections, including those produced by
methicillin- and vancomycin-resistant strains, are provided by the
invention. The methods and compositions of the invention can be
used alone or in combination with traditional antimicrobials and
antibiotics to treat such infections. In addition, the methods and
compositions disclosed herein can be used in settings such as
foreign-body, catheter or endovascular infections, chronic
osteomyelitis, hospital acquired or postoperative infections,
recurrent skin infections, or for S. aureus infections in the
immunocompromised host.
[0025] Carotenoids represent a major class of natural pigments.
More than 600 different carotenoids have been identified in
bacteria, fungi, algae, plants and animals (Staub, 0., In: Pfander,
H. (ed.), Key to Carotenoids, 2.sup.nd ed., Birkhuser Verlag,
Basel). They function as accessory pigments in photosynthesis, as
antioxidants, as precursors for vitamins in humans and animals and
as pigments for light protection and species specific coloration.
Carotenoids have historically been of interest, e.g., for
pharmaceuticals, food colorants, and animal feed and nutrient
supplements. The discovery that these natural products can play an
important role in the prevention of cancer and chronic disease
(mainly due to their antioxidant properties) and, more recently,
that they exhibit significant tumor suppression activity due to
specific interactions with cancer cells, has boosted interest in
their pharmaceutical potential (Bertram, J. S., Nutr. Rev., 1999;
57:182-191; Singh, et al., Oncology, 1998; 12 : 1643-168; Rock, C.
L., Pharmacol. Ther., 1997; 75 : 185-197; Edge, et al., J.
Photochem. Photobiol., 1997; 41 : 189-200).
[0026] Carotenoid can be produced in recombinant microorganisms by
combining biosynthetic genes from different organisms to create
biosynthetic pathways. At present more than 150 genes for 24
carotenogenic enzymes (crt) have been isolated from bacteria,
plants, algae and fungi that can be used to engineer a variety of
diverse carotenoids.
[0027] Complete carotenoid biosynthesis pathways have been cloned
from a number of bacteria, where the biosynthesis enzyme genes are
arranged in gene cluster (reviewed in Armstrong, Ann. Rev.
Microbiol., 1997, 51:629; Sandmann, Eur. J. Biochem., 1994,
223:7).
[0028] The pathways Erwinia and Rhodobacter for the synthesis of
zeaxanthin diglucoside and the acyclic xanthophylls speroidene and
spheroidenone, respectively, were the first from which all involved
enzymes have been identified (Armstrong et al., Mol. & General
Gene., 1989, 216:254; Lang et al., J. Bacteriol., 1995, 177:2064;
Lee and Liu, MoI. Microbiol., 1991, 5:217; Misawa et al., J.
Bacteriol., 1990, 172:6704).
[0029] Various techniques have been applied for cloning of
carotenogenic genes (Hirschberg, J., In: Carotenoids: Biosynthesis
and Metabolism, Vol. 3, Carotenoids, G. Britton, Ed. Basel:
Birkhuser Verlag, 148-194, 1998). Functional color complementation
in E. coli expressing carotenogenic genes from Erwinia has been
used successfully for the cloning of a variety of microbial and
plant carotenogenic genes (Verdoes et al., Biotech, and Bioeng.,
1999, 63:750; Zhu et al., Plant and Cell Physiology, 1997, 38:357;
Kajiwara et al., Plant MoI. Biol., 1995, 29:343; Pecker et al.,
Plant MoI. Biol., 1996, 30:807; plant carotenoid biosynthesis is
reviewed in Hirschberg et al., Pure and Applied Chemistry, 1997,
69:215 1; Cunningham and Gantt, Ann. Rev. of Plant Physiol, and
Plant MoI. Biol., 1998, 49:557).
[0030] Genes encoding the early carotenoid biosynthesis enzymes
GGDP synthase, phytoene synthase and phytoene desaturase account
for more than half of all cloned carotenogenic genes. Different
phytoene desaturase genes are available that introduce two, three,
four or five double bonds into phytoene to produce carotene (plant,
cyanobacteria, algae) (Bartley et al., Eur. J. of Biochem., 1999,
259:396), neurosporene (Rhodobacter) (Raisig et al., J. Biochem.,
1996, 119:559), lycopene (most eubacteria and fungi) (Verdoes, et
al., Biotech, and Bioeng., 1999, 63:750; RuizHidalgo et al., MoI.
& Gen. Genetics, 1997, 253:734) or 3,4-didehydrolycopene
(Neurospora crassa) (Schmidhauser et al., MoI. and Cell Biol.,
1990, 10:5064), respectively. The following are examples of
carotenoid biosynthesis genes have been cloned:
[0031] crtE: GGPP-synthase from R. capsulatus and E. uredovora
[0032] crtB: phytoene synthase from R. capsulatus and E.
uredovora
[0033] crtl: phytoene desaturase from E. uredovora and E.
herbicola
[0034] crtY: lycopene cyclase from E. uredevora and E.
herbicola
[0035] crtA: spheroidene monooxygenase from R. capsulatus and R.
spaeroides crtO: .beta.-C4-ketolase (oxygenase) from Synechocistis
sp.
[0036] crtW: .beta.-C4-ketolase from Algaligenes sp., A.
aurantiacum
[0037] crtD: methoxyneurosporene desaturase from R. capsulatus and
R. spaeroides
[0038] crtX: zeaxanthin glucosyl transferase from E. uredovora and
E. herbicola crtZ: .beta.-carotene hydroxylase from E. uredovora
and E. herbicola
[0039] crtU: .beta.-carotene desaturase from S. griseus
[0040] crtM: dehydrosqualene synthase from S. aureus
[0041] crtN: dehydrosqualene desaturase from S. aureus.
[0042] A leading human pathogen Staphylococcus aureus (S. aureus)
was named secondary to it characteristic golden yellow pigmentation
(aureus=golden, Latin) in comparison to less virulent staphylococci
{e.g. S. epidermidis) that normally colonize the skin surface.
Subsequent research regarding the S. aureus pigment unraveled an
elaborate biosynthetic pathway that produces a series of
carotenoids. Similar carotenoids produced in dietary fruits and
vegetables are well recognized as potent antioxidants by virtue of
their free radical scavenging properties and exceptional ability to
quench singlet oxygen. The S. aureus pigment may have similar
properties. The invention examined whether S. aureus could utilize
its golden carotenoid pigment to resist oxidant based clearance
mechanisms of the host innate immune system. For example,
neutrophils and macrophages kill bacteria by generating an
"oxidative burst" of reactive molecules such as peroxide, bleach
and singlet oxygen that kill bacteria that they have
phagocytosed.
[0043] Golden color imparted by carotenoid pigments is the
eponymous feature of the human pathogen Staphylococcus aureus. A
molecular genetic analysis pairing mutagenesis and heterologous
expression was used to show that this hallmark phenotype is in fact
a virulence factor, serving to protect the bacterium from
phagocytic killing through its antioxidant properties. In the
present era, effective control of this important disease agent is
compromised by rapid evolution of antimicrobial resistance in both
community and hospital settings. The invention demonstrates that
the inhibition of carotenogenesis offers a novel therapeutic
approach to the treatment of complicated S. aureus infections,
effectively rendering the pathogen more susceptible to clearance by
normal host innate immune defenses.
