U.S. patent application number 13/033128 was filed with the patent office on 2011-06-16 for method for identifying drug-sensitizing antisense dna fragments and use thereof.
This patent application is currently assigned to TRIUS THERAPEUTICS, INC.. Invention is credited to Vickie Brown-Driver, John M. Finn, Kedar GC, Robert Haselbeck, Mark Hilgers, Karen Shaw, Mark Stidham.
Application Number | 20110143359 13/033128 |
Document ID | / |
Family ID | 38522866 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143359 |
Kind Code |
A1 |
Haselbeck; Robert ; et
al. |
June 16, 2011 |
Method for Identifying Drug-Sensitizing Antisense DNA Fragments and
Use Thereof
Abstract
The invention provides a method for generating and selecting
drug-sensitizing antisense DNA fragments. In one embodiment, the
method includes identifying a gene of interest using knowledge of
bacterial physiology, biochemistry, genetics, genomics, and other
means. The method includes PCR amplification of a gene of interest
using genomic DNA as a template; fragmentation of the DNA by
sonication or other means; selecting DNA fragments no longer than
400 base pairs; ligating the DNA fragments into a suitable
expression plasmid with a selectable marker; transforming the
plasmids containing the DNA fragments into the organism from which
the gene of interest originated; and selecting clones from
transformed cells that show a phenotypic difference of the clone
grown in the presence of the inducer relative to the phenotype in
the absence of inducer.
Inventors: |
Haselbeck; Robert; (San
Diego, CA) ; Hilgers; Mark; (San Diego, CA) ;
Shaw; Karen; (Poway, CA) ; Brown-Driver; Vickie;
(Solana Beach, CA) ; GC; Kedar; (San Diego,
CA) ; Finn; John M.; (Encinitas, CA) ;
Stidham; Mark; (San Diego, CA) |
Assignee: |
TRIUS THERAPEUTICS, INC.
|
Family ID: |
38522866 |
Appl. No.: |
13/033128 |
Filed: |
February 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11636394 |
Dec 8, 2006 |
7910337 |
|
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13033128 |
|
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60782961 |
Mar 15, 2006 |
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Current U.S.
Class: |
435/6.13 ;
435/6.15 |
Current CPC
Class: |
C12N 15/111 20130101;
C12N 2330/30 20130101; C12Y 205/01031 20130101; C12Y 105/01003
20130101; C12N 15/1137 20130101; C12Y 601/0101 20130101; C12N
2310/11 20130101; C12N 2320/11 20130101; C12Y 101/01158
20130101 |
Class at
Publication: |
435/6.13 ;
435/6.15 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for selecting bacterial strains containing specific
drug-sensitizing DNA fragments, comprising: selecting those strains
wherein the inserted DNA fragment is oriented antisense to some
portion of the gene of interest; selecting those strains from a.
wherein the intact mRNA level expressed from the gene of interest
is reduced at least 25% in the presence of inducer relative to the
absence of inducer; and selecting those strains from b wherein the
magnitude of a discernible phenotype is dependent on the
concentration of the inducer.
2. The method of claim 1, wherein strains are subjected to a wide
range of inducer concentrations to establish a growth response
curve to the inducer, such that: at high concentrations of inducer,
growth is maximally suppressed; at low or no concentrations of
inducer, there is no discernable growth inhibition; or at moderate
and empirically determined concentrations, inducer causes 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, or 90% growth reduction compared to
untreated strains.
3. The method of claim 1, wherein strains are grown in the presence
of a wide range of concentrations of growth-inhibiting compound to
establish a growth response curve and thereby a concentration that
causes a 50% reduction in growth relative to untreated cells
(EC50).
4. The method of claim 1, in which the method is conducted both in
the presence and in the absence of an empirically determined
concentration of inducer that causes between 10% to 50% growth
reduction.
5. The method of claim 3, wherein a decrease in growth inhibitor
EC50 values of at least four fold is evident in inducer-treated
cells relative to cells without inducer treatment.
6. The method of claim 5, wherein the result is an indication that
the antibiotic works as a specific inhibitor of the gene of
interest.
7. The method of claim 3, wherein no significant change in
antibiotic EC50 values is evident in inducer-treated cells relative
to cells without inducer treatment.
8. The method of claim 7, wherein the result is an indication that
the antibiotic does not work as a specific inhibitor of the gene of
interest.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 11/636,394 filed Dec. 8, 2006, now pending;
which claims the benefit under 35 USC .sctn.119(e) to U.S.
Application Ser. No. 60/782,961 filed Mar. 15, 2006, now expired.
The disclosure of each of the prior applications is considered part
of and is incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to bacterial
diseases and more particularly to a methodology for the generation
of bacterial strains useful in detecting the mechanism of action of
antibacterial compounds and to determine genes and gene products
that interact with existing antibiotics.
[0004] 2. Background Information
[0005] Antibiotics are useful agents in curing human and animal
diseases caused by bacterial infections. Currently there are
numerous antibiotics that control bacterial disease by interfering
with a specific bacterial gene product, disrupting the biochemistry
of the organism, and either preventing the organism from growing or
killing the organism. However, antibiotic resistant strains of
bacterial pathogens constantly emerge. Thus, there is a continual
need to discover and develop new antibiotics effective against the
new strains of pathogens. One way to find new agents is to discover
and develop chemical compounds that act at a bacterial gene product
not targeted by any current antibiotics. These potential targets
for new antibiotics have been characterized as `essential` by
various methods that demonstrate the gene or gene product is
required for bacterial growth. With the complete sequencing of the
genomes of pathogens and various biochemical and microbiological
analyses, numerous methodologies are available to define the set of
`essential` proteins in bacteria. Such proteins and the genes that
encode them are prime targets for antibiotic development, as small
molecule inhibitors of these functions can thereby serve to either
stop the growth or to kill pathogenic bacteria in an infection.
