IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

Abstract Mycobacterium tuberculosis (MTB) adopts a special survival strategy to overcome the killing mechanism(s) of host immune system. Amongst the many known factors, small heat shock protein 16.3 (sHSP16.3) of MTB encoded by gene hspX has been reported to be critical for the survival of MTB. In the present study, the effect of recombinant murine interferon-gamma (rmIFN-γ) and recombinant murine interleukin-10 (rmIL-10) on the expression of gene hspX of MTB in murine macrophage RAW264.7 has been investigated. By real-time RT-PCR, it was observed that three increasing concentrations (5, 25 and 50 ng/ml) of rmIFN-γ significantly up-regulated the expression of hspX whereas similar concentrations of rmIL-10 (5, 25 and 50 ng/ml) significantly down-regulated the hspX expression. This effect was not only dependent on the concentration of the stimulus but this was time-dependent as well. A contrasting pattern of hspX expression was observed against combinations of two different concentrations of rmIFN-γ and rmIL-10. The study results suggest that rIL-10 mediated down-regulation of hspX expression, in the presence of low concentration of rIFN-γ, could be used as an important strategy to decrease the dormancy of MTB in its host and thus making MTB susceptible to the standard anti-mycobacterial therapy used for treating tuberculosis. However, as these are only preliminary results in the murine cell line model, this hypothesis needs to be first validated in human cell lines and subsequently in animal models mimicking the latent infection using clinical isolates of MTB before considering the development of modified regimens for humans.

Whole cell & culture filtrate proteins from prevalent genotypes of Mycobacterium tuberculosis provoke better antibody & T cell response than laboratory strain H37Rv.

Background & objectives: The immune responses to different antigens of Mycobacterium tuberculosis H37Rv vary from patient to patient with tuberculosis (TB). Therefore, significant difference might be documented between the H37Rv with long histories of passages and recent clinical isolates of M. tuberculosis. In the present study, immune response of TB patients and healthy controls against 39 clinical M. tuberculosis isolates was correlated with laboratory strain H37Rv. Methods: The antibody response was studied coating whole cell extracts and culture filtrate proteins of M. tuberculosis isolates and laboratory strain H37Rv by enzyme linked immunosorbent assay (ELISA). Lymphoproliferation was studied by incorporation of tritiated thymidine and cytokines (IFN-γ and IL-4) by using commercially available kits. Results: Sero-reactivity to whole cell extract (WCE) of 11 clinical isolates was higher with pooled serum and individual's serum from tuberculosis patients showed significant reactivity (P<0.05) to ten of these isolates using ELISA. Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls. Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response. Interpretation & conclusion: Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H37Rv laboratory strain. Further studies need to be done to purify and identify the dominant protein (s) using whole cell extract and culture filtrates from these immunologically relevant clinical M. tuberculosis isolates, which will be worthwhile to find out pathogenic factors, potential diagnostic markers and protective molecules for tuberculosis.

A simple and rapid method of sample preparation from culture filtrate of M. tuberculosis for two-dimensional gel electrophoresis

Sample preparation for Two-dimensional gel electrophoresis (2DE) is tedious and not sufficient to provide a comparative profile of secreted proteins for various strains of M. tuberculosis. High lipid content in mycobacteria limits the use of common methods as it can hinder the 2DE run. This study highlights the significance of SDS-TCA procedure over common used methods for the preparation of sample from culture filtrate as well as other proteinaceous fluids.

Functional characterization of the phospholipase C activity of Rv3487c and its localization on the cell wall of Mycobacterium tuberculosis.

Mycobacterium tuberculosis survives and persists for prolonged periods within its host in an asymptomatic,latent state and can reactivate years later if the host's immune system weakens. The dormant bacilli synthesize and accumulate triacylglycerol, reputed to be an energy source during latency. Among the phospholipases, phospholipase C plays an important role in the pathogenesis. Mutations in a known phospholipase C, plcC, of M.tuberculosis attenuate its growth during the late phase of infection in mice. Hydrolysis of phospholipids by phospholipase C generates diacylglycerol, a well-known signalling molecule that participates in the activation of extracellular signal-regulated kinases (ERK) through protein kinase C leading to macrophage activation. In the present study, we show that M.tuberculosis possesses an additional cell wall-associated protein, Rv3487c, with phospholipase C activity. The recombinant Rv3487c hydrolyses the substrate phosphatidylcholine and generates diacylglycerol by removing the phosphocholine. Furthermore, Rv3487c is expressed during infection as it exhibits significant humoral immunoreactivity with sera from children with tuberculosis, but not with that from adult patients.