[0044] The invention demonstrates that carotenoid pigment
production contributes to resistance of Group B Streptococcus (GBS)
to macrophage killing. GBS is the leading cause of invasive
bacterial infections such as pneumonia, sepsis and meningitis in
human neonates. A gene required for production of carotenoid
pigment (CyIE) has been identified. The pigment is required for the
organism's production of a hemolysin/cytolysin toxin that have cell
damaging and proinflammatory properties important in disease
pathogenesis. Other bacterial and fungal human pathogens associated
with invasive infections produce carotenoid or melanin-like
pigments with antioxidant properties (e.g. Aspergillus
fumigatus.sub.r Burkholderia cepacia, Serratia marcesens),
suggesting a common pathogenic theme and revealing a potential
selective advantage for pigment production against phagocytic
clearance mechanisms.
[0045] Accordingly, the approach of targeting pigment production
for antimicrobial therapy can be extended to organisms that produce
pigments (e.g., carotenoid pigments or other pigments that confer
oxidative protection). In particular, Aspergillus sp. are an
important cause of mortality in immunocompromised subjects (e.g.,
cancer chemotherapy) and Burkholderia cepacia is an important cause
of mortality in cystic fibrosis. Both Aspergillus and Burkholderia
are commonly multidrug resistant and recalcitrant to existing
antibiotic therapies.
[0046] The invention demonstrates that by using a molecular genetic
approach of targeted gene deletion (to create nonpigmented S.
aureus mutants) and cloning techniques (to express the S. aureus
pigment in other bacteria) to create "living reagents" the true
importance of the pigment in S. aureus disease pathogenesis was
identified. S. aureus carotenoid pigment did indeed protect the
bacterium against peroxide and singlet oxygen and made it more
resistant to killing in mouse and human blood and by purified human
neutrophils. Using a mouse model of S. aureus abscess formation, it
was demonstrated the carotenoid pigment promoted bacterial survival
and disease progression in vivo. Control experiments using mice
impaired in neutrophil oxidant production showed that carotenoid
pigment production contributed to S. aureus virulence by virtue of
its antioxidant properties. The invention demonstrates that
pharmacological inhibition of S. aureus pigment production rendered
the bacterium more susceptible to oxidants and impaired in blood
survival.
[0047] One important mechanism by which phagocytic cells eliminate
pathogens is through release of reactive oxygen species generated
by NADPH oxidase. The invention demonstrates that carotenoids such
as those expressed by S. aureus serve a protective function against
these defense molecules. For example, a .DELTA.CrtM mutant was
killed 10 to 100-fold more efficiently by hydrogen peroxide and 100
to 1,000-fold more efficiently by singlet oxygen than the WT S.
aureus strain. Similarly, heterologous expression of staphylococcal
pigment in S. pyogenes was associated with a 100 to 1,000-fold
decrease in singlet oxygen lethality.
[0048] The invention provides methods and agents that inhibited
carotenogenesis for the treatment of microbial infections and to
facilitate anti-microbial activity. For example, in one aspect, the
mixed function oxidase inhibitor
2-diethylaminoethyl-2.sub./2-diphenyl-valerate (SKF 525-A,
Calbiochem) is shown to inhibit pigment formation in S. aureus, and
a dose-dependent decrease in carotenoid production was demonstrated
in the WT strain of S. aureus grown in the presence of the agent.
Blocking S. aureus pigment formation led to a commensurate
dose-dependent increase in the susceptibility of the organism to
singlet oxygen killing and a decrease in its ability to survive in
human whole blood. As a control, the .DELTA.CrtM mutant was exposed
to SKF 525-A in parallel experiments with no significant effects on
oxidant susceptibility or blood survival.
[0049] The invention provides methods and compositions useful for
treating bacterial infection by inhibiting the production and/or
activity of carotenoids. More particularly, the invention provides
methods of treating a subject having a bacterial infection (e.g., a
bacterial infection of Staphylococcus sp.) comprising contacting a
subject with an agent that inhibits the activity and/or production
of a carotenoid of the Staphylococcus sp. In one aspect, the agent
is a small molecule, a polynucleotide {e.g., a ribozyme or
antisense molecule), a polypeptide (e.g., an antibody) or a
peptidomimetic. In one aspect of the invention, the agent is a
pharmacologic agent that inhibits carotenogenesis. For example, the
agent can be a mixed function oxidase inhibitor such as
2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem),
2,4-dichloro-.beta.-phenylphenoxyethylamine,
2,4-dichloro-6-phenylphenoxyethyldiethylamine, and piperonyl
butoxide.
[0050] In another aspect, the carotenogenesis inhibitor comprises a
oligonucleotide or polynucleotide (e.g., an antisense, ribozyme, or
siRNA). For example, antisense technology is a method of
down-regulating genes where the sequence of the target gene is
known. A large number of carotenoid genes are known in the art
including those of Staphylococcus sp.
[0051] In this aspect, a nucleic acid segment from the desired gene
(e.g., crtM and/or crtN) is cloned and operably linked to a
promoter such that the anti-sense strand of RNA will be
transcribed. This construct is then introduced into a target cell
and the antisense strand of RNA is produced. Antisense RNA inhibits
gene expression by preventing the accumulation of mRNA encoding the
protein of interest. Thus, an antisense molecule against crtM
and/or crtN will result in the reduction of synthesis of
carotenoids a pathogenic microbe, thereby rendering the microbe
susceptible to oxidative damage by phagocytes. A person skilled in
the art will know that certain considerations are associated with
the use of antisense technologies in order to reduce expression of
particular genes. For example, the proper level of expression of
antisense genes may require the use of different chimeric genes
utilizing different regulatory elements known to the skilled
artisan.
[0052] Within the context of the invention, it is apparent that
modulating the expression of bacterial carotenoid biosynthetic
pathways by the use of inhibitory nucleic acids provides a useful
therapeutic method. For example, the invention identifies a number
of genes encoding enzymes in the carotenoid biosynthetic pathway
including crtM and crtN.
Alternatively (or in addition to), it may be desirable to knockout
the crtM/crtN genes leading to the synthesis of C.sub.30
carotenoids. Common molecular biology techniques can be used to
generate inhibitory nucleic acid molecules such as, for example,
antisense molecules that can interact with a crtN and/or M nucleic
acid produced by wild type bacteria (e.g., Staphylococcus sp.).
[0053] As used herein an isolated nucleic acid is substantially
free of proteins, lipids, and other nucleic acids with which an in
vivo-produced nucleic acids naturally associated. Typically, the
nucleic acid is at least 70%, 80%, 90% or more pure by weight, and
conventional methods for synthesizing nucleic acids in vitro can be
used in lieu of in vivo methods. As used herein, "nucleic acid" or
"polynucleotide" or "oligonucleotide" refers to a polymer of
deoxyribonucleotides or ribonucleotides, in the form of a separate
fragment or as a component of a larger genetic construct (e.g., by
operably linking a promoter to a nucleic acid encoding, for
example, an antisense molecule). Numerous genetic constructs (e.g.,
plasmids and other expression vectors) are known in the art and can
be used to produce a desired nucleic acid of the disclosure in
cell-free systems or prokaryotic or eukaryotic (e.g., yeast,
insect, or mammalian) cells. The nucleic acids of the disclosure
can readily be used in conventional molecular biology methods to
produce the peptides of the disclosure.
[0054] Polynucleotides comprising an antisense nucleic acid,
ribozyme, or siRNA can be inserted into an "expression vector." The
term "expression vector" refers to a genetic construct such as a
plasmid, virus or other vehicle known in the art that can be
engineered to contain, for example, an antisense molecule. The
expression vector typically contains an origin of replication, and
a promoter, as well as genes that allow phenotypic selection of the
transformed cells. Various promoters, including inducible and
constitutive promoters, can be utilized in the disclosure.
Typically, the expression vector contains a replicon site and
control sequences that are derived from a species compatible with
the host/target cell.
