Increased virulence of Mycobacterium tuberculosis H37Rv overexpressing LipY in a murine model.

We have investigated the role of Rv3097c-encoded lipase (LipY) on the virulence of Mycobacterium tuberculosis. It has been shown that the overexpression of LipY in strain H37Rv induced increase in virulence of recombinant H37Rv::LipY strain. Compared to H37Rv, infection with H37Rv::LipY caused enhanced mortality, weight loss, bacterial load in lungs, splenomegaly, worsening lung morphology and pathology. Mice immunized with recombinant LipY antigen were protected against challenge with H37Rv::LipY, which correlated with enhanced survival of challenged mice and striking decrease in pathological features observed in unimmunized mice. To probe the cause of increase in virulence of H37Rv::LipY, the immune status of the host infected with H37Rv and H37Rv::LipY was compared. It was found that overexpression of LipY compromised immune responses resulting in attenuation of Th1 and Th17 responses, significant increase in IL-10, decrease in number of macrophages and T cells, and increase in numbers of Treg, and DCs in the lungs whereas in mice immunized with LipY an increased pool of T cells and DCs was observed. This led us to conclude that the increase in the virulence of H37Rv::LipY was due to downregulation of the host's protective immunity and the Rv3097c encoded LipY lipase is a virulence factor of M. tuberculosis.

Identification and characterisation of small-molecule inhibitors of Rv3097c-encoded lipase (LipY) of Mycobacterium tuberculosis that selectively inhibit growth of bacilli in hypoxia.

The mycobacterial Rv3097c-encoded lipase LipY is considered as a true lipase involved in the hydrolysis of triacylglycerol stored in lipid inclusion bodies for the survival of dormant mycobacteria. To date, orlistat is the only known LipY inhibitor. In view of the important emerging role of this enzyme, a search for small-molecule inhibitors of LipY was made, leading to the identification of some new compounds (8a-8d, 8f, 8h and 8i) with potent inhibitory activities against recombinant LipY, with no cytotoxicity [50% inhibitory concentration (CC(50)) ≥ 500 µg/mL]. The compounds 6a, 8c and 8f potently inhibited (>90%) the growth of Mycobacterium tuberculosis H37Rv grown under hypoxia (oxygen-depleted condition) but had no effect on aerobically grown bacilli, suggesting that these new small molecules are highly selective towards the growth inhibition of hypoxic cultures of M. tuberculosis and hence provide new leads for combating latent tuberculosis.

In vivo activity of thiophene-containing trisubstituted methanes against acute and persistent infection of non-tubercular Mycobacterium fortuitum in a murine infection model.

OBJECTIVES: Mycobacterium fortuitum causes opportunist non-tubercular infection in humans. Chronic infection of M. fortuitum has been clinically documented and requires prolonged chemotherapy. The objectives of this study were to characterize acute and persistent infection of M. fortuitum in a murine infection model and to screen thiophene-containing trisubstituted methanes active against both acute and persistent infection. METHODS: A murine infection model of M. fortuitum was used. Bacillary count, bioluminescence, disease symptoms, host immune response, drug susceptibility and mortality were measured. Reactivation of persistent bacilli was induced by dexamethasone. Trisubstituted methanes containing thiophene rings were synthesized and screened in vitro by agar dilution and BACTEC assay and in mice. Cytotoxicity was tested with Vero monkey kidney cells using a resazurin assay. RESULTS: The acute infection in mice was marked by a 3 log rise in viable counts, the appearance of disease symptoms and a rise in the Th1 immune response. Bacilli were susceptible to fluoroquinolones. This was followed by persistent infection, in which disappearance of disease symptoms, a decline in Th1 response and non-susceptibility to fluoroquinolones was observed. When the mice were immunocompromised on day 40 post-infection (persistent state) by dexamethasone, a rise in viable counts, symptoms and susceptibility to fluoroquinolones and a prominent Th1 response reappeared. Two lead compounds were found that cleared the mice of bacilli in acute infection and caused a 2.29-2.99 log reduction in cfu of persistent bacilli. CONCLUSIONS: The study established acute and persistent infection in mice and identified two promising anti-M. fortuitum compounds with a selectivity index >10.