[0006] Prior to genomic-based approaches, discovery and development
of antimicrobial drugs relied upon the vast literature of bacterial
physiology and biochemistry. For example, it has long been known
that beta-lactam antibiotics inhibit and kill bacterial pathogens
by interfering with the formation of the bacterial cell wall. The
specific process inhibited by the beta-lactam antibiotics are the
transpeptidation reactions that link peptidoglycan units in the
final stages of cell wall biosynthesis. Transpeptidation is
catalyzed by penicillin binding proteins (PBPs) encoded by
bacterial genes. Thus, PBPs are validated targets for antibiotics
and remain the subject of antibiotic discovery and development.
[0007] The peptidoglycan monomer unit is made by bacteria in a
conserved biochemical pathway (FIG. 1). Each step of the process is
catalyzed by a different bacterial protein that are encoded by
corresponding genes including GlmU, MurA, MurB, MurC, MurD, MurE,
MurF, MurG, MraY, and UppS. MurA is inhibited by the antibiotic
fosfomycin, thus validating peptidoglycan biosynthesis as an
essential process and qualifying the other individual proteins in
the pathway as potential targets for new antibiotics. Genomic
methods have confirmed the presence of these genes in all bacterial
pathogens, further validating their potential as antibiotic
targets.
[0008] Another set of known antibacterial targets are those enzymes
used for bacterial protein synthesis. Many different useful
antibiotics are known to act through inhibition of various steps of
protein synthesis. One step requires the activity of tRNA
synthetase enzymes that "charge" by means of covalent acylation a
specific amino acid to its cognate tRNA molecule. These charged
tRNAs are basic building blocks in protein synthesis. Inhibition of
the charging process can severely limit protein synthesis. Small
molecule inhibition of tRNA synthetases has been demonstrated, and
certain inhibitors have been commercialized for use in
antibacterial chemotherapy. For example, the commercial antibiotic
mupirocin inhibits bacterial isoleucyl-tRNA synthetase. Many
patents have been filed and granted on other chemical inhibitors of
tRNA synthetase, including methionyl-tRNA synthetase.
[0009] Still another set of known antibacterial targets are those
enzymes used for bacterial DNA synthesis. One biochemical required
for DNA synthesis is tetrahydrofolate, a metabolite produced by the
enzyme dihydrofolate reductase (DHFR). Thus, inhibition of DHFR can
result in an antibiotic effect. The widely used antibiotic
trimethoprim acts through selective inhibition of bacterial DHFR.
Many patents concern the discovery and development of antibiotics
having DHFR as the mechanism of action.
[0010] The present invention concerns the development of strains of
bacteria that can be used to detect specific mechanisms of
antibiotic action and thereby aid in antibiotic discovery and
development. The method of creating the strains of bacteria
involves generating and expressing antisense RNA that confers a
hypersensitive phenotype to the bacteria that is specific for any
particular antibiotic mechanism of action.
[0011] Experimental means have long been sought to modulate gene
expression in bacteria. Conditional reduction in expression of
target essential genes can make the host bacterium more sensitive
to small molecules that inhibit the products of these genes. One
way of doing this is to conditionally express RNA that is
complementary to the mRNA transcribed from target genes. The
formation of a double-stranded RNA species can result in blockage
of translation or to degradation of the targeted mRNA. A
substantial body of research has described the incidence of
naturally occurring antisense post-transcriptional regulation in
bacteria. Antisense regulation has been shown to be involved in the
regulation of plasmid copy number, global regulation of cellular
physiology, and to post-transcriptional regulation of certain
cellular functions. These findings have inspired a number of
efforts to experimentally create and exploit antisense regulation
systems that can attenuate regulation of particular genes of
interest. One experimental system was demonstrated as early as 1984
when researchers reported that portions of the lacZ gene, when
expressed from an inducible promoter in the antisense direction,
could cause specific and substantial attenuation of
beta-glactosidase expressed from the lacZ gene. (Pestka, S. et al.,
(1984). Subsequently, others generated a histidine auxotroph
phenotype by transforming Mycobacterium smegmatis with a plasmid
containing an antisense fragment to its hisD gene ("Development and
use of a conditional antisense mutagenesis system in
mycobacterium." Parish T, and Stoker N G (1997), FEMS Microbiology
Letters 154, 151-157). In another work ("Expression of an Antisense
hla Fragment in Staphylococcus aureus Reduces Alpha-Toxin
Production In Vitro and Attenuates Lethal Activity in a Murine
Model." Kernodle et al., (1997) Infection and Immunity, 65,
179-184) a 600 base pair fragment of the hla gene was cloned in
antisense orientation into a plasmid and transformed into S.
aureus. The resulting strain made 16-fold less alpha-toxin thereby
significantly blocking lethality of these cells in a murine
infection model. A method described for the protist Dictyostelium
discoideum used an un-regulated promoter to drive expression of
random cDNAs in the antisense orientation in order to globally
survey the resulting phenotypes of affected genes. ("Mutagenesis
and gene identification in Dictyostelium by shotgun antisense"
Spann et al. (1996) Proc. Natl. Acad. Sci. USA, 93, 5003-5007). By
this method, numerous genes were functionally cataloged by the
phenotypic effects of specific antisense-expressed cDNAs. These and
other works demonstrate that certain antisense fragments cloned
into suitable vectors can be used to alter bacterial strain
phenotype by lowering the expression of specific genes in essential
and non-essential metabolic pathways. The resulting
inducer-dependent phenotype of the bacteria engineered with
antisense genes includes results from altered metabolism, altered
virulence, and reduced growth.