[0055] Transformation or transfection of a host/target cell with a
polynucleotide of the disclosure can be carried out using
conventional techniques known to those skilled in the art. For
example, where the host cell is E. coli, competent cells that are
capable of DNA uptake can be prepared using the CaCl.sub.2,
MgCl.sub.2 or RbCl methods known in the art. Alternatively,
physical means, such as electroporation or microinjection can be
used. Electroporation allows transfer of a polynucleotide into a
cell by high voltage electric impulse. Additionally,
polynucleotides can be introduced into host cells by protoplast
fusion, using methods well known in the art. Suitable methods for
transforming eukaryotic cells, such as electroporation and
lipofection, also are known.
[0056] A host cell or target cell encompassed by of the disclosure
are any cells in which the polynucleotides of the disclosure can be
used to express an inhibitory nucleic acid molecule. The term also
includes any progeny of a host/target cell. Host/target cells,
which are useful, include bacterial cells, fungal cells (e.g.,
yeast cells), plant cells and animal cells. For example, host cells
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology (1986)). As representative examples of
appropriate hosts, there may be mentioned: fungal cells, such as
yeast; insect cells such as Drosophila S2 and Spodoptera Sf9;
animal cells such as CHO, COS or Bowes melanoma; plant cells, and
the like. The selection of an appropriate host is deemed to be
within the scope of those skilled in the art from the teachings
herein.
[0057] Host cells can be eukaryotic host cells (e.g., mammalian
cells). In one aspect, the host cells are mammalian production
cells adapted to grow in cell culture. Examples of such cells
commonly used in the industry are CHO, VERO, BHK, HeLa, CV1
(including Cos; Cos-7), MDCK, 293, 3T3, C127, myeloma cell lines
(especially murine), PC12 and W138 cells. Chinese hamster ovary
(CHO) cells are widely used for the production of several complex
recombinant proteins, e.g. cytokines, clotting factors, and
antibodies (Brasel et al., Blood 88:2004-2012, 1996; Kaufman et
al., J. Biol Chem 263: 6352-6362, 1988; McKinnon et al., MoI
Endocrinol 6:231-239, 1991; Wood et al., J. Immunol. 145:3011-3016,
1990). The dihydrofolate reductase (DHFR)-deficient mutant cell
lines (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216-4220,
1980) are the CHO host cell lines commonly used because the
efficient DHFR selectable and amplifiable gene expression system
allows high level recombinant protein expression in these cells
(Kaufman, Meth Enzymol. 185:527-566, 1990). In addition, these
cells are easy to manipulate as adherent or suspension cultures and
exhibit relatively good genetic stability. CHO cells and
recombinant proteins expressed in them have been extensively
characterized and have been approved for use in clinical
manufacturing by regulatory agencies.
[0058] The activity of such inhibitory agents (e.g., small
molecules (2-diethylaminoethyl-2,2-diphenyl-valerate) and
inhibitory nucleic acids) can be determined using conventional
methods known to those of skill in the art, such as the assays
described herein (both in vitro and in vivo assays).
[0059] The disclosure also provides a method for inhibiting the
growth of a bacterium by contacting the bacterium with an
inhibiting effective amount of a carotenoid biosynthesis inhibitor
(i.e., a carotenogenesis inhibitor). The term "contacting" refers
to exposing the microbe (e.g., bacterium) to an agent so that the
agent can inhibit, kill, or lyse microbe or render it susceptible
to oxidative destruction. Contacting of an organism with an agent
that inhibits carotenoid biosynthesis can occur in vitro, for
example, by adding the agent to a bacterial culture, or contacting
a bacterially contaminated surface with the agent.
[0060] Alternatively, contacting can occur in vivo, for example by
administering the agent to a subject afflicted with a bacterial
infection or susceptible to infection. In vivo contacting includes
both parenteral as well as topical "Inhibiting" or "inhibiting
effective amount" refers to the amount of agent that is sufficient
to cause, for example, a bacteriostatic or bactericidal effect,
reduce coloration of a particular bacterial cell type, or decrease
the amount of a particular carotenoid produced by the bacteria.
Bacteria that can be affected by the use of a carotenoid inhibitor
include both gram-negative and gram-positive bacteria. For example,
bacteria that can be affected include Staphylococcus aureus,
Streptococcus pyogenes (group A), Streptococcus sp. (viridans
group), Streptococcus agalactiae (group B), S. bovis, Streptococcus
(anaerobic species), Streptococcus pneumoniae, and Enterococcus
sp.; Gram-negative cocci such as, for example, Neisseria
gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis;
Gram-positive bacilli such as Bacillus anthracis, Bacillus
subtilis, P. acne Corynebacterium diphtheriae and Corynebacterium
species which are diptheroids (aerobic and anaerobic), Listeria
monocytogenes, Clostridium tetani, Clostridium difficile,
Escherichia coli, Enterobacter species, Proteus mirablis and other
sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella,
Shigella, Serratia, and Campylobacter jejuni. In particular the
methods and compositions of the invention are useful against any
pathogen that synthesizes a carotenoid the confers protection
against reactive oxygen species (e.g., species produced by NADPH
oxidase). Infection with one or more of these bacteria can result
in diseases such as bacteremia, pneumonia, meningitis,
osteomyelitis, endocarditis, sinusitis, arthritis, urinary tract
infections, tetanus, gangrene, colitis, acute gastroenteritis,
impetigo, acne, acne posacue, wound infections, born infections,
fascitis, bronchitis, and a variety of abscesses, nosocomial
infections, and opportunistic infections. The method for inhibiting
the growth of bacteria can also include contacting the bacterium
with the peptide in combination with one or more antibiotics.
[0061] Fungal organisms may also be affected by the carotenoid
inhibitors (e.g., Microsporum canis and other Microsporum sp.; and
Trichophyton sp. such as T. rubrum, and T. mentagrophytes), yeasts
(e.g., Candida albicans, C. Tropicalis, or other Candida species),
Saccharomyces cerevisiae, Torulopsis glabrata, Epidermophyton
floccosum, Malassezia furfur (Pityropsporon orbiculare, or P.
ovale), Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus
nidulanS.sub.f and other Aspergillus sp., Zygomycetes (e.g.,
Rhizopus, Mucor), Paracoccidioides brasiliensis, Blastomyces
dermatitides, Histoplasma capsulatum, Coccidioides immitis, and
Sporothrix schenckii.
[0062] A carotenoid biosynthesis inhibitor can be administered to
any host, including a human or non-human animal, in an amount
effective to inhibit the production of carotenoids that confer, for
example, resistance to oxidative attack. In one aspect, the
administration results in the inhibition of growth of a bacterium,
virus, and/or fungus. Thus, the methods and compositions are useful
as antimicrobial agents, antiviral agents, and/or antifungal
agents.
[0063] Any of a variety of art-known methods can be used to
administer a carotenoid inhibitory agent either alone or in
combination with other antibiotic agents. For example,
administration can be parenterally by injection or by gradual
infusion over time. The agent (s) can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, by inhalation, or transdermally.
[0064] In another aspect, a carotenoid biosynthesis inhibitor can
be formulated either alone or in combination with other
antibiotics/antifungals for topical administration (e.g., as a
lotion, cream, spray, gel, or ointment). Such topical formulations
are useful in treating or inhibiting microbial, fungal, and/or
viral presence or infections on the eye, skin, and mucous membranes
such as mouth, vagina and the like. Examples of formulations in the
market place include topical lotions, creams, soaps, wipes, and the
like. It may be formulated into liposomes to reduce toxicity or
increase bioavailability. Other methods for delivery include oral
methods that entail encapsulation of the in microspheres or
proteinoids, aerosol delivery (e.g., to the lungs), or transdermal
delivery (e.g., by iontophoresis or transdermal electroporation).