Functional characterization of VC1929 of Vibrio cholerae El Tor: role in mannose-sensitive haemagglutination, virulence and utilization of sialic acid.

The nonadhesive mutant CD11 of Vibrio cholerae El Tor, defective in expression of mannose-sensitive haemagglutinin, lacks a protein when compared with its parent strain. Determination of the amino acid sequence revealed the identity of the protein as the product of VC1929, which is annotated to encode a protein, DctP, involved in the transport of C4-dicarboxylates. We cloned the dctP gene in pUC19 vector and expressed it in mutant CD11. Expression of DctP in the resulting complemented strain restored virulence, adhesive and colonizing capabilities, mannose-sensitive haemagglutination (MSHA) and ability to grow in medium containing sialic acid as a sole carbon source. The mutation in CD11 was caused by insertion of an adenine nucleotide in the reading frame of dctP. Recombinant purified DctP protein showed MSHA of human red blood cells, and protected rabbits against infection by V. cholerae. The protein was localized in membrane and cell wall fractions. The mutant, recombinant CD11 expressing DctP and parent strains were grown in M9 minimal medium in the presence of various carbohydrates (glucose, malate, fumarate, succinate or N-acetylneuraminic acid). The mutant was unable to grow in minimal medium containing N-acetylneuraminic acid (sialic acid) as the sole carbon source whereas the recombinant and parent strains utilized all the sugars tested. It is concluded that DctP is a mannose-sensitive haemagglutinin and a virulence factor and is involved in the utilization of sialic acid.

Overexpression of Rv3097c in Mycobacterium bovis BCG abolished the efficacy of BCG vaccine to protect against Mycobacterium tuberculosis infection in mice.

Rv3097c of Mycobacterium tuberculosis encoding lipase (LipY) was overexpressed in Mycobacterium bovis BCG. Efficacy of recombinant BCG to protect against infection of M. tuberculosis was evaluated in mice. Whereas the parent BCG vaccine protected the mice against infection, recombinant BCG overexpressing LipY offered no protection as judged by viable counts of tubercule bacilli in lungs, weight of infected mice, pathology of lungs and survival of challenged mice. Downregulation of overexpression of LipY by antisense approach considerably restored protection of infected mice as observed with parent BCG vaccine. Overexpression of lipase in BCG caused extensive hydrolysis of triacylglycerol (TG) as identified by TLC, HPLC and NMR spectroscopy. A good correlation could be inferred between hydrolysis of TG and decrease in Th1 secreted IFNγ and IL-2, proinflammatory cytokines and survival of infected mice. Mice immunized with purified LipY antigen were protected and both proinflammatory and Th1 specific cytokines were augmented. TG was found to be a poor vaccine providing no protection, which appears to be due to attenuation of Th1 and proinflammatory immune responses. In conclusion this is the first experimental report to show that immunogenicity of BCG vaccine was impaired by LipY-induced hydrolysis of specific lipids leading to suppression of host immune responses.

2,3-dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular agents: design, synthesis, biological evaluation and SAR studies.

The alarming resurgence of tuberculosis (TB) underlines the urgent need for development of new and potent anti-TB drugs. Towards this goal we herein report the design and synthesis of 2,3-dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular agents. These easily accessible, small molecules were found to exhibit in vitro activity against Mycobacterium tuberculosis H37Rv in a MIC range of 0.78 µg/mL to 25 µg/mL. A detailed SAR study on these hex-2-enopyranosid-4-uloses led to the identification of compound 5g (S007-724) which on the basis of low MIC (0.78 µg/mL-M. tuberculosis H37Rv; 1.56 µg/mL-MDR, SDR strains of M. tuberculosis; 0.78 µg/mL-inhibition of intracellular replication of M. tuberculosis) and SI value of 13.5 has been identified as a promising lead molecule.

Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development.