[0012] The "shotgun antisense" method described above for
Dictyostelium could also be used to globally to survey for
essential genes if an inducible promoter is used. In this way,
resulting cells would not exhibit the resulting growth sensitivity
until expression of the antisense DNA is turned on. Two similar but
independently conducted antisense-based essential gene surveys were
recently described ("Identification of critical Staphylococcal
genes using conditional phenotypes generated by antisense RNA" Ji
et al., (2001), Science 293:2266-2269; "A genome-wide strategy for
the identification of essential genes in Staphylocccus aureus"
Forsyth et al., (2002) Molecular Microbiology 43:1387-1400). In
both experiments, genomic DNA of S. aureus was purified,
fragmented, and then "shotgun cloned" behind a plasmid-borne
inducible promoter. After transformation of this shotgun library
into S. aureus, resulting colonies were surveyed for growth in the
presence or absence of inducer. Those that failed to grow due to
expression of a gene fragment in the antisense orientation were
collected and analyzed by DNA sequencing, and it was found that
this pool of clones was highly enriched for known essential genes
involved in such cellular functions as cell-wall biosynthesis, DNA
replication, protein translation, RNA transcription, and fatty acid
biosynthesis.
[0013] Other publications focused on antisense as a technique
useful in identifying essential bacterial genes from an entire
genome. Two patents were issued to Elitra Pharmaceuticals based on
processes that were used in the Forsyth et al. publication above:
U.S. Pat. No. 6,228,579 and U.S. Pat. No. 6,924,101, describe a
process of surveying fragments of microbial genomic DNA for their
capacity to reduce or block proliferation when expressed in the
microorganism in the antisense orientation. The methods in the
patent include fragmentation of genomic DNA from the organism of
interest, cloning the fragments adjacent to an inducible promoter
sequence on a plasmid, introducing the plasmid into the
microorganism of interest, and comparing growth of the organism in
the presence and absence of an inducer compound or stimulus that
results in a dependent presence or absence of expression of the
cloned fragment. Clones containing an antisense fragment that
showed growth inhibition in the presence of inducer compound are
selected, and the `essential` gene for the clone is deduced by
sequencing the antisense fragment. The presumption of these methods
is that those fragments that cause reduction or blockage of
proliferation of the microorganism do so by a mechanism that is
specific to the function of the gene from which the fragment was
derived, implying that this gene is one whose encoded cellular
function is required for normal proliferation. Both patents state
in their abstract and describe in their claims, "Antisense
fragments that result in lethality when expressed indicate that the
endogenous gene is a proliferation gene." Additional claims in the
antisense patent literature include the method of using
antisense-expression for screening compounds for the purpose of
identifying specific inhibitors of the protein-target of the
antisense, especially U.S. Pat. No. 6,924,101.
[0014] While growth inhibition or lethality resulting in expression
of an antisense fragment may be due to specific mRNA attenuation as
described above, these phenotypes may also be due to unspecific
mechanisms. Even though a gene fragment is expressed in the
antisense orientation, it could still be translated to produce a
toxic "cryptic peptide" that can inhibit growth (Lopes J M, Soliman
N, Smith P K, Lawther R P, Mol. Microbiol. 1989 August; 3(8):
1039-51, "Transcriptional polarity enhances the contribution of the
internal promoter, ilvEp, in the expression of the ilvGMEDA operon
in wild-type Escherichia coli K12"). Indeed, one analysis showed
that substantial open reading frames can occur in the antisense
orientation in many E. coli genes (Merino et al., (1994),
"Antisense overlapping open reading frames in genes from bacteria
to humans." Nucleic Acids Res. 22, 1903-1908). The prevalence of
antisense-oriented coding regions could result in identification of
ostensibly antisense-oriented gene fragments that cause unspecific
growth sensitivity phenotypes. Another way in which RNA fragments
can inhibit growth in ways other than by specific antisense-based
mRNA attenuation is demonstrated by aptamers. (Ellington A D,
Szostak J W, "In vitro selection of RNA molecules that bind
specific ligands." Nature, 1990 Aug. 30; 346(6287):818-22; Blum, J
H, Dove, S L, Hocschild, A, and Melalanos, J J (2000) "Isolation of
peptide aptamers that inhibit intracellular processes'" PNAS 97,
2241-2246). Such RNA fragments, which may be either in the sense or
antisense orientation to the genes that they originated from, may
actually form small molecules that may inhibit cellular functions
entirely unrelated to the gene of origin.
[0015] In any instance of antisense growth inhibition, expression
of antisense RNA fragments can result in bacterial strains that are
hypersensitive to many different classes of chemical compounds
unrelated to the target gene. Therefore, a limitation of current
antisense methodology is that the resulting strains may not be
attenuated for a specific metabolic pathway. A new method is needed
in order to demonstrate a direct cause-and-effect mechanism for any
particular growth-inhibiting antisense fragment.
[0016] Another limitation of current antisense methods involves the
specificity of generating antisense to any given gene. This
limitation is related to artifacts discussed above. Specifically,
it is not possible at present to predict the required sequence and
length of antisense RNA for any given gene that will produce the
desired specific antisense effect. Hasan et al., ("Antisense RNA
does not significantly affect expression of the galK gene of
Escherichia coli or the N gene of coliphage lambda." Gene 72,
247-252) reported numerous failed attempts at producing a bona fide
antisense effect despite three years of effort with many
configurations of the genes and promoters. Hasan concludes that " .
. . clear-cut regulation is more an exception than a rule (with
antisense), and requires the use of a suitable gene and careful
design, combined with strong conviction and good luck." This report
demonstrates that simply cloning and expressing the inverted
sequence of a gene will not reliably result in an antisense
effect.
[0017] Another limitation of existing methodology concerns the use
of genomic DNA as the source of the fragments for generating an
antisense-based strains. First and foremost, existing methods
define proliferation genes as those that generate a growth
inhibited phenotype upon generation of an antisense strain to that
gene. As indicated in the research cited, many antisense fragments
will produce growth inhibition phenotype without connection to the
gene from which it was derived. More importantly, targets for
antibiotics that include these genes have in fact been defined by
the integrated knowledge of bacterial genetics, physiology, and
biochemistry. There have not been reports in the literature of an
essential biochemical process identified using antisense that was
not known through other means. Thus, it is inefficient to use
genomic DNA fragmentation as the starting point for a method of
constructing antisense-based sensitized strains for antibiotic
discovery.
