Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
1998-12-30
2002-04-16
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S004000, C435S007100, C435S007200, C435S007320
Reexamination Certificate
active
06372448
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the use of ylqF, yqeG, yybQ, yerL, and ysxC, essential bacterial genes and polypeptides in identifying antibacterial agents.
BACKGROUND OF THE INVENTION
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system. Most bacteria that are pathogenic to humans are gram positive bacteria. The bacterium
Streptococcus pneumoniae
, for example, typically infects the respiratory tract and can cause lobar pneumonia, as well as meningitis, sinusitis, and other infections.
SUMMARY OF THE INVENTION
The invention is based on the identification of ten genes of the gram positive bacterium
Streptococcus pneumoniae
and of
Bacillus subtilis
as being essential for survival. The
Streptococcus pneumoniae
genes are termed “S-ylqF,” “S-yqeG,” “S-yybQ,” “S-yerL,” and “S-ysxC.” The orthologs of these genes in
Bacillus subtilis
are termed “B-ylqF,” “B-yqeG,” “B-yybQ,” “B-yerL,” and “B-ysxC,” respectively. The terms “ylqF,” “yqeG,” “yybQ,” “yerL,” and “ysxC” genes and polypeptides are used to refer to the
Streptococcus pneumoniae
and
Bacillus subtilis
genes and polypeptides, as well as their homologs and orthologs, collectively. While “homologs” are structurally similar genes contained within a species, “orthologs” are functionally equivalent genes from other species (within or outside of a given genus, e.g., from
E. coli
). These genes are considered “essential” genes, and their polypeptides are considered “essential” polypeptides. Each gene and polypeptide can be used in methods for identifying similar genes and polypeptides in pathogenic and non-pathogenic microorganisms. Each polypeptide can be used to identify compounds that are inhibitors of the pathogens in which the polypeptide (ylqF, yqeG, yybQ, yerL, or ysxC) is expressed. Such inhibitors attenuate bacterial growth by inhibiting the activity of ylqF, yqeG, yybQ, yerL, or ysxC polypeptide, or by inhibiting gene transcription or translation.
The amino acid and nucleic acid sequences of the essential polypeptides described herein are set forth in
FIGS. 1-10
as shown in Table 1.
TABLE 1
Essential Polypeptides Disclosed Herein
SEQ ID NO.
SEQ ID NO.
SEQ ID
of the Coding
of the Non-
NO. of
Strand of
coding Strand
Essential
Amino
the Nucleic
of the
Nucleic Acid or
FIG.
Acid
Acid
Nucleic Acid
Polypeptide
NO.
Sequence
Sequence
Sequence
S-ylqF
1
2
1
3
S-yqeG
2
5
4
6
S-yybQ
3
8
7
9
S-yerL
4
11
10
12
S-ysxC
5
14
13
15
B-ylqF
6
17
18
16
B-yqeG
7
20
21
19
B-yybQ
8
23
22
24
B-yerL
9
26
25
27
B-ysxC
10
29
30
28
Since these genes have been identified and shown to be essential for survival, these genes and polypeptides (including homologs and orthologs of the sequences disclosed herein) can be used to identify antibacterial agents. Such antibacterial agents can readily be identified with high throughput assays to detect inhibition of the metabolic pathway in which these essential polypeptides participate. This inhibition can be caused by small molecules interacting with (e.g., binding directly or indirectly to) the ylqF polypeptide, for example, or other essential polypeptides in that pathway.