Other methods of administration will be known to those skilled in
the art.
[0065] Preparations for parenteral administration of a composition
comprising a carotenoid inhibitor include sterile aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils (e.g., olive oil), and injectable organic esters
such as ethyl oleate. Examples of aqueous carriers include water,
saline, and buffered media, alcoholic/aqueous solutions, and
emulsions or suspensions. Examples of parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's, and fixed oils. Intravenous vehicles
include fluid and nutrient replenishers, electrolyte replenishers
(such as those based on Ringer's dextrose), and the like.
Preservatives and other additives such as, other antimicrobial,
anti-oxidants, cheating agents, inert gases and the like also can
be included.
[0066] The disclosure provides a method for inhibiting a bacterial,
viral and/or fungal-associated disorder by contacting or
administering a therapeutically effective amount of a carotenoid
biosynthesis inhibitor (e.g., a crtM and/or crtN inhibitor) either
alone or in combination with other antimicrobial agents to a
subject who has, or is at risk of having, such a disorder. The term
"inhibiting" means preventing or ameliorating a sign or symptoms of
a disorder {e.g., a rash, sore, and the like). Examples of disease
signs that can be ameliorated include an increase in a subject's
blood level of TNF, fever, hypotension, neutropenia, leukopenia,
thrombocytopenia, disseminated intravascular coagulation, adult
respiratory distress syndrome, shock, and organ failure. Examples
of subjects who can be treated in the disclosure include those at
risk for, or those suffering from, a toxemia, such as endotoxemia
resulting from a gram-negative bacterial infection, venom
poisoning, or hepatic failure. Other examples include subjects
having a dermatitis as well as those having skin infections or
injuries subject to infection with gram-positive or gram-negative
bacteria, a virus, or a fungus. Examples of candidate subjects
include those suffering from infection by E. coli, Neisseria
meningitides, staphylococci, or pneumococci. Other subjects include
those suffering from gunshot wounds, renal or hepatic failure,
trauma, burns, immuno-compromising infections (e.g., HIV
infections), hematopoietic neoplasias, multiple myeloma,
Castleman's disease or cardiac myxoma. Those skilled in the art of
medicine can readily employ conventional criteria to identify
appropriate subjects for treatment in accordance with the
disclosure.
[0067] A therapeutically effective amount can be measured as the
amount sufficient to decrease a subject's symptoms of dermatitis or
rash by measuring the frequency of severity of skin sores.
Typically, the subject is treated with an amount of a therapeutic
composition of the invention sufficient to reduce a symptom of a
disease or disorder by at least 50%, 90% or 100%. Generally, the
optimal dosage will depend upon the disorder and factors such as
the weight of the subject, the type of bacteria, virus or fungal
infection, the weight, sex, and degree of symptoms. Nonetheless,
suitable dosages can readily be determined by one skilled in the
art. Typically, a suitable dosage is 0.5 to 40 mg/kg body weight,
e.g., 1 to 8 mg/kg body weight.
[0068] As mentioned previously, the compositions and methods of the
invention can include the use of additional (e.g., in addition to a
carotenoid biosynthesis inhibitor) therapeutic agents (e.g., an
inhibitor of TNF, an antibiotic, and the like). The carotenoid
biosynthesis inhibitor, other therapeutic agent (s), and/or
antibiotic (so can be administered, simultaneously, but may also be
administered sequentially. Suitable antibiotics include
aminoglycosides (e.g., gentamicin), beta-lactams (e.g., penicillins
and cephalosporins), quinolones (e.g., ciprofloxacin), and
novobiocin. Generally, the antibiotic is administered in a
bactericidal, antiviral and/or antifungal amount.
[0069] The methods and compositions of the invention utilizing
carotenoid biosynthesis inhibitors are useful as a broad-spectrum
antimicrobials suitable for tackling the growing problem of
antibiotic-resistant bacteria strains, and for treating and/or
preventing outbreaks of infectious diseases, including diseases
caused by bioterrorism agents like anthrax, plague, cholera,
gastroenteritis, multidrug-resistant tuberculosis (MDR TB).
[0070] A pharmaceutical composition comprising a carotenogenesis
inhibitor according to the disclosure can be in a form suitable for
administration to a subject using carriers, excipients, and
additives or auxiliaries. Frequently used carriers or auxiliaries
include magnesium carbonate, titanium dioxide, lactose, mannitol
and other sugars, talc, milk protein, gelatin, starch, vitamins,
cellulose and its derivatives, animal and vegetable oils,
polyethylene glycols and solvents, such as sterile water, alcohols,
glycerol, and polyhydric alcohols. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial, anti-oxidants, chelating agents, and inert gases.
Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed., Easton: Mack Publishing Co.,
1405-1412, 1461-1487 (1975), and The National Formulary XIV., 14th
ed., Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's, The Pharmacological Basis for
Therapeutics (7th ed.).
[0071] The pharmaceutical compositions according to the disclosure
may be administered locally or systemically. A "therapeutically
effective dose" is the quantity of an agent according to the
disclosure necessary to prevent, to cure, or at least partially
arrest the symptoms of a bacterial infection. Amounts effective for
this use will, of course, depend on the severity of the disease and
the weight and general state of the subject. Typically, dosages
used in vitro may provide useful guidance in the amounts useful for
in situ administration of the pharmaceutical composition, and
animal models may be used to determine effective dosages for
treatment of infections. Various considerations are described,
e.g., in Langer, Science, 249: 1527, (1990); Gilman et al. (eds.)
(1990), each of which is herein incorporated by reference.
[0072] As used herein, "administering a therapeutically effective
amount" is intended to include methods of giving or applying a
pharmaceutical composition of the disclosure to a subject that
allow the composition to perform its intended therapeutic function.
The therapeutically effective amounts will vary according to
factors, such as the degree of infection in a subject, the age,
sex, and weight of the individual. Dosage regimes can be adjusted
to provide the optimum therapeutic response. For example, several
divided doses can be administered daily or the dose can be
proportionally reduced as indicated by the exigencies of the
therapeutic situation.
[0073] The pharmaceutical composition can be administered in a
convenient manner, such as by injection (subcutaneous, intravenous,
etc.), oral administration, inhalation, transdermal application, or
rectal administration. Depending on the route of administration,
the pharmaceutical composition can be coated with a material to
protect the pharmaceutical composition from the action of enzymes,
acids, and other natural conditions that may inactivate the
pharmaceutical composition. The pharmaceutical composition can also
be administered parenterally or intraperitoneally. Dispersions can
also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof, and in oils. Under ordinary conditions of storage
and use, these preparations may contain a preservative to prevent
the growth of microorganisms.
[0074] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. In all cases, the
composition should be sterile and should be fluid to the extent
that easy syringability exists. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size, in the case of dispersion, and by the use
of surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chloro-butanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be typical to
include isotonic agents, for example, sugars, polyalcohols, such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent that delays
absorption, for example, aluminum monostearate and gelatin.
[0075] Sterile injectable solutions can be prepared by
incorporating the pharmaceutical composition in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
pharmaceutical composition into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from
those enumerated above.
[0076] The pharmaceutical composition can be orally administered,
for example, with an inert diluent or an assimilable edible
carrier. The pharmaceutical composition and other ingredients can
also be enclosed in a hard or soft-shell gelatin capsule,
compressed into tablets, or incorporated directly into the
individual's diet. For oral therapeutic administration, the
pharmaceutical composition can be incorporated with excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% by weight
of active compound. The percentage of the compositions and
preparations can, of course, be varied and can conveniently be
between about 5% to about 80% of the weight of the unit.
