RESUMEN
Tuberculosis (TB) is the deadly infectious disease challenging the public health globally and its impact is further aggravated by co-infection with HIV and the emergence of drug resistant strains of Mycobacterium tuberculosis. In this study, we attempted to characterise the Rv2004c encoded protein, a member of DosR regulon, for its role in drug resistance. In silico docking analysis revealed that Rv2004c binds with streptomycin (SM). Phosphotransferase assay demonstrated that Rv2004c possibly mediates SM resistance through the aminoglycoside phosphotransferase activity. Further, E. coli expressing Rv2004c conferred resistance to 100µM of SM in liquid broth cultures indicating a mild aminoglycoside phosphotransferase activity of Rv2004c. Moreover, we investigated the role of MSMEG_3942 (an orthologous gene of Rv2004c) encoded protein in intracellular survival, its effect on in-vitro growth and its expression in different stress conditions by over expressing it in Mycobacterium smegmatis (M. smegmatis). MSMEG_3942 overexpressing recombinant M. smegmatis strains grew faster in acidic medium and also showed higher bacillary counts in infected macrophages when compared to M. smegmatis transformed with vector alone. Our results are likely to contribute to the better understanding of the involvement of Rv2004c in partial drug resistance, intracellular survival and adaptation of bacilli to stress conditions.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Macrófagos/microbiología , Mycobacterium smegmatis/efectos de los fármacos , Proteínas Quinasas/genética , Estreptomicina/farmacología , Proteínas de Unión al ADN , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Humanos , Kanamicina Quinasa/metabolismo , Simulación del Acoplamiento Molecular , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Unión Proteica , Regulón , Células THP-1RESUMEN
Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κß. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.
Asunto(s)
Proteínas Bacterianas/genética , Citocinas/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Nitrorreductasas/genética , Proteínas Quinasas/genética , Regulón , Transducción de Señal , Receptor Toll-Like 2/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ADN , Regulación Bacteriana de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 2/química , Receptor Toll-Like 2/genéticaRESUMEN
We report whole-genome sequences of two clinical isolates of Mycobacterium tuberculosis isolated from patients in Odisha, India. The sequence analysis revealed that these isolates are of an ancestral type and might represent some of the "pristine" isolates in India that have not admixed with other lineages.