[0018] Second, the use of genomic DNA does not ensure that any
particular gene will be adequately represented. The use of genomic
DNA is biased towards genes with sequences that are more amenable
to inhibition by antisense. Genes of equal value as antibiotic
targets are each different in their susceptibility to antisense
inactivation because of their different sequences and length.
Because the gene fragment required for generating a specific
antisense strain for a given gene cannot be specified, there is a
significant risk that a useful antisense fragment will not be
generated for any particular gene. To generate the number and
diversity of fragments required to ensure a specific antisense
strain will be represented for any particular gene, the use of
genomic DNA is inappropriate. A bacterial genome may consist of
several thousand genes, thus requiring millions of clones to be
generated and examined.
[0019] Another limitation when using genomic DNA as a source for
generating the fragments is that bacterial genomes often contain
two or more copies of a gene encoding an essential function.
Antisense to only one of these genes may not be sufficient to
demonstrate a phenotype. For example, MurA in peptidoglycan
biosynthesis is encoded by two different genes in many
Gram-positive pathogens. The mode of action of the antibiotic
fosfomycin is through inhibiting the MurA protein, demonstrating
that MurA is an essential process. Antisense methodology as
described in the literature on the whole genome would erroneously
exclude MurA as an essential process.
[0020] One important process of antibiotic discovery is the
determination of the mode of action for any given antibacterial
compound. Mode of action determination can help differentiate those
compounds with new and specific mechanisms of action from those
compounds with non-specific or old modes of action. The utility of
antisense in creating strains depleted in a particular target
protein and thus sensitized to inhibitors of that target have been
published. However, given that a particular target has been
pre-selected for antibiotic discovery, it is inconvenient,
inefficient, and incomplete to create any particular
antisense-based strain using DNA from the entire genome. A new
process is needed for specific work.
[0021] The identity of "proliferation" genes and other "essential"
genes in bacteria has been accomplished a number of different ways
and precede the use of antisense for this purpose.
[0022] Prior to genomics-based methods, microbial processes such as
cell wall biosynthesis, protein synthesis, isoprenoid biosynthesis,
and tetrahydrofolate synthesis were known to be useful sites of
antibiotic action. Tools to track mode of action for antibiotics
targeting these processes are useful in antibiotic discovery and
development.
[0023] The presence of a gene fragment in antisense orientation can
cause inducer-dependent growth-inhibited phenotype by mechanisms
other than post-transcriptional reduction in mRNA. The potential
for artifacts makes the selection of growth-inhibited phenotype an
insufficient criterion for selecting specific antisense
strains.
[0024] Many limitations exist for the methods of creating
antisense-based hypersensitive strains for antibiotic discovery,
including gene-specific requirements for antisense structure
(sequence and length), an inconvenient cloning requirement to
ensure adequate representation for any given gene, and multiple
genes encoding essential processes.
[0025] Current published methods for generating antisense-based
strains for detecting mode of action are subject to artifact, and
are inconvenient, inefficient, and incomplete for use in antibiotic
drug discovery.
SUMMARY OF THE INVENTION
[0026] The invention provides a method for generating and selecting
drug-sensitizing antisense DNA fragments. In one embodiment, the
method includes identifying a gene of interest using knowledge of
bacterial physiology, biochemistry, genetics, genomics, and other
means. The method includes PCR amplification of a gene of interest
using genomic DNA as a template; fragmentation of the DNA by
sonication or other means; selecting DNA fragments no longer than
400 base pairs; ligating the DNA fragments into a suitable
expression plasmid with a selectable marker; transforming the
plasmids containing the DNA fragments into the organism from which
the gene of interest originated; and selecting clones from
transformed cells that show a phenotypic difference of the clone
grown in the presence of the inducer relative to the phenotype in
the absence of inducer.
[0027] In another embodiment, the invention provides that the
discernible phenotype is growth rate; relative sensitivity to a
growth inhibiting compound; requirement for addition of a nutrient
to the growth medium; morphology such as shape; relative
sensitivity to osmotic stress, or colony size, for example.
[0028] In another embodiment, the invention provides a method for
selecting bacterial strains containing specific drug-sensitizing
DNA fragments including selecting those strains wherein the
inserted DNA fragment is oriented antisense to some portion of the
gene of interest selecting those strains wherein the mRNA level of
the organism in the presence of inducer is less than 25% of the
levels in the absence of inducer; selecting those strains according
to the method of the invention wherein the magnitude of the
phenotype is dependent on the concentration of the inducer.
BRIEF DESCRIPTION OF FIGURES
[0029] FIG. 1 shows a peptidoglycan biosynthetic pathway in
bacteria.
[0030] FIG. 2 shows a process for identification of specific
antisense fragments.
[0031] FIG. 3 shows a validation process for specific sensitized
antisense strains.
[0032] FIG. 4A shows a EC50 Shift Sensitivity of MetRSl antisense
clones to the specific MetRS inhibitor Rxl9. (a) Characterization
of growth inhibition curve in the presence and absence of xylose.
One of the antisense clones (HI) was characterized as to its
sensitivity to the MetRS-specific inhibitor Rxl9. Upper line: no
xylose added. Lower line: +60 mM xylose. The EC50 concentration for
each condition was determined. The "EC50 shift" is defined as the
ratio of the EC50 in the absence and in the presence of xylose. The
EC50 shift in this experiment is calculated to be 711/85.8=8.3. (b)
EC50 shift for Rxl9 in six different MetRS antisense clones. Clones
HI, H2, H6, and E4 showed EC50 shifts for Rxl9 greater than 4.
Clones A3 and A5 showed EC50 shifts less than 4 even though all six
clones had xylose-dependent growth inhibited phenotype and inserts
with fragments antisense to the MetRS gene.
