Heterogeneity among Homologs of Cutinase-Like Protein Cut5 in Mycobacteria.

The study of genomic variability within various pathogenic and non-pathogenic strains of mycobacteria provides insight into their evolution and pathogenesis. The mycobacterial genome encodes seven cutinase-like proteins and each one of these exhibit distinct characteristics. We describe the presence of Cut5, a member of the cutinase family, in mycobacteria and the existence of a unique genomic arrangement in the cut5 gene of M. tuberculosis (Mtb) strains. A single nucleotide (T) insertion is observed in the cut5 gene, which is specific for Mtb strains. Using in silico analysis and RT-PCR, we demonstrate the transcription of Rv3724/cut5 as Rv3724a/cut5a and Rv3724b/cut5b in Mtb H37Rv and as full length cut5 in M. bovis. Cut5b protein of Mtb H37Rv (MtbCut5b) was found to be antigenically similar to its homologs in M. bovis and M. smegmatis, without any observed cross-reactivity with other Mtb cutinases. Also, the presence of Cut5b in Mtb and its homologs in M. bovis and M. smegmatis were confirmed by western blotting using antibodies raised against recombinant Cut5b. In Mtb H37Rv, Cut5b was found to be localized in the cell wall, cytosol and membrane fractions. We also report the vast prevalence of Cut5 homologs in pathogenic and non pathogenic species of mycobacteria. In silico analysis revealed that this protein has three possible organizations in mycobacteria. Also, a single nucleotide (T) insertion in Mtb strains and varied genomic arrangements within mycobacterial species make Rv3724/Cut5 a potential candidate that can be exploited as a biomarker in Mtb infection.

Recombinant E. coli expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions: a proteome-level study.

Vitreoscilla haemoglobin (VHb) expression in heterologous host was shown to enhance growth and oxygen utilization capabilities under oxygen-limited conditions. The exact mechanism by which VHb enhances the oxygen utilization under oxygen-limiting conditions is still unknown. In order to understand the role of VHb in promoting oxygen utilization, changes in the total protein profile of E. coli expressing the vgb gene under its native promoter was analysed. Two-dimensional difference gel electrophoresis (2D DIGE) was employed to quantify the differentially expressed proteins under oxygen-limiting conditions. Overexpression of proteins involved in aerobic metabolic pathways and suppression of proteins involved in non-oxidative metabolic pathways shown in this study indicates that the cells expressing VHb prefer aerobic metabolic pathways even under oxygen limitation. Under these conditions, the expression levels of proteins involved in central metabolic pathways, cellular adaptation and cell division were also found to be altered. These results imply that Vitreoscilla haemoglobin expression alters aerobic metabolism specifically, in addition to altering proteins involved in other pathways, the significance of which is not clear as of now.

Intracellular growth and survival of Salmonella enterica serovar Typhimurium carrying truncated hemoglobins of Mycobacterium tuberculosis.

Two distantly related truncated hemoglobins (trHbs), HbN and HbO, are produced at different growth stages of Mycobacterium tuberculosis. Oxygen and nitric oxide (NO) binding properties of these trHbs suggest their vital role(s) in adaptation of tubercle bacillus under hypoxic and nitrosative stress conditions. Here, we have demonstrated that HbN of M. tuberculosis provides distinct advantage over HbO in supporting intracellular growth and survival of the heterologous host, Salmonella enterica serovar Typhimurium, during macrophage infection specifically against toxicity of NO. HbN and HbO encoding genes of M. tuberculosis have been expressed in a NO-sensitive hmp mutant of S. enterica serovar Typhimurium that exhibits attenuated growth within the macrophages. Presence of HbN and HbO conferred distinct oxygen dependent NO metabolizing activity to the mutant S. enterica serovar Typhimurium. However, the HbN carrying cells exhibited nearly 2-3-fold higher NO metabolizing activity than the isogenic strain having HbO under aerobic condition. More than half of the NO uptake activity of HbN carrying cells was retained when oxygen level dropped to microaerobic condition. In comparison, NO uptake activity of HbO carrying cells of mutant S. enterica dropped drastically (90%) under similar hypoxic conditions. When internalized by mice peritoneal macrophages, HbN carrying cells exhibited 3- and 4-fold higher survival compared to similarly bound and internalized HbO carrying and control cells, respectively. The protective effect of HbN persisted even after activation of macrophages in the presence of IFN-gamma, whereas, HbO did not show any significant effect on survival of the NO-sensitive hmp mutant of Salmonella. These results provide strong experimental evidence in support of the protective role of HbN against nitrosative stress inside macrophages and suggest that intracellular protection conferred by HbN of M. tuberculosis might not be restricted to its native host only.