[0077] The tablets, troches, pills, capsules, and the like can also
contain the following: a binder, such as gum gragacanth, acacia,
corn starch, or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid, and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin, or a
flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring. When the dosage unit form is a capsule, it can contain,
in addition to materials of the above type, a liquid carrier.
Various other materials can be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules can be coated with shellac, sugar, or both. A
syrup or elixir can contain the agent, sucrose as a sweetening
agent, methyl and propylparabens as preservatives, a dye, and
flavoring, such as cherry or orange flavor. Of course, any material
used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic/biocompatible in the amounts
employed. In addition, the pharmaceutical composition can be
incorporated into sustained-release preparations and
formulations.
[0078] Thus, a "pharmaceutically acceptable carrier" is intended to
include solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the pharmaceutical
composition, use thereof in the therapeutic compositions and
methods of treatment is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0079] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form" as used herein, refers to
physically discrete units suited as unitary dosages for the
individual to be treated; each unit containing a predetermined
quantity of pharmaceutical composition is calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the disclosure are related to the characteristics of the
pharmaceutical composition and the particular therapeutic effect to
be achieve.
[0080] The principal pharmaceutical composition is compounded for
convenient and effective administration in effective amounts with a
suitable pharmaceutically acceptable carrier in an acceptable
dosage unit. In the case of compositions containing supplementary
active ingredients, the dosages are determined by reference to the
usual dose and manner of administration of the said
ingredients.
[0081] An agent useful to inhibit carotenogenesis in a bacterial
organism [e.g., Staphylococcus sp.) maybe used in combination with
commonly used antibiotics and/or antimicrobials. Accordingly, a
pharmaceutical composition of the invention can comprise a
carotenogenesis inhibitor and one or more additional antimicrobials
or antibiotics.
[0082] The following example (s) are provided in illustration of
the invention and should not be construed in any way as
constituting a limitation thereof.
EXAMPLES
[0083] The biosynthetic pathway for S. aureus carotenoids includes
the essential functions of genes crtM and crtN, encoding
dehydrosqualene synthase and dehydrosqualene desaturase,
respectively. To probe the biological activities of the S. aureus
pigment, an isogenic mutant of a golden-colored human clinical
isolate by allelic replacement of crtM was generated. The
.DELTA.CrtM mutant was nonpigmented and lacked the characteristic
triple-peak spectral profile of wild-type carotenoid at 440, 462
and 491 nM wavelengths. No differences in growth rate, stationary
phase density, surface charge, buoyancy or hydrophobicity were
observed between WT and .DELTA.CrtM S. aureus. S. aureus crtM and
crtN together are sufficient for production of
4,4'-diaponeurosporene. To facilitate gain of function analyses,
both genes were expressed in the nonpigmented Streptococcus
pyogenes, a human pathogen associated with a disease spectrum
similar to that of S. aureus. When transformed with a pCrtMN
plasmid, S. pyogenes gained yellow pigmentation with the spectral
characteristics of a carotenoid. Complementation of the S. aureus
.DELTA.CrtM mutant with the same pCrtMN vector also partially
restored pigmentation.
[0084] Bacteria, mice, human CGD patient and chemical reagents:
Wild-type S. aureus strain (Pig1), isolated from the skin of a
child with atopic dermatitis. Streptococcus pyogenes strain 5448 is
a well-characterized serotype M1T1 clinical isolate.sup.18. CD1 and
C57B1/6 mice were purchased from Charles River. The
gp91.sup.p{acute over (.eta.)}oW.about. mice were bred at the
Veteran's Administration Medical Center, San Diego and maintained
on trimethoprim/sulfamethoxazole prophylaxis until 3 d prior to
experiments. S. aureus and S. pyogenes were propagated in
Todd-Hewitt broth (THB) or on THB agar (Difco, Detroit, Mich.).
Unless otherwise indicated, all experiments were performed with
bacteria derived from S. aureus 36-48 h stationary phase cultures
or S. pyogenes 24 h stationary phase cultures, a point when
pigmentation phenotypes were readily apparent.
[0085] Human CGD patient: The patient was an 18 y.o. female with a
gp47.sup.phox deficiency (homozygous .DELTA.GT deletion in exon 2).
At the time of study she was in good health and her only medication
was interferon-.gamma. (50 mcg/m.sup.2) administered three times
weekly by subcutaneous injection.
[0086] Generation of the carotenoid-deficient S. aureus mutant,
.DELTA.CrtM. Precise, in-frame allelic replacement of the S. aureus
crtM gene with a chloramphenicol acetyltransferase (cat) cassette
was performed using PCR-based methods as described for S.
pyogenes.sup.19 or Streptococcus agalactiae.sup.20, with minor
modifications. Primers were designed based on the published S.
aureus crtMN sequence.sup.6 cross-referenced to genome S. aureus
strain N315.sup.21. PCR was used to amplify -500 bp upstream of
crtM with primers crtMupF 5'-TTAGGAAGTGCATATACTTCAC-S' (SEQ ID NO:
1) and crtMstartR
5'-GGTGGTATATCCAGTGATTTTTTTCTCCATACTAGTCCTCCTATATTGAAATG-S' (SEQ ID
NO:2), along with approximately 500 bp of sequence immediately
downstream of crtM with primers: crtMendF
5'-TACTGCGATGAGTGGCAGGGCGGGGCGTAACAAAGTATTTAGTATTGAAGC-3' (SEQ ID
NO: 3) and crtMdownR 5'-GGCACCGTTATACGATCATCGT-3' (SEQ ID NO: 4).
The crtMstartR and crtMendF primers were constructed with 25 bp 5'
extensions corresponding to the 5' and 3' ends of the cat gene,
respectively. The upstream and downstream PCR products were then
combined with a 650 bp amplicon of the complete cat gene (from
pACYC184) as templates in a second round of PCR using primers
crtMupF and crtMdownR. The resultant PCR amplicon, containing an
in-frame substitution of crtM with cat, was subcloned into
temperature-sensitive vector pHY304 to create the knockout plasmid.
This vector was transformed initially into permissive S. aureus
strain RN4220 (provided by Dr. Paul Sullam) and then into S. aureus
strain Pig1 by electroporation. Transformants were grown at
30.degree. C., shifted to the nonpermissive temperature for plasmid
replication (4O.degree. C.), and differential antibiotic selection
and pigment phenotype were used to identify candidate mutants.
Allelic replacement of the crtM allele in was confirmed
unambiguously by PCR reactions documenting targeted insertion of
cat and absence of crtM in chromosomal DNA isolated from the final
mutant .DELTA.CrtM.
[0087] Complementation and heterologous expression studies.
Primers CrtF 5'-CAGTCTAGAAATGGCATTTCAATATAGGAG-S' (SEQ ID NO: 5)
and
[0088] CrtR 5'-ATCGAGATCTCTCACATCTTTCTCTTAGAC-S' (SEQ ID NO: 6)
were used to amplify the contiguous CrtM and CrtN genes from the
chromosome of WT S. aureus strain Pig1. The fragment was
directionally cloned into the shuttle expression vector
pDCerm.sup.19 and the recombinant plasmid (pCrtMN) used to
transform by electroporation the S. aureus .DELTA.CrtM mutant and
S. pyogenes strain 5448.
[0089] Spectral profile of the S. aureus carotenoid. Stationary
phase (48 h) cultures of WT S. aureus Pig1 and its isogenic
.DELTA.CrtM mutant were subjected to methanol extraction. The
absorbance profile of the extracts was measured with a MBA 2000
spectrophotometer (Perkin Elmer).
