Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-05-25
2003-07-08
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023400, C536S023500, C536S023700, C435S320100, C435S069100, C435S252300, C435S252100
Reexamination Certificate
active
06590087
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to genes which control sporulation in mycobacteria. In particular the invention relates to the whm gene family. The invention also relates to compositions and methods for the prophylaxis, diagnosis and treatment of diseases caused by mycobacteria.
2. Background Information
The unusually slow growth rate of mycobacteria is significant to both the pathogenesis and treatment of mycobacterial infections, such as tuberculosis and leprosy. The slow growth rate necessitates protracted chemotherapeutic regimens which leads to poor patient compliance and the emergence of drug-resistant organisms. Little is known about the mechanism of cell division and its regulation in these bacteria.
Multiple proteins, including the highly conserved tubulin-like protein FtsZ, are known to participate in assembly of the prokaryotic cell division apparatus (1-3). The interactions among these proteins and the temporal and spatial regulation of their functions in
Escherichia coli, Bacillus subtilis,
and
Caulobacter crescentus
are the focus of intensive investigation (4). Doubling times for mycobacteria are significantly longer than in these better characterized species: 18-24 hours in
Mycobacterium tuberculosis,
14 days in
Mycobacterium leprae,
and 3 hours in
Mycobacterium smegmatis
(a saprophyte). The basis for these prolonged cell division times is unknown, although the limited number of rRNA operons (one in
M. tuberculosis,
two in
M. smegmatis
) and the metabolic costs of maintaining the complex cell wall that characterizes this genus have been cited as possible contributing factors (5-7).
WhiB is an 87 amino acid protein in
Streptomyces coelicolor
that is dispensible for normal growth but is required for the maturation of aerial hyphae during sporulation (8,9).
S. coelicolor
whiB mutants fail to assemble FtsZ rings in their aerial hyphae leading to the arrest of hyphal development prior to crosswall formation (10). The WhiB protein contains an acidic N-terminus with 4 cysteine residues, a helix-turn-helix motif, and a basic C terminus. The mechanism by which WhiB regulates spore formation has not yet been established.
Southern blot surveys suggested the existence of a mycobacterial gene closely related to whiB of
S. coelicolor
(11). This suggested to the present inventors that such a gene, if present, would be valuable in the diagnosis and treatment of mycobacterial diseases such as tuberculosis.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a DNA sequence encoding a protein required for cell division in mycobacterium. Such mycobacteria include, but are not limited to
M. tuberculosis, M. bovis
and
M. smegmatis.
The invention provides mycobacterial homologues of the whiB gene from
Streptomyces coelicolor.
In particular, the invention provides a homologue to the whiB gene, whmD, which is useful for diagnosis and therapy of mycobacterial diseases such as tuberculosis,
Mycobacterium avium
complex (MAC) infection, leprosy, and infection by atypical mycobacteria.
The whmD gene can be used to screen for drugs which interrupt cell division.
The whmD gene can be crystallized, its 3-dimensional structure determined, and rational drug design employed to develop antimicrobials which are cell division inhibitors for mycobacteria.
The whmD gene can also be used to find useful proteins which interact or bind to it. Among such proteins are likely to be found proteins which are involved in cell division and may also serve as targets for antimicrobial drugs.
The whmD gene may be used as a probe to elucidate cell division in pathogenic mycobacteria. These organisms divide unusually slowly, and may enter a non-replicating state during latent human tuberculosis. Understanding latent tuberculosis is essential for controlling tuberculosis, and should lead to improved vaccines, diagnostics and therapeutic compositions and methods.
The whmD gene may be used to construct a conditionally lethal mutant strain of
M. tuberculosis
which might be used as a novel live attenuated vaccine against TB. The vaccine strain would be viable in the presence of a chemical inducer which would be co-administered with the vaccine. At an appropriate time after effective immunity had been elicited, the administration of the exogenous chemical inducer would be stopped and the conditionally lethal mutant would die within the host. Such mutants would have advantages over the existing TB vaccine, bacille Calmette-Guerin.
In one embodiment of the invention an isolated and purified subgenomic DNA segment is provided. Its nucleotide sequence is shown in
FIG. 2
(SEQ ID NO:1).
In another embodiment of the invention, an isolated and purified amino acid sequences of the whmD protein is provided. Sequences from
M. tuberculosis
and
M. smegmatis
are shown in
FIG. 3
(SEQ ID NO: 2 and SEQ ID NO:3).
In another embodiment of the invention, a preparation of an isolated polypeptide is provided which comprises at least twelve, preferably at least 20, and most preferably at least 30 contiguous amino acids of the sequence shown in SEQ ID NO:2 or SEQ ID NO:3. Peptides comprising contiguous residues 1-60 of the N-terminal region (MSYES . . . AEEDQ); 61-99 of the central cysteine-rich region (WQER . . . CEVRDAC); 100-126 of the central helix-turn-helix region are considered to be of particular interest.
It is yet another object of the invention to provide a method for screening potential therapeutic agents for the ability to regulate the growth of mycobacteria, particularly
M. tuberculosis.
In yet another embodiment of the invention a reporter construct is provided. The reporter comprises a whmD transcription regulatory region covalently linked in a cis configuration 5′ of a gene encoding an assayable product, wherein transcription of the gene is regulated by the whmD transcription regulatory region.
In another embodiment of the invention a method is provided for screening potential therapeutic agents for the ability inhibit the growth of mycobacterium by inactivating or inhibiting the expression of whmD. The method comprises the steps of: incubating a potential therapeutic agent with a cell which contains a whmD reporter construct, said reporter construct comprising a whmD transcription regulatory region covalently linked in a cis configuration to a downstream gene encoding an assayable product; and measuring the production of the assayable product, a potential therapeutic agent which decreases the production by the cell of the assayable product being an agent which will inhibit the growth of the mycobacterium by inactivating the expression of whmD.
In still another embodiment of the invention a method is provided for screening potential therapeutic agents for use in modulating the growth of a mycobacterium by regulating the activity of whmD. The method comprises the steps of: measuring in vitro transcription from the transcription construct incubated with whmD in the presence or absence of a test compound, the transcription construct comprising a gene coding sequence and a promoter which is responsive to whmD, the promoter being upstream from and adjacent to the gene, the in vitro transcription being effected in the presence and absence of a test substance; and determining whether transcription of the gene is altered by the presence of said test substance, a test substance which alters the transcription of the gene being a candidate for use in regulating the growth of mycobacterium.
In another embodiment of the invention a method is provided for screening potential therapeutic agents for the ability to inhibit the growth of mycobacterium by inhibiting the expression of whmD. The method comprises the steps of: incubating a potential therapeutic agent with a cell which contains a whmD reporter construct, said reporter construct comprising a whmD transcription regulatory region covalently linked in a cis configuration to a downstream gene encoding an assayable product; and measuring the production of the assayable product, a potent
Bishai William R.
Gomez James E.
Hobbs Ann S.
Johns Hopkins University
Minnifield Nita
Venable LLP
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