Parvez Akhtar

Publications (5)16.07 Total impact

• Article: Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis.

  Parvez Akhtar, Saleem A Khan
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  ABSTRACT: The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid.
  Plasmid 01/2012; 67(2):111-7. · 1.52 Impact Factor
• Article: Variable-number tandem repeat 3690 polymorphism in Indian clinical isolates of Mycobacterium tuberculosis and its influence on transcription.

  Parvez Akhtar, Sarman Singh, Pablo Bifani, Satinder Kaur, Brahm S Srivastava, Ranjana Srivastava
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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. · 2.50 Impact Factor
• Article: The tubulin-like RepX protein encoded by the pXO1 plasmid forms polymers in vivo in Bacillus anthracis.

  Parvez Akhtar, Syam P Anand, Simon C Watkins, Saleem A Khan
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  ABSTRACT: Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.
  Journal of bacteriology 03/2009; 191(8):2493-500. · 3.94 Impact Factor
• Article: GTP-dependent polymerization of the tubulin-like RepX replication protein encoded by the pXO1 plasmid of Bacillus anthracis.

  Syam P Anand, Parvez Akhtar, Eowyn Tinsley, Simon C Watkins, Saleem A Khan
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  ABSTRACT: RepX protein encoded by the pXO1 plasmid of Bacillus anthracis is required for plasmid replication. RepX harbours the tubulin signature motif and contains limited amino acid sequence homology to the bacterial cell division protein FtsZ. Although replication proteins are not known to polymerize, here we show by electron microscopy that RepX undergoes GTP-dependent polymerization into long filaments. RepX filaments assembled in the presence of GTPgammaS were more stable than those assembled in the presence of GTP, suggesting a role for GTP hydrolysis in the depolymerization of the filaments. Light scattering studies showed that RepX underwent rapid polymerization, and substitution of GTP with GTPgammaS stabilized the filaments. RepX exhibited GTPase activity and a mutation in the tubulin signature motif severely impaired its GTPase activity and its polymerization in vitro. Unlike FtsZ homologues, RepX harbours a highly basic carboxyl-terminal region and exhibits GTP-dependent, non-specific DNA binding activity. We speculate that RepX may be involved in both the replication and segregation of the pXO1 plasmid.
  Molecular Microbiology 03/2008; 67(4):881-90. · 5.01 Impact Factor
• Article: Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice.

  Parvez Akhtar, S Srivastava, Anil Srivastava, Mukesh Srivastava, Brahm S Srivastava, Ranjana Srivastava
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  ABSTRACT: 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.
  Microbes and Infection 8(14-15):2855-62. · 3.10 Impact Factor