Essential bacterial genes and their use

Details Essential bacterial genes and their use Essential bacterial genes and their use

1999-09-09

2003-09-30

Graser, Jennifer E. (Department: 1645)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S252300, C435S243000, C435S069100, C424S244100, C536S024320

Reexamination Certificate

active

06627747

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to essential bacterial genes and their use 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 discovery of two genes in the gram positive bacterium
Streptococcus pneumoniae
that are essential for the survival of this and other bacteria. For convenience, these genes, yphC and yqjK, are collectively referred to herein as “essential” genes and the polypeptides that these genes encode are referred to as “essential” polypeptides since
Streptococcus pneumoniae
cells lacking functional yphC or yqjK genes are unable to survive.
The yphC and yqjK genes are useful molecular tools for identifying similar genes in pathogenic microorganisms. The essential polypeptides that these genes encode are useful targets for identifying compounds that are inhibitors of the pathogens in which the essential polypeptides are expressed. Such compounds diminish bacterial growth by inhibiting the activity of an essential protein, or by inhibiting transcription of an essential gene or translation of the mRNA transcribed from the essential gene.
The invention, therefore, features an isolated yphC polypeptide having the amino acid sequence set forth in SEQ ID NO:2, as depicted in
FIG. 1
, or conservative variations thereof. An isolated nucleic acid encoding yphC also is included within the invention. In addition, the invention includes (a) an isolated nucleic acid having the sequence of SEQ ID NO:1, as depicted in
FIGS. 1A-B
, or degenerate variants thereof; (b) an isolated nucleic acid having the sequence of SEQ ID NO:1, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions, as described below, to genomic DNA encoding the polypeptide of SEQ ID NO:2. The yphC polypeptide depicted in
FIGS. 1A-B
is a partial sequence of the full-length polypeptide, which is depicted in
FIGS. 2A-2B
. The invention also features an isolated yphC polypeptide having the amino acid sequence set forth in SEQ ID NO:5, as depicted in
FIGS. 2A-2B
, or conservative variations thereof. An isolated nucleic acid encoding full-length yphC also is included within the invention. In addition, the invention includes (a) an isolated nucleic acid having the sequence of SEQ ID NO:4, as depicted in
FIGS. 2A-2B
, or degenerate variants thereof; (b) an isolated nucleic acid having the sequence of SEQ ID NO:4, or degenerate variants thereof, wherein T is replace by U; and (c) nucleic acids complementary to (a) and (b).
As described above for yphC, the invention includes an isolated nucleic acid encoding yqjK. In addition, the invention includes (a) an isolated nucleic acid having the sequence of SEQ ID NO:7, as depicted in
FIGS. 3A-B
, or degenerate variants thereof; (b) an isolated nucleic acid having the sequence of SEQ ID NO:7, or degenerate variants thereof, wherein T is replaced by U; (c) nucleic acids complementary to (a) and (b); and (d) fragments of (a), (b), and (c) that are at least 15 base pairs in length and that hybridize under stringent conditions, as described below, to genomic DNA encoding the polypeptide of SEQ ID NO:8. These sequences are summarized in Table 1.
TABLE 1
Essential Nucleic Acids and Polypeptides
SEQ ID
SEQ ID
NO. OF
NO. OF
AMINO
SEQ ID NO.
NON-
Essential Nucleic
FIG.
ACID
OF CODING
CODING
Acid or Polypeptide
NO.
SEQUENCE
STRAND
STRAND
yphC-partial
1
2
1
3
sequence
yphC-full-length
2A-2B
5
4
6
yqjK
3
8
7
9
Identification of these essential genes allows homologs of the essential genes to be found in other strains within the species, and it allows orthologs of the essential genes to be found in other organisms (e.g., Bacillus sp.,
H. influenzae, H. pylori
, and
E. coli
). While “homologs” are structurally similar genes contained within the Streptococcus species, “orthologs” are functionally equivalent genes from other species, as determined, for example, in a standard complementation assay. Thus, the essential polypeptides can be used not only as a model for identifying similar genes in other Streptococcus strains, but also to identify homologs and orthologs of essential genes in other species (e.g., other gram positive bacteria, particularly those bacteria that are pathogenic to humans, and other bacteria generally). Such orthologs can be identified, for example, in a conventional complementation assay. In addition, or alternatively, such orthologs can be expected to exist in bacteria in the same branch of the phylogenetic tree, as set forth, for example, at ftp://ftp.cme.msu.edu/ pub/RDP/SSU_rRNA/SSU/Prok.phylo. For example,
B. subtilis
is in the
B. subtilis
subgroup of the
B. subtilis
group in the Bacillus-Lactobaccillus-Streptococcus Subdivision of the Gram positive phylum. Likewise,
S. pneumoniae
belong to the Stc. pneumonia subgroup of Streptococci, which also are in the Bacillus-Lactobacillus-Streptococcus subdivision of the Gram positive phylum.
E. coli
belong to the Escherichia Salmonella group of the Enterics and relatives within the Gamma subdivision of the Purple bacteria. Other bacteria within the same phylum (particularly, bacteria within the same subdivision, group, or subgroup) can be expected to contain an ortholog of the yphC and/or yqjK genes described herein.
Examples of orthologs of the Streptococcus yphC and yqjK genes are summarized in Table 2. As shown in Table 2, the Streptococcus gene yphC has an ortholog in
B. subtilis
, termed “B-yphC,” and an ortholog in
E. coli
, termed “yfgK,” which is also known as “f503.” The Streptococcus gene yqjK also has an ortholog in
B. subtilis
, termed “B-yqjK,” and an ortholog in
E. coli
, termed “elaC,” which is also known as “o311.” As discussed below, orthologs of essential genes may themselves be essential or non-essential in the organism in which they are found.
As determined by the experiments described below, the B-yphC, yfgK, and B-yqjK orthologs are essential for survival of the bacteria in which they are found. Thus, these essential orthologous genes and the polypeptides encoded by these orthologs can be used to identify compounds that inhibit the growth of the host organism (e.g., compounds that inhibit the activity of an essential protein, or inhibit transcription of an essential gene).
TABLE 2
Orthologs of yphC and yqjK
SEQ ID
SEQ ID
SEQ ID
NO. of
NO. of
NO. of
Amino
Nucleic
Non-
Nucleic
Figure
Acid
Acid
Coding
Acid or
number
Sequence
Sequence
Strand
Poly-
of
of
of
of
peptide
Ortholog
Ortholog
Ortholog
Ortholog
Ortholog
yphC
B. subtilis
4A-4B
11
10
12
B-yphC
GenBank
Accession
No. Z99115
yphC
E. coli
5A-5B
14
13
15
yfgK
GenBank
Accession
No. AE000337
yqjK
B. subtilis
6
17
16
18
B-yqjK
GenBank
Accession
No. Z99116
yqjK
E. coli
7
20
19
21
elaC
GenBank
Accession
No. AE000316
The yphC polypeptides and genes described herein include the polypeptides and genes set forth in
FIGS. 1A-B
and
2
A-
2
B herein, as well as isozymes, variants, and conservative variations of the sequences set forth in
FIGS. 1A-B
and
2
A-
2
B. The invention includes various isozymes of yphC and yqjK. For example, the invention includes a gene that encodes an essential polypeptide but which gene includes one or more point mutations, deletions, or promoter variants, provided that the resulting essential polypeptide retains a biological function of an essential polypeptide.
The yphC polypeptide has structural characteristics of known GTPases. Using BLAST analysis, the yphC polypeptide has been shown to contain t

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