Acetohydroxyacid synthase (AHAS) is a biosynthetic enzyme essential for de novo synthesis of branched-chain amino acids. The genome sequence of Mycobacterium tuberculosis revealed genes encoding four catalytic subunits, ilvB1 (Rv3003c), ilvB2 (Rv3470c), ilvG (Rv1820) and ilvX (Rv3509c), and one regulatory subunit, ilvN (Rv3002c), of AHAS. All these genes were found to be expressed in M. tuberculosis growing in vitro. Each AHAS subunit gene was cloned and expressed in Escherichia coli. AHAS activity of IlvB1 and IlvG was found in cell-free lysates and with recombinant purified proteins. Kinetic studies with purified IlvG revealed positive cooperativity towards substrate and cofactors. To understand the role of the catalytic subunits in the biology of M. tuberculosis, expression of AHAS genes was analysed in different physiological conditions. ilvB1, ilvB2 and ilvG were differentially expressed. The role of ilvB1 in persistence is known, but the upregulation of ilvB2 and ilvG in extended stationary phase, ex vivo, and in acid stress and hypoxic environments, suggests the relevance of AHAS enzymes in the metabolism and survival of M. tuberculosis by functioning as catabolic AHAS. These enzymes are therefore potential targets for drug development.

Downregulation of Rv0189c, encoding a dihydroxyacid dehydratase, affects growth of Mycobacterium tuberculosis in vitro and in mice.

Dihydroxyacid dehydratase (DHAD), a key enzyme involved in branched-chain amino acid (BCAA) biosynthesis, catalyses the synthesis of 2-ketoacids from dihydroxyacids. In Mycobacterium tuberculosis, DHAD is encoded by gene Rv0189c, and it shares 40% amino acid sequence identity and conserved motifs with DHAD of Escherichia coli encoded by ilvD. In this study, Rv0189c was overexpressed in E. coli and the resultant protein was characterized as a homodimer (~155 kDa). Functional characterization of Rv0189c was established by biochemical testing and by genetic complementation of an intron-disrupted ilvD-auxotrophic mutant of E. coli to prototrophy. Growth of M. tuberculosis, E. coli BL21(DE3) and recombinant E. coli BL21(DE3) ΔilvD carrying Rv0189c was inhibited by transient nitric oxide (NO) exposure in minimal medium but growth was restored if the medium was supplemented with BCAA (isoleucine, leucine and valine). This suggested that inactivation of Rv0189c by NO probably inhibited bacterial growth. The role of Rv0189c in M. tuberculosis was elucidated by antisense and sense RNA constructs. Growth of M. tuberculosis transformed with a plasmid encoding antisense mRNA was markedly poor in the lungs of infected mice and in Middlebrook 7H9 broth compared to that of sense and vector-alone transformants, but growth was normal when the medium was supplemented with BCAA. Upregulation of Rv0189c was observed during the early exponential phase of growth, under acid stress and ex vivo, suggesting that Rv0189c has a role in the survival of M. tuberculosis during normal and stress conditions. It may be concluded that the DHAD encoded by Rv0189c is essential for the survival of M. tuberculosis and could be a potential drug/vaccine target, as it is absent in mammals.

Comparative expression analysis of rpf-like genes of Mycobacterium tuberculosis H37Rv under different physiological stress and growth conditions.

Mycobacterium tuberculosis H37Rv possesses five resuscitation-promoting factors, RpfA-E, which are required for the resuscitation of dormancy in mycobacteria induced by prolonged incubation of the culture in stationary phase. This study explores the transcriptional profile of all the rpf-like genes of M. tuberculosis H37Rv in the exponential phase, stationary phase, non-culturable phase and Rpf-mediated resuscitation phase. The relative expression was also monitored under acid stress, nutrient starvation and low-oxygen (hypoxia) conditions by real-time quantitative PCR. We show differential relative expression of the rpf genes during different stages of growth and under stress. During early resuscitation, all rpf genes were expressed with maximal expression ratios for rpfA and rpfD. rpfC was consistently expressed during all stages of growth and nutrient starvation. Acid stress induced higher relative expression of rpfD and rpfE and hypoxia of rpfC and rpfE. These results therefore provide further evidence that the rpf genes perform distinct roles during cell growth and cell survival under different physiological stresses, and are consistent with the rpf-like genes being differentially regulated.

Fast-growing, non-infectious and intracellularly surviving drug-resistant Mycobacterium aurum: a model for high-throughput antituberculosis drug screening.