[0033] FIG. 4B shows an EC50 shift for Rxl9 in six different MetRS
antisense clones. Clones HI, H2, H6, and E4 showed EC50 shifts for
Rxl9 greater than 4. Clones A3 and A5 showed EC50 shifts less than
4 even though all six clones had xylose-dependent growth inhibited
phenotype and inserts with fragments antisense to the MetRS
gene.
[0034] FIG. 5 shows an EC50 shifts for MetRS antisense clone HI on
a panel of antibiotics. The sensitivity of MetRS antisense clone to
various antibiotics was determined in the absence and presence of
xylose. With one exception, the clone showed the same sensitivity
to antibiotics in the absence or presence of xylose. For Rxl9, the
clone was about 8 fold more sensitive in the presence of xylose
relative to the sensitivity in the absence of xylose.
[0035] FIGS. 6A and 6B show the response of murB2 antisense clone
to cefotaxime in the presence and absence of a subinhibitory
concentration antisense inducer. Upper line: no inducer. Lower
line: +40 mM xylose, (a): murB2 antisense clone, (b) metRS
antisense clone. The ratio of the IC50+xylose/EC50 (-xylose) gives
the `IC50 shift`. In (a) the shift is computed to be
(2096/6.1)=343. In (b), the metRS antisense clone showed no
difference in sensitivity to cefotaxime, indicating that the
cefotaxime mechanism of action is unrelated to metRS.
[0036] FIGS. 7A and 7B show B. anthracis murB-2 antisense strain
sensitivity to cephalosporins and other antibiotics, (a) antibiotic
panel including cefotaxime, ceftriaxone, cefepime, and cefoxitin,
all of which showed greater than 100 fold shift in 1050 in the
presence of xylose, (b) antibiotic panel showing greater than
4-fold shift in 1050 for cloxacillin, oxacillin, dicloxacillin, and
cefaclor.
[0037] FIGS. 8A through 8C show an antibiotic dose response in the
presence and absence of xylose for S. aureus engineered with murB
antisense. (a) Cefoxitin IC50 shift is computed to be
1012/129.1=7.8. (b) Dicloxacillin IC50 shift is computed to be
49.5/8.6=5.8. (c) Antibiotic panel IC50 shift for S. aureus murB
antisense strain showing 4-fold or greater sensitivity to
cloxacillin, oxacillin, dicloxacillin, pipericillin, cefotaxime,
ceftriaxone, cefepime, cefoxitin, and cefozolin.
[0038] FIG. 9 is a table of antisense fragments.
[0039] FIG. 10 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis metRS-1
(metS) gene open reading frame (ORF).
[0040] FIG. 11 shows a graphic map of xylose-responsive growth
inhibitory antisense fragments corresponding to the S. aureus metRS
(metS) gene open reading frame (ORF).
[0041] FIG. 12 shows a graphic map of xylose-responsive growth
inhibitory antisense fragments corresponding to the B. anthracis
murB-2 gene open reading frame (ORF).
[0042] FIG. 13 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis glmU gene
open reading frame (ORF).
[0043] FIG. 14 shows a graphic map of xylose-responsive growth
inhibitory antisense fragments corresponding to the B. anthracis
murAl gene open reading frame (ORF).
[0044] FIG. 15 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis dxs gene
open reading frame (ORF).
[0045] FIG. 16 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis dxr-2 gene
open reading frame (ORF).
[0046] FIG. 17 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis gcpE gene
open reading frame (ORF).
[0047] FIG. 18 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis gyrB andgyrA
gene open reading frames (ORF).
[0048] FIG. 19 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis fabF gene
open reading frame (ORF).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0049] "Beta-lactam" A beta-lactam (p-lactam) is a lactam with a
heteroatomic ring structure, consisting of three carbon atoms and
one nitrogen atom. The beta-lactam ring is part of several
antibiotics, such as penicillin, which are therefore also called
beta-lactam antibiotics. These antibiotics work by inhibiting the
bacterial cell wall synthesis. Examples of beta-lactam antibiotic
classes include penicillins, cephalosporins, and cabapenems.
[0050] "Antibiotic" is a compound used to control infections.
Examples of antibiotics include:
[0051] Cell wall inhibitors include: Beta-lactams include
(penicillin G, penicillin V, nafcillin, methicillin, oxacillin,
cloxacillin, dicloxacillin, flucloxacillin, ampicillin,
amoxicillin, carbenicillin, ticarcillin, azocillin, mezlocillin,
pipericillin, Cephalosporins, cephalothin, cefazolin, cefalexin,
cefuroxime, cefamandole, cefoxitin, cefaclor, moxalactam,
cefaperazone, ceftazidime, ceftriaxone, clavulanic acid, sulbactam,
imipenem, aztreonam, Cefoxitin, cefozolin, Cefaclor).
[0052] Inhibitors of peptidoglycan biosynthesis, Inhibitors of
GlmU, MurA (Fosfomycin) Inhibitors of MurB (dihydropyrrolones),
Inhibitors of Undecaprenyl pyrophosphate Synthetase (hydantoins,
sulfonamides), Inhibitors of LpxC (UDP-3-O-acyl N-acetylglycosamine
deacetylase), Lipopeptides (daptomycin), Isoprenoid biosynthesis
inhibitors (bacitracin, phosmidomycin), Fatty acid biosynthesis
inhibitors (cerulenin, triclosan, isoniazid) Protein synthesis
inhibitors, aminoglycosides (streptomycin, gentamicin and
kanamycin), tetracyclines, chloramphenicol, macrolides
(erythromycin, azithromycin, clarithromycin), lincosamides
(lincomycin and clindamycin), and oxazolidinones (linezolid) tRNA
synthetase inhibitors such as mupirocin, MetRS inhibitors
(catechols, prolines, quinolones), Peptide deformylase inhibitors
(hydroxamates), RNA synthesis inhibitors such as rifampicin, DNA
gyrase inhibitors such as quinolones (nalidixic acid,
ciprofloxacin) Antifolates (trimethoprim and sulfamethoxazole),
"EC50" and "IC50" mean the concentration of compound resulting in
50% of the growth rate of the organism compared to the untreated
control.
