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Molecular Characterization of Mycobacterium tuberculosis H37Rv Protein(s) involved in Persistence/Latency

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dc.contributor.author Kaur, Satinder
dc.contributor.author Srivastava, Ranjana (Guide)
dc.date.accessioned 2015-10-30T05:41:03Z
dc.date.available 2015-10-30T05:41:03Z
dc.date.issued 2010
dc.identifier.uri http://hdl.handle.net/123456789/1594
dc.description Guide- Dr. Ranjana Srivastava, Ph.d Thesis Submitted to JNU, New Delhi in 2010. en
dc.description.abstract Tuberculosis is the world‘s longest running catastrophe killing more than two hundred people every hour and more than five thousand everyday. According to WHO, someone is infected every second (Dye et al., 1999). Mycobacterium tuberculosis, the causative agent of TB, in most cases gets cleared immediately by the host immune response in macrophages. Active TB may develop within a finite time frame (1-3 years), while in the rest clinically latent infection is maintained that can be reactivated under immuno-compromised conditions as generated during HIV infection, malnutrition, use of steroids or immuno-suppressive medications or advanced age (Flynn et al., 2001). Persistence of TB despite prolonged chemotherapy is due to a subpopulation of non-replicating or ‗dormant‘ bacilli. Dormant mycobacteria are phenotypically drug resistant and are responsible for latent TB infection (Wayne, 1994). Latent mycobacteria and drug resistant mycobacteria are two important aspects of life threatening disease (Pai et al., 2000). Latency may be defined as a reversible state of low metabolic activity in which cells can persist for extended periods without division. It is a survival strategy, a state of equilibrium in which the bacilli establishes a persistent infection and resists elimination by host immune system. Latent TB infection presents one of the major obstacles in gaining control over TB worldwide. Lack of information about the state of the bacilli during clinical latency hinders our ability to model latent TB in laboratory settings. However both in vitro (Waynes and Hayes 1996; Betts et al., 2002) and in vivo systems have been developed which contribute to our current understanding of latency. Animal models of infected mice which develop chronic infection similar to latent human TB are being explored (McCune et al., 1956). These models are helpful in identifying new drug targets for persistent M. tuberculosis in the recent past (Cunnigham and Spreadbury, 1998; Hu and Coats, 1999; Boon et al., 2001). Results of a worldwide Survey showed that 2 percent of M. tuberculosis isolates were extensively drug resistant (XDR-TB) i.e., strain resistant to at least rifampicin and isoniazid, a fluoroquinolone and one or more of the second line drugs also (Karklina et al., 1996) and 4.3 percent cases were multidrug resistant (MDR-TB), i.e., strains resistant to first line drugs- rifampicin and isoniazid (Flanders, 1994). Widespread use of Bacilli Calmette Guerin (BCG) vaccine, which is the only available TB vaccine, has only limited impact on the global burden of TB. Vaccination with BCG induces variable 0-80% protection against pulmonary TB. Therefore there is a need to boost any pre-existing immunity that BCG vaccination has provided. These problems require new measures for effective TB control. The search for new anti-TB compounds and targets for drug development are urgently required. These targets could be bacterial virulence factors or factors essential for normal growth and survival of bacteria. The study of virulence determinants associated with persistence and reactivation has assumed great significance. Although a good deal of information is known about host factors limiting M. tuberculosis growth during latent stages; but little is known about the bacterial determinants required for persistence infection. A substantial barrier in our understanding of these factors has been an inability to experimentally recreate conditions encountered by M. tuberculosis during periods of latency in the host i.e. lack of an ideal in-vitro model of persistence that can be used for identification of virulence determinants. In future some of these genes and the proteins they encode, as well as new ones, should provide new bacterial targets that can be used for creating vaccines and drugs as well as more selective diagnostic reagents. There are a number of questions which remain to be answered like- What are the factors which help mycobacteria to survive in face of a strong immune response? What are the mechanism(s) and molecular factors that contribute to the establishment of latent/ persistent infection? Several approaches have been used to identify the possible therapeutic targets. One of the technologies called ―Transposon insertion mutagenesis‖ was used in our laboratory for identification of gene affecting virulence. A library of TnphoA insertion mutants of M. fortuitum was constructed by using a broad host range plasmid pRT291 (kmr) carrying transposon TnphoA A mutant was selected on the basis of attenuation in virulence and persistence through in-vivo screening of library of mutants in mice (Parti et