OBJECTIVES: Enoyl acyl-carrier-protein reductase (InhA), the primary endogenous target for isoniazid and ethionamide, is crucial to type-II fatty acid biosynthesis (FAS-II). The objectives of this study were first to generate InhA mutants of Mycobacterium aurum, secondly to characterize InhA-mediated isoniazid and ethionamide resistance mechanisms across those mutants and finally to investigate the interaction of InhA with enzymes in the FAS-II pathway in M. aurum. METHODS: Spontaneous mutants were generated by isoniazid overdose and limited broth dilution, while for genetically modified mutants sense-antisense DNA technology was used. Southern hybridization and immunoprecipitation were both used to identify the InhA homologue in M. aurum. The latter method was further used to compare the level of InhA expression in M. aurum with that in corresponding mutants. Isoniazid/ethionamide susceptibility modulation was examined in vitro and ex vivo using a resazurin assay as well as by cfu counting. In addition, circular dichroism and the bacterial two-hybrid system were exploited to investigate the interaction of InhA with other enzymes of the FAS-II pathway. RESULTS: A Mycobacterium tuberculosis InhA homologue was detected in M. aurum. Susceptibility to isoniazid/ethionamide was significantly altered in genetically modified mutants and simultaneously InhA was overexpressed in both spontaneous and genetically modified mutants. InhA interacts with other FAS-II enzymes of M. aurum in vivo. CONCLUSION: Close resemblance of isoniazid/ethionamide action on InhA between M. tuberculosis and M. aurum further supports the use of fast-growing and intracellularly surviving drug-resistant M. aurum to substitute for highly virulent, extremely slow-growing M. tuberculosis strains in the early stage of antituberculosis inhibitor screening.

Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge.

Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli. Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera.

Variable-number tandem repeat 3690 polymorphism in Indian clinical isolates of Mycobacterium tuberculosis and its influence on transcription.

Variable-number tandem repeat (VNTRs) occur throughout the chromosome of Mycobacterium tuberculosis. Although these polymorphic VNTRs, also known as mycobacterial interspersed repetitive units (MIRUs), have proved to be useful tools in molecular epidemiology, their biological significance is less well understood. This study investigated the polymorphism of the VNTR 3690 locus located in the intergenic region between rv3304 and rv3303c (encoding the gplD2 and lpdA genes, respectively) and its possible function in the regulation of gene expression. The copy number of VNTR 3690 was found to vary among Indian clinical isolates of M. tuberculosis (one to twelve copies), M. tuberculosis H37Rv TMC102 (four copies), M. tuberculosis H37Ra (two to four copies), Mycobacterium bovis BCG (one copy). The expression of lpdA as measured by quantitative RT-PCR was 12-fold higher in M. tuberculosis H37Rv than in M. bovis BCG. Using a GFP reporter system in which the 5'-flanking region of lpdA was fused to the gfp gene, the effect of VNTRs on gene expression was measured in an M. bovis BCG host background by real-time PCR. Compared with one VNTR repeat, a 12.5-fold upregulation of GFP expression was found with a flanking region containing four VNTR 3690 repeats, indicating that there is a good correlation between VNTR copy number and transcription of lpdA.

A transposon insertion mutant of Mycobacterium fortuitum attenuated in virulence and persistence in a murine infection model that is complemented by Rv3291c of Mycobacterium tuberculosis.

Mycobacterium fortuitum is a non-tubercular fast growing pathogenic mycobacteria whose virulence factors have not been studied. Infection of M. fortuitum ATCC 6841 in a murine infection model leads to spinning of the head in 8-12 days after infection, 20-25% mortality and a constant bacillary load in the kidney of mice, suggesting persistence. From a TnphoA insertion library, a mutant MT13 was isolated which was attenuated in virulence with lesser bacterial burden, milder and delayed spinning of the head and no mortality of mice. The significant feature of the mutant was its failure to persist in kidney and thus the persistent bacillary load characteristic exhibited by the wild type strain was not observed. The insertion of transposon in MT13 was mapped in a region of the genome, which showed homology to Rv3291c of M. tuberculosis, annotated as a transcriptional regulatory factor and reported to be up regulated in nutrient starvation and anaerobic persistent states. Complementation of MT13 with rv3291c resulted in restoration of wild type characteristics including persistence in kidney suggesting the role of a Rv3291c homolog in the virulence and persistence of M. fortuitum.