[0090] Oxidant susceptibility assays. Tests for susceptibility to
oxidants were performed either in PBS (S. aureus) or THB (S.
pyogenes). Hydrogen peroxide (H.sub.2O.sub.2) was added to 1.5%
final concentration, 2.times.10.sup.9 bacteria incubated at
37.degree. C. for 1 h, then 1,000 U/ml of catalase (Sigma) added to
quench residual H.sub.2O.sub.2. Dilutions were plated on THA for
enumeration of surviving cfu. For the singlet oxygen assay,
10.sup.8 S. aureus or 4.times.10.sup.8 S. pyogenes were incubated
at 37.degree. C. in individual wells of a 24-well culture plate in
the presence or absence of 1-6 .mu.g/ml methylene blue and situated
exactly 10 cm from a 100-watt light source. Bacterial viability was
assessed after 1-3 h by plating dilutions on THA. Control plates
handled identically but wrapped in foil or exposed to light in the
absence of methylene blue did not show evidence of bacterial
killing.
[0091] Whole blood killing assays. Bacteria were washed twice in
PBS, diluted to an inoculum of 10.sup.4 cfu in 25 .mu.l PBS, and
mixed with 75 .mu.l of freshly drawn human or mouse blood in
heparinized tubes. The tubes were incubated at 37.degree. C. for 4
h with agitation, at which time dilutions were plated on THA for
enumeration of surviving cfu.
[0092] Neutrophil intracellular survival assay. Neutrophils were
purified from healthy human volunteers using a Histopaque gradient
(Sigma) per manufacturer's directions. Intracellular survival
assays were performed as follows. Bacterial cultures were washed
twice in PBS, diluted to a concentration of 4.5.times.10.sup.6 cfu
in 100 .mu.l RPMI+10% FCS, and mixed with 3.times.10.sup.5
neutrophils in the same media (multiplicity of infection,
MOI=15:1), centrifuged at 700.times.g for 5 min, then incubated at
37.degree. C. in a 5% CO.sub.2 incubator. Gentamicin (Gibco) (final
concentration 400 .mu.g/ml for S. aureus and 100 .mu.g/ml for S.
pyogenes) was added after 10 min to kill extracellular bacteria. At
specified time points, the contents of sample wells were withdrawn,
centrifuged to pellet the neutrophils, and washed to remove the
antibiotic medium. Neutrophils were then lysed in 0.02% triton-X,
and cfu calculated by plating on THA. Several assays were repeated
with addition of a step involving preincubation of the bacterial
inoculum with 10% autologous human serum for 15 min on ice.
[0093] Murine model of subcutaneous infection. Ten to 16 week old
CD-I or gp91.sup.Phox-/- mice were injected subcutaneously in one
flank (chosen randomly) with the bacterial test strain, and
simultaneously in the opposite flank with a different strain for
direct comparison. Bacterial cultures were washed, diluted and
resuspended in PBS mixed 1:1 with sterile Cytodex beads (Amersham)
at the specified inoculum, following an established protocol for
generating localized S. aureus and S. pyogenes subcutaneous
infection. Lesion size, as assessed by the maximal
length.times.width of the developing ulcers, was recorded daily.
Cumulative lesion size represents the total sum of lesion sizes
from all animals in each treatment group on a given day. At day 8
(S. aureus) or day 5 (S. pyogenes), animals were euthanized, skin
lesions excised, homogenized in PBS, and plated on THA for
quantitative culture.
[0094] Statistics. The significance of experimental differences in
oxidant sensitivity, blood killing, and neutrophil survival were
evaluated by unpaired Students t test. Results of the mouse in vivo
challenge studies were evaluated by paired Student's t test.
[0095] Assurances. All animal experiments were approved by the UCSD
Committee on the Use and Care of Animals and performed using
accepted veterinary standards. Experimentations using human blood
were approved by the Dual Tracked UCSD Human Research Protection
Program/CHSD IRB. Prior informed consents were obtained from the
human subjects.
[0096] Assays for buoyancy, surface charge, and hydrophobicity. To
measure buoyancy, sequential overlay gradients of 1 ml each 70%,
60% and 50% Percoll were prepared in 5 ml glass test tubes. One ml
of overnight bacterial culture was placed on top of the Percoll
layers, the tubes centrifuged in a swinging bucket centrifuge for 8
min at 500.times.g, and the migration of bacteria to various
Percoll interphases recorded. To measure surface charge, bacteria
were harvested by centrifugation and washed in
morpholinepropanesulfonic acid (MOPS) buffer (2O mM, pH=7.0). One
ml of culture was resuspended in 0.5 ml MOPS and the OD600
measured. Bacteria cells were incubated at room temperature or 15
min with cytochrome C (Sigma, St. Louis, Mo.) at a final
concentration of 0.5 mg/ml. Samples were centrifuged
(13,000.times.g for 5 min) and the amount of cytochrome C remaining
in the supernatant quantitated at 530 nm. Cytochrome C values were
adjusted to reflect binding per culture OD.sub.6oo=1.0. To measure
hydrophobicity, 0.5 ml of S, aureus culture was washed and
resuspended in 1.0 ml PBS, 300 .mu.l of n-hexadecane layered on top
of the cell suspension, and tubes vortexed for 60 sec. Samples were
incubated at RT for 30 min to allow for phase separation. The
aqueous phase was removed and the ratio of the OD.sub.600 of the
aqueous phase versus the OD.sub.600 of the culture in PBS
determined.
[0097] Protease and cathelicidin sensitivity assays. Human
neutrophil elastase, and cathepsin G were purchased from
Calbiochem. The antimicrobial peptide mCRAMP was synthesized at the
Louisiana State University Protein Facility (Martha Juban,
Director). S. aureus cultures were diluted (1:2,000) in 10 mM
phosphate buffer (pH 7.2)+0.5% LB to .about.1.times.10.sup.6
CFU/ml. Ninety .mu.l of this bacterial suspension was added to
replicate wells in a 96-well plate. Dilutions of the cathepsin G
(20 and 100 mU/ml), human neutrophil elastase (12.5 and 50
.mu.g/ml), and murine CRAMP (0.4-3 .mu.M) were prepared in 10 mM
phosphate buffer and added to wells in 10 .mu.l volume; 1O mM
phosphate buffer alone was used as a negative control. After 2 h
incubation at 37.degree. C., 25 .mu.l aliquots of each well were
serially diluted in PBS and plated on THB. Each experiment was
performed in duplicate and repeated.
[0098] Phagocytotic uptake assay. S. aureus were labeled with
SYTOR9, a component of BacLight.TM. kit (Invitrogen, Carlsbad,
Calif.), for 15 min at RT per manufacturer's guidelines. Labeled
bacteria were washed.times.3 times to remove excess dye, then
preopsonized in 10% autologous human serum for 10 min on ice.
Bacteria were added to 3.times.106 purified human neutrophils at
MOI=15, incubated at 37.degree. C. for 5 min, then centrifuged at
500.times.g for 6 min to pellet neutrophils. The supernatant was
discarded and the cell pellet resuspended in 15 ml of 0. L mg/ml
ethidium bromide in PBS to quench fluorescence of extracellular
bacteria. The percentage of neutrophils with intracellular bacteria
was enumerated by direct visualization under fluorescent
microscopy. Experiments were performed in duplicate and
repeated.
[0099] Representative images were captured on using Delta Vision
Deconvolution Microscope System (Nikon TE-200 Microscope) at the
UCSD Digital Imaging Core Facility.