[0053] A general protocol for generating and selecting
antisense-encoding DNA fragments is shown in FIG. 2. A general
protocol for validating those clones with specific antisense
response is shown in FIG. 3.
[0054] Our interest in antisense has been in development of
specific antisense strains of Bacillus anthracis and of
Staphylococcus aureus for use in tracking the mechanism of action
of new antibiotic candidates. To overcome limitations in the
current methods antisense methods, we developed the method outlined
in FIGS. 2 and 3. Unlike previous methods that used a limited
number of antisense fragments for any given gene to generate
antisense strains, we select a single gene based on its validated
utility as an antibiotic target site. We then generate a population
of fragments of limited length for cloning into an expression
vector. This process results in a large number of transformants,
only a small subset of which has the inducer-dependent
growth-inhibited phenotype. This subset requires additional
selection using (1) inducer-dependent decrease in the level of mRNA
for the target and (2) inducer-dependent hypersensitivity to a
growth-inhibiting compound as a second criterion. Finally, the
selective inducer concentration (SIC) is determined that gives the
maximal sensitivity to the antibiotic. The resulting strains that
are selected after passing all of the criteria are selectively
hypersensitive to compounds specifically inhibiting the target gene
or gene product of the antisense or to compounds inhibiting
processes directly linked to the target gene or gene product.
[0055] It is not obvious that such a method should be required for
producing such useful mode of action tools, because previous
publications indicate that many antisense fragments should work and
that the only criterion necessary for selection is an
inducer-dependent growth inhibition. Thus, the frequency of
artifact in antisense is not appreciated. This method is novel
because it begins with the target gene identified in advance and
requires the generation of thousands of fragments from which to
begin selection of those that will generate the desired drug
sensitized strain.
EXAMPLES
[0056] We have built specifically-sensitized antisense strains
using validated antibiotic targets. Four example targets are MurB
(UDP-N-acetylenolpyruvoylglucosamine reductase [EC: 1.1.1.15 8]) in
peptidoglycan biosynthesis (FIG. 1), methionyl-tRNA synthetase [EC:
6.1.1.10] in protein synthesis, UppS in peptidoglycan biosynthesis
(undecaprenyl diphosphate synthase [EC:2.5.1.31]), and DHFR
(dihydrofolate reductase [EC:1.5.1.3]) in DNA biosynthesis. The DNA
sequences of these genes in B. anthracis, S. aureus, and many other
microbial genomes are widely available (for example, in the Kyoto
Encyclopedia of Genes and Genomes (KEGG),
http://www.genome.jp/kegg/genes.html.
[0057] Each of these four genes was subjected to the process
outlined in FIGS. 2 and 3. Table 1 shows the outcome of the process
for each gene, including the number of transformants selected, the
number of transformants demonstrating growth inhibition phenotype
in the presence of the inducer (xylose) relative to the growth in
the absence of inducer, the number of clones with inducer-dependent
growth-inhibited phenotype, and the number of growth-inhibited
phenotype clones with plasmid inserts in the "antisense" and the
"sense" orientation relative to the promoter. Most of the strains
with a growth-inhibited phenotype in the presence of inducer had
inserts in the antisense orientation, but there were a significant
number of strains that had inserts in the sense orientation. This
result demonstrates that the method for selecting gene fragments
for an essential gene requires process steps not previously known
to be required.
[0058] Table 1. B. anthracis strain performance featuring fragments
from B. anthracis genes subjected to process in FIGS. 2 and 3. PCR
amplification of DNA for each gene was performed using
oligonucleotides sequences based on the published sequences for the
corresponding B. anthracis gene. The identities of the isolated
genes were verified by sequencing and then subjected to sonication.
The fragment sizes were monitored by agarose gel electrophoresis to
verify the fragments were less than 200 bp in length. The DNA
fragments were endpolished, ligated into the SmaI site of pBAX-2,
and rescued by transformation in E. coli DH5. Resulting libraries
were amplified in dam-/dcm-E. coli INV110 (Invitrogen). Amplified
library DNA was electroporated into B. anthracis plasmid-less
strain UM23C1-1. Randomly selected transformants were tested for
insert size by PCR. The range of insert sizes was 100-400 bp. About
2000 resulting colonies (CFU) per library were screened for growth
sensitivity in BHI medium with or without added xylose inducer at
2% final concentration. Plasmid DNA from these colonies was
analyzed by DNA sequencing to determine the sequence and
orientation relative to the xylose inducible promoter and to the
gene of interest.
TABLE-US-00001 TABLE 1 Number of Inserts of growth- Inserts of
colonies growth sensitive clones growth-sensitive inhibited in 2%
with "antisense" clones with Gene Enzyme Total CFU xylose medium
orientation "Sense" orientation murB-2 MurB 2024 26 26 0 metRSl
MetRS 2208 40 39 1 uppS UppS 2150 26 23 3 dfrA DHFR 2304 21 15
6
[0059] Table 1 includes results for methionyl-tRNA synthetase
(MetRS) encoded by the metRSl gene. Subjecting this gene to the
protocols in FIG. 2 resulted in approximately 2,000 strains
containing DNA fragments of the metRSl gene. Of these, 40 showed
growth inhibited phenotype upon exposure to the inducer xylose. Of
these, 39 contained inserts antisense to the metRSl gene. The
antisense strains were then characterized in terms of the
inducer-dependence of their growth attenuation and their
sensitivity to a specific inhibitor of MetRS (Rxl9) (FIG. 3), and
their sensitivity to a panel of antibiotics (FIG. 4A). Many of the
strains were selectively hypersensitive to Rxl9. RT-PCR experiments
verified that in the presence of inducer, the mRNA for metRSl was
reduced in level compared to the mRNA level for metRSl in the
absence of inducer. Levels of other mRNAs were not reduced. Some of
the strains showed no difference in sensitivity to antibiotics.