Identification of genes of Mycobacterium tuberculosis upregulated during anaerobic persistence by fluorescence and kanamycin resistance selection.

Molecular mechanisms involved in maintaining the latent infection of Mycobacterium tuberculosis are least understood. We have applied principles of in vivo expression technology (IVET) to identify upregulated genes in an in vitro simulated condition of anaerobic persistence likely to be encountered by the pathogen in lung granulomas. A promoter library of M. tuberculosis constructed in plasmid pLL192 was subjected to hypoxic condition (dissolved oxygen <1%) in a controlled fermenter. On the basis of green fluorescent protein fluorescence and kanamycin resistance the upregulated promoters were selected, identified by nucleotide sequence and the genes were confirmed by RT-PCR. The upregulated genes include Rv0050 (penicillin binding protein), Rv1511 (GDP-d-mannose dehydratase), Rv1489, Rv2257, Rv2258 (all conserved hypothetical proteins), Rv0467 (isocitrate lyase) and Rv2031c (alpha-crystalline homolog). The involvement of the last four genes in latency has been suggested before. The functional role of Rv0050 and Rv1511 may be important in determining cell wall characteristics controlling permeability of nutrients and antibiotics.

Selection of genes of Mycobacterium tuberculosis upregulated during residence in lungs of infected mice.

In sequel to previous report [Srivastava V, Rouanet C, Srivastava R, Ramalingam B, Locht C, Srivastava BS. Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection. Microbiology 2007;153:659-66], the genes of Mycobacterium tuberculosis upregulated during residence in lungs of infected mice were identified in an in vivo expression system based on kanamycin resistance. A promoter library of M. tuberculosis was constructed in a promoter trap shuttle vector pLL192 containing an artificial bicistronic operon composed of promoterless green fluorescent protein gene followed by kanamycin resistance gene. The library was introduced in M. bovis BCG and then infected in mice by intravenous route. Mice were treated twice daily with 40 mg/kg dose of kanamycin by intramuscular route for 21 days. Recombinant BCG recovered from the lungs were reinfected in mice to enrich clones surviving kanamycin treatment in the lung but sensitive to killing by kanamycin in vitro. After nucleotide sequencing of inserts from these clones, 20 genes belonging to fatty acids metabolism, membrane transport, nitric oxide defence and PE_PGRS/PPE family were identified. Real-time PCR analysis using RNA isolated from M. tuberculosis grown in vitro and from the lungs, confirmed upregulation of genes from 2 to 20-fold in vivo compared to growth in vitro. Several of these select 20 genes were also found upregulated ex vivo in macrophage-like cell line J774A.1, thus, suggesting a correlation in mycobacterial gene expression between ex vivo and in vivo conditions.

C-3 alkyl/arylalkyl-2,3-dideoxy hex-2-enopyranosides as antitubercular agents: synthesis, biological evaluation, and QSAR study.

A series of C-3 alkyl and arylalkyl 2,3-dideoxy hex-2-enopyranoside derivatives were synthesized by Morita-Baylis-Hillman reaction using enulosides 4, 5, and 6 and various aliphatic and aromatic aldehydes. The compounds were evaluated in vitro for the complete inhibition of growth of Mycobacterium tuberculosis H37Rv. They exhibited moderate to good activity in the range of 25-1.56 mug/mL. Among these, 4d, 4h, 5c, and 4hr showed activity at minimum inhibitory concentrations, 3.12, 6.25, 1.56, and 1.56 mug/mL, respectively. These compounds were safe against cytotoxicity in VERO cell line and mouse macrophage cell line J 744A.1. A QSAR analysis by CP-MLR with alignment-free 3D-descriptors indicated the relevance of structure space comparable to the minimum energy conformation (from conformational analysis) of 5c to the activity. The study indicates that the compounds attaining the conformational space of 5c and reflecting some symmetry, minimum eccentricity, and closely placed geometric and electronegativity centers therein are favorable for activity.

Macrophage-specific Mycobacterium tuberculosis genes: identification by green fluorescent protein and kanamycin resistance selection.