[0100] The biosynthetic pathway for S. aureus carotenoids.sup.6
includes the essential functions of genes crtM and crtN, encoding
dehydrosqualene synthase and dehydrosqualene desaturase,
respectively (FIG. 1a). To probe the biological activities of the
S. aureus pigment, an isogenic mutant of a golden-colored human
clinical isolate by allelic replacement of crtM was generated (FIG.
1a). Consistent with previous reports, pigmentation of the
wild-type (WT) strain became apparent in early stationary phase of
growth and continued to intensify before reaching a plateau at
36-48 h (FIG. 1b). The .DELTA.CrtM mutant was nonpigmented and
lacked the characteristic triple-peak spectral profile of wild-type
carotenoid at 440, 462 and 491 nM wavelengths (FIG. 1b). No
differences in growth rate, stationary phase density, surface
charge, buoyancy or hydrophobicity were observed between WT and
.DELTA.CrtM S. aureus (FIGS. 5a-d). S. aureus crtM and crtN
together are sufficient for production of
4,4'-diaponeurosporene.sup.3. To facilitate gain of function
analyses, both genes were expressed in the nonpigmented
Streptococcus pyogenes, a human pathogen associated with a disease
spectrum similar to that of S. aureus. When transformed with the
pCrtMN plasmid, S. pyogenes gained yellow pigmentation (FIG. 1b)
with the spectral characteristics of a carotenoid. Complementation
of the S. aureus .DELTA.CrtM mutant with pCrtMN vector also fully
restored pigmentation (FIG. 1b).
[0101] One important mechanism by which phagocytic cells eliminate
pathogens is through release of reactive oxygen species generated
by NADPH oxidase.sup.7. It has been suggested that bacterial
carotenoids such as those expressed by S. aureus could serve a
protective function against these defense
molecules.sup.8'.sup.9'.sup.10. To test this experimentally, the
susceptibility of WT and .DELTA.CrtM S. aureus to oxidants in vitro
was compared. As shown in FIGS. 1c and 1d, the .DELTA.CrtM mutant
was killed more efficiently by hydrogen peroxide and singlet oxygen
compared to the WT S. aureus strain. Complementation with pCrtMN
restored the ability of the .DELTA.CrtM mutant to resist singlet
oxygen killing (FIG. 1d). Similarly, heterologous expression of
staphylococcal pigment in S. pyogenes led to a significant decrease
in susceptibility to singlet oxygen (FIG. 1e).
[0102] It was then determined whether the observed antioxidant
activity of the S. aureus carotenoid translated to increased
bacterial resistance to innate immune clearance using two ex vivo
assay systems: human or mouse whole blood survival and coculture
with purified human neutrophils. WT S. aureus survived
significantly better than the nonpigmented .DELTA.CrtM
intracellularly within human neutrophils (FIG. 2a, FIG. 6f) and in
whole blood of normal mice or human donors (FIGS. 2b, e). The
former effect was not explained by differences in the rate of
phagocytosis, since uptake of the WT S. aureus and .DELTA.CrtM
mutant was comparable (FIG. 6a). Nor were differences attributable
to changes in the magnitude of neutrophil oxidative burst, since
uptake of WT and mutant strains produced similar results in a
nitroblue tetrazolium (NBT) reduction assay (FIG. 6b).
Complementation of the S. aureus .DELTA.CrtM mutant with pCrtMN
restored resistance to killing by mouse whole blood (FIG. 2b).
Likewise, the pigmented S. pyogenes expressing staphylococcal
carotenoid showed enhanced survival in human neutrophils versus the
parent strain (FIG. 2c).
[0103] To verify the association of S. aureus carotenoid expression
with enhanced phagocyte resistance was a direct consequence of its
antioxidant properties, assays were repeated in the presence of the
oxidative burst inhibitor diphenyleneiodonium (DPI). WT and
.DELTA.CrtM S. aureus survived equally well in human neutrophils
(FIG. 2d) and mouse blood (FIG. 6c) when oxidative burst was
inhibited by DPI. The gp91.sup.Phox-/- mouse represents a model of
human X-linked chronic granulomatous disease, an inherited defect
in phagocyte oxidative burst function. The survival advantage of WT
over nonpigmented .DELTA.CrtM S. aureus was evident only in the
blood of normal mice (CD1 or C57B1/6), and not in the blood of
91.sup.Phox-/- mice lacking NADPH oxidase activity (FIG. 2e,
f).
[0104] It was recently reported that the apparent neutrophil
killing of pathogens by reactive oxygen species could largely
reflect the activation of granule proteases mediated through
changes in potassium flux. There was no difference in the
susceptibility of WT and .DELTA.CrtM S. aureus to the antimicrobial
action of cathepsin G, and both strains were resistant to human
neutrophil elastase as previously observed for S. aureus.sup.13
(FIG. 6d). Other effector molecules of mammalian neutrophils
critical to innate immune defense are the cathelicidin family of
antimicrobial peptides.sup.14. The carotenoid-deficient S. aureus
mutant was equally susceptible to killing by the murine
cathelicidin mCRAMP when compared to the WT strain (FIG. 6e). These
results support a primary role for the free-radical scavenging
antioxidant properties of the S. aureus carotenoid in resistance to
neutrophil-mediated killing.
[0105] The in vitro and ex vivo results demonstrate that S. aureus
carotenoid is both necessary and sufficient to promote oxidant
resistance and phagocyte survival. To assess the significance of
these observations to disease pathogenesis, a murine subcutaneous
challenge model was developed. In these studies, individual animals
were injected simultaneously in one flank with the WT S. aureus
strain and the opposite flank with the .DELTA.CrtM mutant. At the
site of WT injection (10.sup.6 cfu), mice developed sizeable
abscess lesions reaching a cumulative size of 80 mm.sup.2 by day 4;
injection of an equivalent inoculum of the carotenoid-deficient
mutant on the contralateral flank failed to produce visible lesions
(FIG. 3a). Quantitative culture from skin lesions at two different
challenge doses (10.sup.6 cfu to 10.sup.7 cfu) consistently
demonstrated significantly higher numbers of surviving WT S. aureus
compared to the .DELTA.CrtM mutant in the individual mice (FIG.
3a). To corroborate that an antioxidant effect is key to the
mechanism of protection afforded by the S. aureus carotenoid in
vivo, the subcutaneous infection experiment was repeated in
gp91.sup.Phox-/- mice. In the absence of host NADPH oxidase
function, WT and .DELTA.CrtM mutant S. aureus produced lesions of
similar cumulative size and no survival advantage was detected on
quantitative abscess culture (FIG. 3b). It was then determined that
S. aureus carotenoid was sufficient to enhance bacterial virulence
by comparing the course of infection produced by S. pyogenes
expressing CrtMN to controls transformed with vector alone. In FIG.
3c, lesions generated by the carotenoid-expressing strain were
significantly larger and contained greater numbers of surviving
bacteria than those produced by the WT strain. Raw data from the in
vivo experiments is provided in Table 1.
TABLE-US-00001 TABLE 1 Lesion size and bacterial culture counts
from in vivo mouse challenge studies. Mouse Bacterial Dose No.
