Devendra Singh Chauhan

Proteogenomic Analysis of Mycobacterium tuberculosis By High Resolution Mass Spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ∼80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ∼250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

Predominance of Ancestral Lineages of Mycobacterium tuberculosis in India

Molecular epidemiologic findings suggest an ancient focus of TB.

Microarray Analysis of Efflux Pump Genes in Multidrug-Resistant Mycobacterium tuberculosis During Stress Induced by Common Anti-Tuberculous Drugs

Treatment of multidrug-resistant tuberculosis has become one of the major problems in public health. Understanding the molecular mechanisms of drug resistance has been central to tuberculosis research in recent times. DNA microarray technology provides the platform to study the genomic variations related to these mechanisms on a comprehensive level. To investigate the role of efflux pumps in drug resistance, we have constructed a custom DNA microarray containing 25 drug efflux pump genes of Mycobacterium tuberculosis (Indian Patent file no. 2071/DEL/2007) and monitored changes in the expression of these genes on exposure of common anti-tuberculous drugs. Expression profiling of efflux pump genes in multidrug-resistant M. tuberculosis isolates showed overexpression of 10 genes following exposure to various anti-tuberculous drugs. Although two of these genes (Rv3065 and Rv2938) have already been reported to be active drug efflux pumps in M. tuberculosis in earlier studies, the increased activities of other eight efflux pump genes (Rv1819, Rv2209, Rv2459, Rv2477c, Rv2688, Rv2846, Rv2994, and Rv3728) have been demonstrated in multidrug-resistant isolates by us for the first time. After confirmation of differential expressions of these genes by real-time reverse transcription polymerase chain reaction, it was observed that a simultaneous overexpression of efflux pump genes Rv2459, Rv3728, and Rv3065 was associated with resistance to the combination of isoniazid and ethambutol, and these drugs, along with streptomycin, were identified to group together, where efflux-mediated drug resistance appears to be important in M. tuberculosis and follows a constant pattern of induction in multidrug-resistant isolates. Isoniazid and ethambutol combination was also found to be affected in 10% (6/60) of the clinical isolates in the presence of carbonyl cyanide m-chloro phenylhydrazone in resazurin microtitre plate assay, supporting the role of efflux pumps in the resistance to these drugs. Overexpression of two of the genes (Rv2477 and Rv2209) has also been observed with ofloxacin stress in M. tuberculosis.

Current understanding on micro RNAs and its regulation in response to Mycobacterial infections

MicroRNAs (miRNAs) are evolutionarily conserved, naturally abundant, small, regulatory non-coding RNAs that inhibit gene expression at the post-transcriptional level in a sequence-specific manner. Due to involvement in a broad range of biological processes and diseases, miRNAs are now commanding considerable attention. Although much of the focus has been on the role of miRNAs in different types of cancer, recent evidence also points to a critical role of miRNAs in infectious disease, including those of bacterial origin. Now, miRNAs research is exploring rapidly as a new thrust area of biomedical research with relevance to deadly bacterial diseases like Tuberculosis (caused by Mycobacterium tuberculosis). The purpose of this review is to highlight the current developments in area of miRNAs regulation in Mycobacterial diseases; and how this might influence the diagnosis, understanding of disease biology, control and management in the future.

Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing

Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian-Delhi family (CAS1_Del) (37%), East African-Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India.

Typing of drug resistant isolates of Mycobacterium tuberculosis from India using the IS6110 element reveals substantive polymorphism

We investigated IS6110 polymorphism in clinical isolates of Mycobacterium tuberculosis from patients attending the outpatient department at various hospitals in northern India. DNA fingerprinting of 126 clinical isolates of M. tuberculosis was carried out using restriction fragment length polymorphism (RFLP) associated with the IS6110 element in M. tuberculosis genomes. A substantive degree of polymorphism was evident in the MDR M. tuberculosis isolates. The number of bands in the fingerprints varied from 0 to 19. However, the lack of common bands made it difficult to cluster the majority of these isolates. We were also unable to associate drug resistance with IS6110 copy number. Specific regions of the gyrA and katG genes from a representative number of these isolates were sequenced to determine the genotype. The majority of the isolates analyzed were found to belong to Group 1, indicating that these strains were evolutionarily older. We find no evidence of the W strain, prevalent in the US, in our study. The epidemiological patterns of the various strains in India seem to be very complex, as reflected by the presence of a large number of different strains (types) within north India.

Identification of Mycobacterium tuberculosis genes preferentially expressed during human infection.