Mycobacterium tuberculosis survives and multiplies inside macrophages of its host by modulating the expression of several genes essential for in vivo survival. An in vivo expression system has been developed, based on green fluorescent protein and kanamycin resistance, to identify M. tuberculosis genes which appear to be up-regulated in infected macrophages. A promoter-trap shuttle vector, pLL192, was constructed, containing a streptomycin resistance gene as selection marker and an artificial bicistronic operon composed of the promoterless green fluorescent protein (gfp) gene, followed by the kanamycin resistance gene. A unique BamHI site upstream of the gfp gene allowed for insertion of promoter libraries. The vector was validated by the use of known regulated or constitutive M. tuberculosis promoters. In addition, an M. tuberculosis genomic DNA library was inserted into pLL192 and then introduced into Mycobacterium bovis BCG. The recombinant BCG cells were then used to infect the J774A.1 murine macrophage-like cell line in the presence of kanamycin. Several recombinant BCG cells were thereby selected that were resistant to kanamycin within infected macrophages, but were sensitive to kanamycin when grown in vitro. The kanamycin resistance phenotype was paralleled by the fluorescence phenotype. After nucleotide sequencing, the corresponding genes were identified as mce1A, PE_PGRS63(RV3097c), Rv2232, Rv1026, Rv1635c, viuB, Rv2231(cobC) and Rv0997. Real-time PCR analysis using RNA isolated at various time points from M. tuberculosis and M. bovis BCG grown in vitro and within macrophages, confirmed the up-regulation of these genes. The level of up-regulation varied from 2- to 40-fold in macrophages compared to growth in vitro.

Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice.

Ability of Mycobacterium tuberculosis to survive under oxidative stress in vivo is an important aspect of pathogenesis. Rv3303c gene from M. tuberculosis encodes an NAD(P)H quinone reductase. These enzymes have been shown to manage oxidative stress in other pathogenic bacteria. We have hypothesized that Rv3303c protein will remove reactive oxygen species released by the host and hence reduce oxidative stress to M. tuberculosis. rv3303c was PCR cloned and the purified recombinant enzyme reduced superoxide generator menadione. Antisense and sense RNA constructs of rv3303c were electroporated in M. tuberculosis H37Rv. The transformants were characterized by difference in expression of specific mRNA and protein. Antisense transformants were markedly reduced in virulence as compared to sense transformants as judged by several parameters such as weight and survival of infected mice, growth in vivo, colonization and histopathology of lungs. In the presence of menadione, the sense transformant was more resistant to killing in vitro than the antisense transformant. It may be concluded that the rv3303c gene contributes to virulence of M. tuberculosis in vivo and this might be mediated in part by increased resistance to reactive oxygen intermediates thereby enhancing intracellular growth and colonization.

Identification of a repetitive sequence belonging to a PPE gene of Mycobacterium tuberculosis and its use in diagnosis of tuberculosis.

A repetitive sequence specific to Mycobacterium tuberculosis was isolated from a lambda gt11 library of M. tuberculosis by DNA-DNA hybridization using genomic DNA of M. tuberculosis as probe followed by subtractive hybridization with a cocktail of other mycobacterial DNA. This led to identification of CD192, a 1291 bp fragment of M. tuberculosis containing repetitive sequences, which produced positive hybridization signals with M. tuberculosis DNA within 30 min. Nucleotide sequencing revealed the presence of several direct and inverted repeats within the 1291 bp fragment that belonged to a PPE family gene (Rv0355) of M. tuberculosis. The use of CD192 as a DNA probe for the identification of M. tuberculosis in culture and clinical samples was investigated. The 1291 bp sequence was present in M. tuberculosis, Mycobacterium bovis and M. bovis BCG, but was not present in many of the other mycobacterial strains tested, including M. tuberculosis H37Ra. More than 300 clinical isolates of M. tuberculosis were probed with CD192, and the presence of the 1291 bp sequence was observed in all the clinical strains, including those lacking IS6110. The sequence displayed RFLP among the clinical isolates. A PCR assay was developed which detected M. tuberculosis with 100% specificity from specimens of sputum, cerebrospinal fluid and pleural effusion from clinically diagnosed cases of tuberculosis.