B S Srivastava

Central Drug Research Institute, Lakhnau, Uttar Pradesh, India

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Publications (67)164.17 Total impact

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    ABSTRACT: 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.
    Tuberculosis (Edinburgh, Scotland) 01/2014; · 2.54 Impact Factor
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    ABSTRACT: 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 (CC50) ≥500μg/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.
    International journal of antimicrobial agents 05/2013; · 3.03 Impact Factor
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    ABSTRACT: 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. 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. 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. The study established acute and persistent infection in mice and identified two promising anti-M. fortuitum compounds with a selectivity index >10.
    Journal of Antimicrobial Chemotherapy 02/2012; 67(5):1188-97. · 5.34 Impact Factor
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    ABSTRACT: Authors' response: on sialic acid transport and utilization by Vibrio cholerae.
    Microbiology 10/2011; · 3.06 Impact Factor
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    ABSTRACT: 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.
    Microbiology 08/2011; 157(Pt 11):3180-6. · 3.06 Impact Factor
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    ABSTRACT: 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.
    Vaccine 06/2011; 29(29-30):4754-60. · 3.77 Impact Factor
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    ABSTRACT: 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.
    European journal of medicinal chemistry 03/2011; 46(6):2217-23. · 3.27 Impact Factor
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    ABSTRACT: 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.
    Microbiology 01/2011; 157(Pt 1):38-46. · 3.06 Impact Factor
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    ABSTRACT: 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.
    Microbiology 09/2010; 157(Pt 1):29-37. · 3.06 Impact Factor
  • Ravi Kr Gupta, Brahm S Srivastava, Ranjana Srivastava
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    ABSTRACT: 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.
    Microbiology 09/2010; 156(Pt 9):2714-22. · 3.06 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Journal of Antimicrobial Chemotherapy 09/2009; 64(4):774-81. · 5.34 Impact Factor
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    ABSTRACT: 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.
    Journal of Medical Microbiology 07/2009; 58(Pt 6):798-805. · 2.30 Impact Factor
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    ABSTRACT: 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.
    FEMS Immunology & Medical Microbiology 06/2009; 56(2):179-84. · 2.68 Impact Factor
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    ABSTRACT: 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.
    Microbial Pathogenesis 10/2008; 45(5-6):370-6. · 1.97 Impact Factor
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    ABSTRACT: The present invention relates to the use of extracts of Salicornia species for the manufacture of an agent having anti-�tubercular activity. The present invention also refers to a process for the preparation of novel extracts of Salicornia species with anti-�tubercular activity and also to its use for treatment of tuberculosis.
    Ref. No: EP 1,684,778 B1, Year: 07/2008