The identification of Mycobacterium tuberculosis genes, specifically expressed during infection is a key step in understanding molecular mechanism of mycobacterial pathogenesis. Such genes likely encode proteins required for mycobacteriums survival and progressive infection within the host. In this study, we applied in-vivo-induced antigen technology (IVIAT) to M. tuberculosis and identified 11 putative in-vivo induced genes encoding for immunogenic proteins of diverse functions; these included transcriptional regulators (Rv1460 and Rv2565), biosynthesis and macromolecule metabolism (leuD, guaB1, plcC, hupB and glyS), polyketide synthases (pks6 and pks9), cell processes (ctpA) and one with unknown function (Rv3701c). Quantitative real time-PCR analysis of these genes in the specimens obtained from TB patients demonstrated induced expression of eight genes as compared with bacteria grown in-vitro. In addition, distribution of these genes in different strains of M. tuberculosis was analyzed using PCR and their nucleotide sequence alignments and they were found to be widely distributed among M. tuberculosis isolates including multiple-drug resistant (MDR) and extensively-drug resistant (XDR). This study identified several antigenic determinants of M. tuberculosis expressed during infection, which might help pathogens adapt to or counter hostile environments and suggesting their role during disease process.

Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India

ABSTRACT For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.

Mycobacterium indicus pranii as stand-alone or adjunct immunotherapeutic in treatment of experimental animal tuberculosis.

Background & objectives: Mycobacterium w (M.w) is a saprophytic cultivable mycobacterium and shares several antigens with M. tuberculosis. It has shown good immunomodulation in leprosy patients. Hence in the present study, the efficacy of M.w immunotherapy, alone or in combination with multi drug chemotherapeutic regimens was investigated against drug sensitive M. tuberculosis H37Rv and three clinical isolates with variable degree of drug resistance in mice. Methods: BALB/c mice were infected with M. tuberculosis H37Rv (susceptible to all first and second line drugs) and three clinical isolates taken from the epository of the Institute. The dose of 200 bacilli was used for infection via respiratory route in an aerosol chamber. Chemotherapy (5 days/wk) was given one month after infection and the vaccinated group was given a dose of 1×107 bacilli by subcutaneous route. Bacterial load was measured at 4 and 6 wk after initiation of chemotherapy. Results: M.w when given along with chemotherapy (4 and 6 wk) led to a greater reduction in the bacterial load in lungs and other organs of TB infected animals compared to. However, the reduction was significantly (P<0.05) more in terms of colony forming units (cfu) in both organs (lungs and spleen). Conclusion: M.w (as immunomodulator) has beneficial therapeutic effect as an adjunct to chemotherapy.

Determination of drug susceptibility patterns and genotypes of Mycobacterium tuberculosis isolates from Kanpur district, North India.

BACKGROUND AND OBJECTIVES Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of tuberculosis epidemiology. This study was done to characterize prevalent M. tuberculosis genotypes in a defined area of Kanpur district, North India by spoligotyping and IS6110-Restriction Fragment Length Polymorphism (RFLP) and to correlate the genotypes identified with their drug susceptibility patterns. METHODS Ninety-eight patients had clinical features suggestive of pulmonary tuberculosis (PTB) and out of them, 22 were new smear positive PTB (CAT I DOTS), 48 smear positive re-treatment, defaulters and CAT I failure PTB (CAT II DOTS) and 28 new smear negative PTB (CAT III). Out of them, sputum culture was positive for M. tuberculosis in 74 cases. DNA was extracted from growth on Lowenstein-Jensen slants and subjected to spoligotyping. Clusters were subsequently analyzed with IS6110 RFLP. Drug susceptibility testing was done for rifampicin, isoniazid, ethambutol, ofloxacin, streptomycin and kanamycin. RESULTS Thirty-seven spoligo patterns were observed. Predominant spoligotypes belonged to Central Asian Delhi family (33.78%), Beijing family (10.8%), East African-Indian family (5.4%), T1 family (5.4%) and U family (4.1%). RFLP analysis revealed 66% isolates had more than 10 IS6110 copies while 17% isolates each had low (1-5) and intermediate (6-9) copy numbers. All the isolates clustered by spoligotyping were identified unique by RFLP. Resistance to at least one drug was present in 35 (47.3%), out of which 8 patients belonged to CAT I and 27 to CAT II. Eleven (14.86%) were multi drug-resistant (MDR) and out of them, 6 (54.5%) isolates were of ST1/Beijing family. MDR-TB was significantly higher in Beijing strain than others (p<0.0001), however, most (83%) were from previously treated cases and thus can not be linked with recent transmission. CONCLUSION This approach of molecular typing appears promising and merits further evaluation to study dynamics of TB transmission specially in India.