Lesion size (mm.sup.2) Lesion size (mm.sup.2) Lesion culture (cfu)
Lesion culture (cfu) Strain Strain (cfu) tested median/mean (Range:
low-high) median/mean (Range: low-high) CD1 S. aureus WT 10 10
5.5/8.2 0-42 4.8 .times. 10 /7.0 .times. 10 1.2 .times. 10 -3.2
.times. 10 CD1 .DELTA.CrtM mutant 10 10 0.0/0.2 0-2 1.7 .times. 10
/1.9 .times. 10 6.0 .times. 10 -7.5 .times. 10 CD1 S. aureus WT 10
10 13.0/17.9 0-55 4.0 .times. 10 /1.2 .times. 10 4.4 .times. 10
-7.9 .times. 10 CD1 .DELTA.CrtM mutant 10 10 2.5/6.6 0-32 3.8
.times. 10 /5.3 .times. 10 7.0 .times. 10 -1.6 .times. 10 Phox S.
aureus WT 10 11 4.0/7.0 0-18 7.7 .times. 10 /2.1 .times. 10 1.6
.times. 10 -1.2 .times. 10 Phox .DELTA.CrtM mutant 10 11 4.0/6.6
0-50 4.1 .times. 10 /1.7 .times. 10 1.5 .times. 10 -1.0 .times. 10
CD1 S. pyogenes + 10 14 20.0/20.4 0-75 6.4 .times. 10 /1.0 .times.
10 6.0 .times. 10 -1.3 .times. 10 vector only CD1 S. pyogenes + 10
14 6.0/7.0 0-21 1.6 .times. 10 /6.3 .times. 10 8.0 .times. 10 -6.0
.times. 10 pCrtMN indicates data missing or illegible when
filed
[0106] Given the protective effect provided to the bacteria by the
golden yellow pigments, a pharmacologic agent that inhibits
carotenogenesis might render S. aureus more susceptible to immune
clearance. The mixed function oxidase inhibitor
2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem)
was previously shown to inhibit pigment formation in S. aureus,
though a moderate residual accumulation of 6 carotenoid
intermediate was noted in those experiments. Shown in FIG. 4a, a
dose-dependent decrease in pigment production in the WT strain of
S. aureus grown in the presence of this agent was obtained.
Blocking S. aureus pigment formation led to a dose-dependent
increase in the susceptibility of the organism to singlet oxygen
killing (FIG. 4b), and a decrease in its ability of WT S. aureus to
survive in human whole blood (FIG. 4c). As a control, the
.DELTA.CrtM mutant was exposed to SKF 525-A in parallel experiments
with no significant effects on oxidant susceptibility or blood
survival (FIG. 4b, c).
[0107] Golden color imparted by carotenoid pigments is the
eponymous feature of the human pathogen Staphylococcus aureus. A
molecular genetic analysis pairing mutagenesis and heterologous
expression was performed to show that this hallmark phenotype is in
fact a virulence factor, serving to protect the bacterium from
phagocytic killing through its antioxidant properties. In the
present era, effective control of this important disease agent is
compromised by rapid evolution of antimicrobial resistance in both
community and hospital settings. In principle, the inhibition of
carotenogenesis may offer a novel therapeutic approach to the
treatment of complicated S. aureus infections, effectively
rendering the pathogen more susceptible to clearance by normal host
innate immune defenses.
[0108] In addition, WT S. aureus survived significantly better than
the nonpigmented .DELTA.CrtM in whole blood of human donors or
normal mice and intracellularly within human neutrophils. The
latter effect was not explained by differences in the rate of
phagocytosis, since uptake of the WT S. aureus and .DELTA.CrtM
mutant was comparable. Nor were differences attributable to changes
in the magnitude of neutrophil oxidative burst, since uptake of WT
and mutant strains produced similar results in a nitroblue
tetrazolium (NBT) reduction assay. Complementation of the S. aureus
.DELTA.CrtM mutant with pCrtMN restored resistance to killing by
mouse whole blood or human neutrophils. Likewise, the pigmented S.
pyogenes expressing staphylococcal carotenoid showed enhanced
survival in human neutrophils compared to the parent strain.
[0109] To verify the association of S. aureus carotenoid expression
with enhanced phagocyte resistance was a direct consequence of its
antioxidant properties, assays were repeated in the presence of the
oxidative burst inhibitor diphenyleneiodonium (DPI). WT and
.DELTA.CrtM S. aureus survived equally well in human neutrophils
and mouse blood when oxidative burst was inhibited by DPI. The
gp91.sup.Phox-/- mouse represents a model of human X-linked chronic
granulomatous disease, an inherited defect in phagocyte oxidative
burst function. The survival advantage of WT over nonpigmented
.DELTA.CrtM S. aureus was evident only in the blood of normal mice
(CD1 or C57B1/6), and not in the blood of gp91.sup.Phox-/- mice
lacking NADPH oxidase activity.
[0110] It was recently reported that the apparent neutrophil
killing of pathogens by reactive oxygen species could largely
reflect the activation of granule proteases mediated through
changes in potassium flux. No difference was identified in the
susceptibility of WT and .DELTA.CrtM S. aureus to the antimicrobial
action of cathepsin G, and both strains were resistant to human
neutrophil elastase as previously observed for S. aureus. Other
effector molecules of mammalian neutrophils critical to innate
immune defense are the cathelicidin family of antimicrobial
peptides. The carotenoid-deficient S. aureus mutant was equally
susceptible to killing by the murine cathelicidin mCRAMP when
compared to the WT strain. These results support a primary role for
the free-radical scavenging antioxidant properties of the S. aureus
carotenoid in resistance to neutrophil-mediated killing.
[0111] The in vitro and ex vivo results demonstrate that S. aureus
carotenoid is both necessary and sufficient to promote oxidant
resistance and phagocyte survival. To assess the significance of
these observations to disease pathogenesis, a murine model for
subcutaneous challenge was developed. In these studies, individual
animals were injected simultaneously in one flank with the WT S.
aureus strain and the opposite flank with the .DELTA.CrtM mutant.
At the site of WT injection mice developed sizeable subcutaneous
abscesses, injection of an equivalent inoculum of the
carotenoid-deficient mutant on the contralateral flank failed to
produce visible abscesses. Quantitative culture from skin lesions
consistently demonstrated significantly higher numbers of surviving
WT S. aureus compared to the .DELTA.CrtM mutant in the individual
mice. To corroborate that an antioxidant effect is key to the
mechanism of protection afforded by the S. aureus carotenoid in
vivo, the subcutaneous infection experiment was repeated in
gp91.sup.p{acute over (.eta.)}ox''/.about. mice. In the absence of
host NADPH oxidase function, WT and .DELTA.CrtM mutant S. aureus
produced lesions of similar cumulative size and no survival
advantage was detected on quantitative abscess cultures.
Furthermore, while 5. pyogenes infection was associated with
development of necrotic ulcers rather than abscess formation,
lesions generated by the carotenoid-expressing strain were
significantly larger and contained greater numbers of surviving
bacteria than those produced by the WT strain.
[0112] Given the protective effect provided to the bacteria by the
golden yellow pigments, a pharmacologic agent that inhibited
carotenogenesis was tested to determine whether the agent might
render S. aureus more susceptible to immune clearance. The mixed
function oxidase inhibitor
2-diethylaminoethyl-2,2-diphenyl-valerate (SKF 525-A, Calbiochem)
was previously shown to inhibit pigment formation in S. aureus, and
a dose-dependent decrease in carotenoid production was demonstrated
in the WT strain of S. aureus grown in the presence of the agent.
Blocking S. aureus pigment formation led to a commensurate
dose-dependent increase in the susceptibility of the organism to
singlet oxygen killing and a decrease in its ability to survive in
human whole blood. As a control, the .DELTA.CrtM mutant was exposed
to SKF 525-A in parallel experiments with no significant effects on
oxidant susceptibility or blood survival.
[0113] Although a number of embodiments and features have been
described above, it will be understood by those skilled in the art
that modifications and variations of the described embodiments and
features may be made without departing from the teachings of the
disclosure or the scope of the invention as defined by the appended
claims. The appendices attached hereto are provided to further
illustrate but not limit the invention.
* * * * *