In an exemplary assay, but not the only assay, a promoter that responds to depletion of the ylqF, yqeG, yybQ, yerL, or ysxC polypeptide by upregulation or downregulation is linked to a reporter gene. To identify a promoter that is up- or down-regulated by the depletion of the polypeptide, the gene encoding the polypeptide is deleted from the genome and replaced with a version of the gene in which the sequence encoding the polypeptide (ylqF, yqeG, yybQ, yerL, or ysxC) is operably linked to a regulatable promoter. The cells containing this regulatable genetic construct are kept alive by the essential polypeptide produced from the genetic construct containing the regulatable promoter. However, the regulatable promoter allows the expression of the polypeptide to be reduced to a level that causes growth inhibition. Total RNA prepared from bacteria under such growth-limiting conditions is compared with RNA from wild-type cells. Standard methods of transcriptional profiling can be used to identify mRNA species that are either more or less abundant (i.e., up- or down-regulated) when expressed under the limiting conditions. Genomic sequence information, e.g., from GenBank, can be used to identify a promoter that drives expression of the identified RNA species. Such promoters are up- or down-regulated by depletion of the ylqF, yqeG, yybQ, yerL, or ysxC polypeptide.
Having identified a promoter(s) that is up- or down-regulated by depletion of the essential polypeptide (ylqF, yqeG, yybQ, yerL, or ysxC), the promoter(s) is operably linked to a reporter gene (e.g., &bgr;-galactosidase, gus, or green fluorescent protein (GFP)). A bacterial strain containing this reporter gene construct is then exposed to test compounds. Compounds that inhibit the ylqF, yqeG, yybQ, yerL, or ysxC polypeptide (or other polypeptides in the essential pathway in which the polypeptide participates) will cause a functional depletion of the polypeptide and therefore lead to an upregulation or downregulation of expression of the reporter gene. Because the polypeptides described herein are essential for the survival of bacteria, compounds that inhibit these polypeptides in such an assay are expected to be antibacterial and can be further tested, if desired, in standard susceptibility assays.
Another suitable method for identifying antibacterial compounds involves screening for small molecules that specifically interact with (i.e., bind directly or indirectly to) the ylqF, yqeG, yybQ, yerL, or ysxC polypeptide. A variety of suitable interaction and binding assays are known in the art as described, for example, in U.S. Pat. Nos. 5,585,277 and 5,679,582, incorporated herein by reference. For example, in various conventional assays, test compounds can be assayed for their ability to interact with a ylqF polypeptide by measuring the ability of the small molecule to stabilize the ylqF polypeptide in its folded, rather than unfolded, state. More specifically, one can measure the degree of protection against unfolding that is afforded by the test compound. Test compounds that bind, for example, the ylqF polypeptide with high affinity cause, for example, a large shift in the temperature at which the polypeptide is denatured. Test compounds that stabilize the ylqF polypeptide in a folded state can be further tested for antibacterial activity in a standard susceptibility assay. Similar assays can be performed with the other essential polypeptides described herein.
In a related method for identifying antibacterial compounds, the ylqF, yqeG, yybQ, yerL, or ysxC polypeptide, is used to isolate peptide or nucleic acid ligands that specifically bind the polypeptide. These peptide or nucleic acid ligands are then used in a displacement screen to identify small molecules that interact with the polypeptide. Such assays can be carried out essentially as described above.
Another suitable method for identifying inhibitors of the ylqF, yqeG, yybQ, yerL, or ysxC polypeptides involves identifying a biochemical activity of the polypeptide and then screening for small molecule inhibitors of the activity using, for example, a high throughput screening method.
The ylqF, yqeG, yybQ, yerL, and ysxC polypeptides can be used, separately or together, in assays to identify test compounds that interact with these polypeptides. Test compounds that interact with these polypeptides then can readily be tested, in conventional assays, for their ability to inhibit bacterial growth. Test compounds that interact with the ylqF, yqeG, yybQ, yerL, or ysxC polypeptides are candidate antibacterial agents, in contrast to compounds that do not interact with the ylqF, yqeG, yybQ, yerL, or ysxC polypeptides. As described herein, any of a variety of art-known methods can be used to assay for the interaction of test compounds with the ylqF, yqeG, yybQ, yerL,
Fritz Christian
Guzman Luz-Maria
Youngman Philip
Graser Jennifer E.
Millennium Pharmaceuticals Inc.
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