These strains are likely growth-attenuated due to a mechanism other
than specific post-transcriptional mRNA reduction. These strains
are not useful for detecting antibacterial compounds.
[0060] Table 1 also shows results for the MurB target, the second
step of peptidoglycan biosynthesis (FIG. 1). All bacterial
pathogens require MurB; however, there are currently no antibiotics
that act by inhibiting MurB. Subjecting the functional B. anthracis
murB gene {murB-2) to the protocols in FIG. 2 resulted in
approximately 2,000 strains containing DNA fragments of the murB
gene. Of these, 26 showed growth inhibited phenotype upon exposure
to the inducer xylose. Of these, 26 contained inserts antisense to
the murB-2 gene. The strains were then characterized in terms of
the inducer-dependence of their growth attenuation and their
sensitivity to a panel of antibiotics. Many of the strains were
selectively hypersensitive to cell wall inhibiting antibiotics
(FIGS. 5 and 6A). RT-PCR experiments verified that in the presence
of inducer, the mRNA for murB-2 was reduced in level compared to
the mRNA level for murB-2 in the absence of inducer. Levels of
other mRNAs were not reduced. Some of the strains showed no
difference in sensitivity to antibiotics. These strains are likely
growth-attenuated due to a mechanism other than specific
post-transcriptional mRNA reduction. These strains are not useful
for detecting mechanism of action of antibacterial compounds.
Example 1
[0061] FIG. 10 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis metRS-1
(metS) gene open reading frame (ORF). All fragments were found to
be in the antisense orientation (leftward pointing arrow) relative
to the corresponding gene (rightward pointing arrow). Numbers after
the name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 2
[0062] FIG. 11 shows a graphic map of xylose-responsive growth
inhibitory antisense fragments corresponding to the S. aureus metRS
(metS) gene open reading frame (ORF). All fragments were found to
be in the antisense orientation (leftward pointing arrow) relative
to the corresponding gene (rightward pointing arrow). Numbers after
the name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 3
[0063] FIG. 12 shows a graphic map of xylose-responsive growth
inhibitory antisense fragments corresponding to the B. anthracis
murB-2 gene open reading frame (ORF). All fragments were found to
be in the antisense orientation (leftward pointing arrow) relative
to the corresponding gene (rightward pointing arrow). Numbers after
the name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 4
[0064] FIG. 13 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis glmU gene
open reading frame (ORF). All fragments were found to be in the
antisense orientation (leftward pointing arrow) relative to the
corresponding gene (rightward pointing arrow). Numbers after the
name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 5
[0065] FIG. 14 shows a graphic map of xylose-responsive growth
inhibitory antisense fragments corresponding to the B. anthracis
murAl gene open reading frame (ORF). All fragments were found to be
in the antisense orientation (leftward pointing arrow) relative to
the corresponding gene (rightward pointing arrow). Numbers after
the name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 6
[0066] FIG. 15 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis dxs gene
open reading frame (ORF). All fragments were found to be in the
antisense orientation (leftward pointing arrow) relative to the
corresponding gene (rightward pointing arrow). Numbers after the
name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 7
[0067] FIG. 16 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis dxr-2 gene
open reading frame (ORF). All fragments were found to be in the
antisense orientation (leftward pointing arrow) relative to the
corresponding gene (rightward pointing arrow). Numbers after the
name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 8
[0068] FIG. 17 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis gcpE gene
open reading frame (ORF). All fragments were found to be in the
antisense orientation (leftward pointing arrow) relative to the
corresponding gene (rightward pointing arrow). Numbers after the
name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 9
[0069] FIG. 18 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis gyrB andgyrA
gene open reading frames (ORF). All fragments were found to be in
the antisense orientation (leftward pointing arrow) relative to the
corresponding gene (rightward pointing arrow). Numbers after the
name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
Example 10
[0070] FIG. 19 shows a graphic map of xylose-responsive growth
inhibitory fragments corresponding to the B. anthracis fabF gene
open reading frame (ORF). All fragments were found to be in the
antisense orientation (leftward pointing red arrow) relative to the
corresponding gene (rightward pointing green arrow). Numbers after
the name of each antisense DNA fragment correspond to the span and
position of the fragment relative to the gene sequence.
[0071] The method described in FIGS. 2 and 3 can produce antisense
strains in S. aureus similar to those in B. anthracis. FIGS. 8 A-C
shows growth sensitivity of a S. aureus strain selected using the
method outlined in FIGS. 2 and 3 using the murB gene from S.
aureus. One selected strain that showed growth-inhibited phenotype
in the presence of xylose relative to the absence of inducer was
characterized in terms of the inducer-dependence of growth
attenuation and sensitivity to a panel of antibiotics. The strain
showed selective hypersensitivity to antibiotics with mode of
action at the cell wall.
[0072] In addition to genes corresponding to cell wall
biosynthesis, protein synthesis, and DNA synthesis, we have built
antisense strains using the method described in FIGS. 2 and 3 to
obtain antisense strains in B. anthracis for the individual steps
in non-mevalonate isoprenoid biosynthesis; for gyrA and gyrB,
enzymes involved in DNA replication and the target proteins for the
fluoroquinoline antibiotics such as novobiocin, ciprofloxacin,
levofloxacin, and trovafloxacin; and for fabF mdfabH, steps in
fatty acid biosynthesis. These results further illustrate the
general applicability and utility in the method described in FIGS.
2 and 3 as pertaining to the variety of cellular metabolic
processes relevant to this process.
[0073] Examples 1 and 2 are genetic maps of the regions of the
metRSl and metRS genes that generated xylose-responsive growth
inhibitory antisense fragments from B. anthracis and S. aureus.
These examples illustrate that the region of the gene and the
fragment length producing useful xylose responsive growth
inhibitory fragments are efficiently generated using the method
described in FIGS. 2 and 3. These examples also illustrate that
each gene has characteristic regions and fragment lengths. These
fragments are found efficiently using the method described in FIGS.
