Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Reexamination Certificate
2001-02-23
2003-12-16
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
C435S006120, C435S007100, C435S252300, C514S012200, C514S014800
Reexamination Certificate
active
06664074
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the use of yacM and yqeJ, which are essential bacterial genes, in identifying antibacterial agents.
BACKGROUND OF THE INVENTION
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections can be life threatening, 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 that the yacM and yqeJ genes of the gram positive bacterium
Streptococcus pneumoniae,
termed “S-yacM” and “S-yqeJ,” are essential for survival. Thus, the essential polypeptides that these genes encode are useful targets for identifying compounds that are inhibitors of the bacteria in which the polypeptides are expressed. Such inhibitors can inhibit 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 amino acid and nucleic acid sequences of the Streptococcus yacM and yqeJ polypeptides and genes are set forth in
FIGS. 1 and 2
, as summarized in Table 1.
TABLE 1
Essential Nucleic Acids and Polypeptides
Essential
SEQ ID
SEQ ID
SEQ ID
Nucleic
NO. OF
NO. OF
NO. OF
Acid or
FIG
CODING
AMINO ACID
NON-CODING
Polypeptide
NO.
STRAND
SEQUENCE
STRAND
S-yacM
1
1
2
3
S-yqeJ
2
4
5
6
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 ssp. and
E. coli
). The terms “yacM” and “yqeJ” refer to the S-yacM and S-yqeJ 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). The yacM and yqeJ genes and polypeptides can be used in methods for identifying similar genes in pathogenic and non-pathogenic microorganisms. In particular, S-yacM and S-yqeJ genes can be used to identify orthologs of yacM and yqeJ genes in other species (e.g., other gram positive bacteria, and other bacteria generally). Examples of orthologs of these Streptococcus genes are summarized in Table 2. As shown in Table 2, the Streptococcus yacM gene has an ortholog in
B. subtilis,
termed “B-yacM,” and an ortholog in
E. coli,
termed “ygbP.” The Streptococcus yqeJ gene also has an ortholog in
B. subtilis,
termed “B-yqeJ,” and an ortholog in
E. coli,
termed “ybeN.” Having identified such orthologous genes as essential, these 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 Essential Nucleic Acids and Polypeptides
SEQ ID
SEQ ID
Essential
FIG.
SEQ ID
NO.
NO.
Nucleic
Num-
NO. of
of Amino
of Non-
Acid or
ber of
Coding
Acid Se-
coding
Poly-
Ortho-
Strand of
quence of
Strand of
peptide
Ortholog
log
Ortholog
Ortholog
Ortholog
yacM
B. subtilis
3
7
8
9
B-yacM
Subtilist
Accession No.
BG10152
Swissprot
Accession No.
Q06755
yacM
E. coli
4
10
11
12
ygbP (new
name ISPD)
GenBank
Accession No.
g1789104
Colibri
Accession No.
EG13110
yqeJ
B. subtilis
5
13
14
15
B-yqeJ
Subtilist
Accession No.
BG11638
Swissprot
Accession No.
P54455
yqeJ
E. coli
6
16
17
18
ybeN (new
name NADD)
GenBank
Accession No.
g1786856
Colibri
Accession No.
EG13241
The yacM and yqeJ polypeptides and genes described herein include the polypeptides and genes set forth in
FIGS. 1 and 2
herein, as well as isozymes, allelic variants, and conservative variants of the sequences set forth in
FIGS. 1 and 2
. For example, the invention includes a gene that encodes a yacM or yqeJ 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 a yacM or yqeJ polypeptide as determined, for example, in a conventional complementation assay. Also encompassed by the terms yacM gene and yqeJ gene are degenerate variants of the nucleic acid sequences set forth in
FIGS. 1-6
. Degenerate variants of a nucleic acid sequence exist because of the degeneracy of the amino acid code; thus, those sequences that vary from the sequences shown in
FIGS. 1-6
, but which nonetheless encode a yacM or yqeJ polypeptide are degenerate variants.
Likewise, because of the similarity in the structures of amino acids, conservative variations (as described herein) can be made in the amino acid sequence of the yacM or yqeJ polypeptide while retaining the function of the polypeptide (e.g., as determined in a conventional complementation assay). Other yacM and yqeJ polypeptides and genes identified in additional bacterial strains may be such conservative variations or degenerate variants of the particular yacM or yqeJ polypeptide and nucleic acid set forth in
FIGS. 1-6
(SEQ ID NOs:1-18). The yacM genes and polypeptides share at least 80%, e.g., 90% or 95%, sequence identity with the sequences shown in
FIGS. 1
,
3
, or
4
. Regardless of the percent sequence identity between the yacM sequence and the sequence shown in
FIGS. 1
,
3
or
4
, the yacM genes and polypeptides that can be used in the methods of the invention preferably are able to complement for the lack of yacM function (e.g., in a temperature-sensitive mutant) in a standard complementation assay.
Additional yacM genes that are identified and cloned from additional bacterial strains, and pathogenic, gram positive strains in particular, can be used to produce yacM polypeptides for use in the various methods described herein, e.g., for identifying antibacterial agents. Likewise, the term “yqeJ” encompasses isozymes, variants, and conservative variations of the sequences depicted in
FIGS. 2
,
5
, or
6
.
In various embodiments, the essential polypeptide used in the assays described herein is derived from a non-pathogenic or pathogenic gram-positive bacterium. For example, the polypeptide can be derived from a Streptococcus strain, such as
Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus endocarditis, Streptococcus faecium, Streptococcus sangus, Streptococcus viridans,
and
Streptococcus hemolyticus.
Suitable orthologs of the essential genes can be derived from a wide spectrum of bacteria, such as
E. coli
and
Bacillus subtilis.
Because the genes described herein have been shown to be essential for survival, the essential genes and polypeptides encoded by these essential genes, as well as their homologs and orthologs, 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 the essential polypeptide participates. This inhibition can be caused by small molecules interacting with (e.g., binding directly or indirectly to) the essential polypeptide or other essential polypeptides in that pathway.
In an exemplary assay, but not the only assay, a promoter that responds to depletion of the essential polypeptide by upregulation or downregulation is linked to a reporter gene (e.g., &bgr;-galactosidase, gus, or GFP), as described herein. A bacterial strain containing this reporter gene construct is then exposed to test compounds. Compounds that inhibit the yacM or yqeJ polypeptide (or other polypeptides in the essential pathway in which the polypeptide participates) will cause a functional depletion of the yacM or yqeJ polypeptide and therefore lead to an upregulation or downregulation of expression of the reporter gene. Because the polypeptides d
Fritz Christian
Guzman Luz-Maria
Youngman Philip
Fish & Richardson P.C.
Millennium Pharmaceuticals Inc.
Shahnan-Shah Khatol S
Swartz Rodney P
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