al., 2005). This transposon insertion segment was cloned and sequenced and was confirmed that the disrupted ORF in mutant (MT12) was homologous to rv3291c gene of M. tuberculosis (Srivastava et al., 2009). MT12 of M. fortuitum showed 99% sequence homology to the Rv3291c of M. tuberculosis H37Rv (Parti et al., 2009). The MT12 mutant could be complemented by rv3291c of M. tuberculosis illustrating its role in virulence and persistence in M. fortuitum (Srivastava et al., 2009). The objective of present study was to characterize the mycobacterial protein(s) involved in persistence or latency, the gene chosen for this study was rv3291c (lrpA) – which codes for a leucine responsive regulatory protein. Rv3291c (LrpA) is annotated as a probable transcription factor in the M. tuberculosis H37Rv genome database (www.sanger.ac.uk). Genome analysis has revealed that members of the Lrp and AsnC family of transcriptional regulators are widely distributed among prokaryotes, both bacteria and archaea (Brinkman et al., 2003). The Lrp/AsnC family has also been referred to as feast famine regulatory proteins (FFRPs), because of their potential role in detecting and adapting to starvation conditions (Suzuki, 2003). Like most prokaryotic transcriptional regulators, Lrp-like regulators consists of a DNA-binding domain and a ligand- binding domain. The Escherichia coli Lrp was the best studied member of the Lrp family. It has been reported that Lrp affects transcription of 10% of all E. coli genes, among which the most of them are expressed upon entrance into stationary phase (Tani et al., 2002). To characterize the Rv3291c of M. tuberculosis H37Rv, we initiated studies using molecular and web based in-silico approaches. In order to study its expression in different growth conditions, the gene was PCR amplified from genomic DNA of M. tuberculosis H37Rv and cloned in T7 expression vector to over express the recombinant protein with His-tag. Conditions were optimized to produce recombinant protein in soluble fraction and recombinant protein was purified by affinity chromatography using Ni++-NTA column. Polyclonal antibody against recombinant protein was raised in rabbit to evaluate the expression of the gene under different culture and stress conditions. Further the results of expression analysis were validated by real-time PCR. In order to evaluate the expression analysis of rv3291c at promoter level, a promoter less E.coli–mycobacterial shuttle vector containing the reporter gene β-galactosidase was constructed and validated by cloning the known mycobacterial promoters (e.g. Phsp60 cassette from M. bovis BCG). Construction of transcriptional fusion of rv3291c upstream promoter regions (deletion constructs) in mycobacterial- E.coli shuttle vector pMV206lacZ was carried out to define the minimum regulatory region (P3291c) which is required for expression of β-galactosidase. 5‘- RACE was carried out to map the TSP (Transcription Start Point) of rv3291c operon. By knowing the TSP (+1), we can define the other core promoter elements (-10 region, -35 region etc.) and explore them further. Another study was proposed related to VNTR 3690 (locus located in intergenic region between rv3304 and rv3303c) polymorphism in Indian clinical isolates of M. tuberculosis and its possible function in regulation of gene expression. Variable-number tandem repeat (VNTRs) occur throughout the chromosome of M. tuberculosis. Although these polymorphic VNTRs have proved to be useful tools in molecular epidemiology, their biological significance is less well understood. The presence and variability of VNTR 3690 in the intergenic region led us to investigate the hypervariability of this VNTR locus in Indian clinical isolates of M. tuberculosis and to address the functional role of the repeat variability in transcription by quantitative RT-PCR (qRT-PCR) in M. tuberculosis H37Rv which contain four copies of repeat and M. bovis BCG with one copy of repeat and by fusing intergenic regions containing four and one repeats of VNTR 3690 with a reporter GFP gene (gfp) and assaying for GFP expression. It is assumed that the data obtained through present work will add up a little to the existing knowledge about survival strategies of mycobacteria during persistent state of infection. The thesis is divided into following chapters: Chapter I: Describes the review of literature. Chapter II: Describes various materials and methods used in present investigation. Chapter III: Results obtained during the investigation. Chapter IV: Contains a comprehensive discussion based on the results obtained. Chapter V: Lists the complete references cited in the thesis, followed by an appendix. en
dc.format.extent 2122019 bytes
dc.format.mimetype application/pdf
dc.language.iso en en
dc.relation.ispartofseries CSIR-CDRI Thesis no. K-116 (2010) en
dc.subject Mycobacterium tuberculosis en
dc.subject H37Rv Protein(s) en
dc.title Molecular Characterization of Mycobacterium tuberculosis H37Rv Protein(s) involved in Persistence/Latency en
dc.type Thesis en

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    Ph D Theses submitted by the Research Scholars of CDRI, Lucknow

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