2 and 3.
[0074] Example 3 is a genetic map of the regions of the murB2 gene
from B. anthracis that generated xylose-responsive growth
inhibitory antisense fragments. This example illustrates how
certain genes have regions that are more or less likely to generate
useful fragments.
[0075] Examples 4 and 5 are genetic maps of the regions of the glmU
gene and murAl gene from B. anthracis that generated
xylose-responsive growth inhibitory antisense fragments. These
examples illustrate that each gene in the peptidoglycan
biosynthesis pathway can be used in the method described in FIGS. 2
and 3. We have similarly generated useful strains for the other
steps in the peptidoglycan pathway.
[0076] Examples 6, 7, and 8 are genetic maps of the regions of the
dxs, dxr-2, and gcpE genes from B. anthracis that generated
xylose-responsive growth inhibitory antisense fragments. These
examples illustrate that genes from the non-mevalonate isoprenoid
biosynthesis pathway can be used in the method described in FIGS. 2
and 3. We have similarly generated useful strains for the other
steps in the non-mevalonate isoprenoid biosynthesis pathway.
[0077] Example 9 are genetic maps of the regions of the gyrA and
gyrB genes from B. anthracis that generated xylose-responsive
growth inhibitory antisense fragments. These examples illustrate
that genes involved in DNA replication can be used in the method
described in FIGS. 2 and 3.
[0078] Example 10 is the genetic map of the regions of the fabF
gene from B. anthracis that generated xylose-responsive growth
inhibitory antisense fragments. This example illustrates that genes
involved in fatty acid biosynthesis can be used in the method
described in FIGS. 2 and 3.
[0079] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
151113DNABacillus anthracis Ba-murB2-C1 1ccgccaactg aacctggaat
accacaagcg aactcaagac ccgttaagtt atggtctaac 60gcaatacgtg atacgtcaat
aattgctgca ccgcactgtg ctacaattgt cgt 1132124DNABacillus anthracis
Ba-murB2-H1 2ctcttcaact gttttttgta cgaagtgaat taaatcgatg taatcttgtg
ctgttccgtt 60atcaacattt accataaatc cagcgtgttt taaagaaacg gatcgaatgt
cattattaaa 120gacg 1243174DNABacillus anthracis Ba-murB2-D1
3tgcattcata tataatgctc cgccaactga acctggaata ccacaagcga actcaagacc
60cgttaagtta tggtctaacg caatacgtga tacgtcaata attgctgcac cgcactgtgc
120tacaattgtc gttcctgtta cagtaacacc tgtaatatga attaaactta ctgt
1744145DNABacillus anthracis Ba-murB2-D2 4aagttatggt ctaacgcaat
acgtgatacg tcaataattg ctgcaccgca ctgtgctaca 60attgtcgttc ctgttacagt
aacacctgta atatgaatta aacttactgt aatcccgcga 120attccaccgt
ctttaataat gacat 145596DNABacillus anthracis Ba-metRS1-H1
5aatataactg gatctactac atttcctttt gacttactca tctttccatc cttcattaaa
60atccaaccgt gagcaaagac ttttttcgga agaggt 966107DNABacillus
anthracis Ba-metRS1-H2 6aacttatctg ccttttttac aggttcagca gatagtactt
cagctacacg caattctact 60ttaaagaaat catcaattgt aatttcttct gccttcggtc
cttcttc 1077103DNABacillus anthracis Ba-metRS1-H6 7atccttcatt
aaaatccaac cgtgagcaaa gacttttttc ggaagaggta aatctaatgc 60cattaaaatg
attggccaat aaattgtatg gaaacgaacg att 103893DNABacillus anthracis
Ba-metRS1-E4 8ggttgtcctt tttctacttt tgttccagct ggaatacagc
cgattgtaga taggcttccc 60caagatgtat gtgcttcatc agtaaggcca agc
93999DNABacillus anthracis Ba-uppS-UG9 9tttaacgcga aattaagaat
taatcccgta ttctctttcg tttcttccat ggccttctcc 60atcgctctgc gtgtatgcgt
aggaagacga tcttgttgc 991051DNABacillus anthracis Ba-uppS-UA3
10cctctctttc tacacgcctc cgaatctgcg ccctctatgt tgaaagtctg t
5111103DNABacillus anthracis Ba-dfrA-2G1 11ccaggcagtg gtctaccaat
cgcttcatag ttttttcttc ccataataag cgggtgaccc 60atcgttgttt tctttacata
ctgcaattca ctcggtaaac gcc 10312107DNABacillus anthracis Ba-dfrA-2G6
12caggcagtgg tctaccaatc gcttcatagt tttttcttcc cataataagc gggtgaccca
60tcgttgtttt ctttacatac tgcaattcac tcggtaaacg ccaaggt
10713198DNAStaphylococcus aureus Sa-murB-E9 13ccaccttcac ggataataat
atttgagcca tttcctaaat atgtaacagg aatctcattt 60tgataggcat atttaacaac
tgcttgtact tcttcatttt tagtaggggt aatgtaaaag 120tcggcattac
cacctgtttt agtataagtg tatcgtttta aaggttcatc aactttaatt
180ttttcatttg ggataagt 19814169DNAStaphylococcus aureus Sa-murB-F7
14cttgcaaatt agaatcttgt atcaatttac ctgcaaaatg accaggcggt ctttggaata
60cactaccaca tgaaggatac tctaaaggtt gtttagattc tctacgttct gttaaatcat
120ccattttagc ttgtatttca gtcattttac caggagctaa agtaaatgc
16915146DNAStaphylococcus aureus Sa-murB-B9 15accaattgaa cctggaatac
cacatgcaaa ttcaaggcca gtaagtgcgt aatcacgagc 60aacacgtgag acatcaataa
ttgcagcgcc gctaccggct attatcgcat catcagatac 120ttcgatatga
tctagtgata ataaac 146
* * * * *
References