Localized cutaneous leishmaniasis (LCL) in India is due mostly to Leishmania tropica. It is mainly endemic in the deserts of Rajasthan. Recently, Himachal Pradesh has been identified as a new endemic focus for the disease. In the last few years, the number of new cases has been increasing almost to epidemic proportions. This report presents the preliminary findings of clinico-epidemiologic and investigative results of 161 new localized cases of LCL seen between May 2001 and December 2003. The study populaton was composed of 80 males and 81 females between 10 months and 75 years of age. All were indigenous to the sub-alpine valley along the Satluj River in the mountainous region of the Kinnaur District (altitude = 700-2,900 meters). Most patients were seen from April to September and had 1-8 lesions (duration = 1-6 months) that involved mainly the face. Tissue smears were positive for amastigotes in 37% and histopathology showed non-caseating epitheloid cell granuloma in 77% of the cases. Analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ribosomal gene region of 10 biopsy specimens showed amplicons indistinguishable from L. donovani in eight cases and L. tropica in two cases. Leishmania was cultured on modified Nicole-Novy-McNeal (NNN) medium containing RPMI 1640 medium and heat-inactivated fetal bovine serum from 13 of 38 biopsy samples. Three of these isolated strains were identified as L. donovani while a fourth was L. tropica by PCR-RFLP of the ribosomal internal transcribed spacer region. One strain had a gp63 sequence identical to that of east African strains. Another strain had a unique gp63 sequence that has not been found in L. donovani complex strains. Sand flies trapped in the cattle sheds of a few patients were identified as Phlebotomus longiductus (Parrot 1928). Treatment with intralesional sodium stibogluconate was effective in all patients without any major side effects. One patient developed lupoid leishmaniasis that responded to higher dose of sodium stibogluconate. Though rarely reported as a cause of LCL, L. donovani seems to be the predominant pathogen in this new focus of cutaneous leishmaniasis. Phlebotomus longiductus is a possible vector, albeit based on circumstantial evidence.

PE and PPE proteins appear to be important for virulence and immunopathogenicity in mycobacteria, yet the functions of the PE/PPE domains remain an enigma. To decipher the role of these domains, we have characterized the triacylglycerol (TAG) hydrolase LipY from Mycobacterium tuberculosis, which is the only known PE protein expressing an enzymatic activity. The overproduction of LipY in mycobacteria resulted in a significant reduction in the pool of TAGs, consistent with the lipase activity of this enzyme. Unexpectedly, this reduction was more pronounced in mycobacteria overexpressing LipY lacking the PE domain [LipY(⌬PE)], suggesting that the PE domain participates in the modulation of LipY activity. Interestingly, Mycobacterium marinum contains a protein homologous to LipY, termed LipY mar , in which the PE domain is substituted by a PPE domain. As for LipY, overexpression of LipY mar in Mycobacterium smegmatis significantly reduced the TAG pool, and this was further pronounced when the PPE domain of LipY mar was removed. Fractionation studies and Western blot analysis demonstrated that both LipY and LipY(⌬PE) were mainly present in the cell wall, indicating that the PE domain was not required for translocation to this site. Furthermore, electron microscopy immunolabeling of LipY(⌬PE) clearly showed a cell surface localization, thereby suggesting that the lipase may interact with the host immune system. Accordingly, a strong humoral response against LipY and LipY(⌬PE) was observed in tuberculosis patients. Together, our results suggest for the first time that both PE and PPE domains can share similar functional roles and that LipY represents a novel immunodominant antigen.

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.

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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 ϳ80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ϳ250 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.