Mycobacterium tuberculosis persists primarily in macrophages after infection and manipulates the host defence pathways in its favour. 2D gel electrophoresis results showed that vimentin, an intermediate filament protein, is downregulated in macrophages infected with live Mycobacterium tuberculosis H37Rv when compared to macrophages infected with heat- killed H37Rv. The downregulation was confirmed by Western blot and quantitative RT-PCR. Besides, the expression of vimentin in avirulent strain, Mycobacterium tuberculosis H37Ra- infected macrophages was similar to the expression in heat-killed H37Rv- infected macrophages. Increased expression of vimentin in H₂O₂- treated live H37Rv-infected macrophages and decreased expression of vimentin both in NAC and DPI- treated heat-killed H37Rv-infected macrophages showed that vimentin expression is positively regulated by ROS. Ectopic expression of ESAT-6 in macrophages decreased both the level of ROS and the expression of vimentin which implies that Mycobacterium tuberculosis-mediated downregulation of vimentin is at least in part due to the downregulation of ROS by the pathogen. Interestingly, the incubation of macrophages with anti-vimentin antibody increased the ROS production and decreased the survival of H37Rv. In addition, we also showed that the pattern of phosphorylation of vimentin in macrophages by PKA/PKC is different from monocytes, emphasizing a role for vimentin phosphorylation in macrophage differentiation.

Anaplasma phagocytophilum is an obligate intracellular Gram-negative bacterium that causes human granulocytic anaplasmosis. Assessing the number of these bacteria is important for in vitro and in vivo infection studies. Colony count is used to set references for the multiplicity of infections in the case of culturable bacteria. However, the number of bacteria present inside the host cells, in which the bacteria are maintained, can be considered in the case of obligate intracellular bacteria. McFarland standards are a series of turbidity-based standards used to visually assess the approximate number of culturable bacteria. The turbidity of each standard can be related to their respective absorbances or optical densities (ODs). In this study, we describe a simple method to assess the approximate number of A. phagocytophilum based on McFarland standards. The ODs of cell-free crude extracts of A. phagocytophilum were used to assess the approximate number of bacteria while considering that the cell debris also contributes to the ODs. The consistency of this method was also tested using the bacterial cultures grown at different times. In summary, we provide a simple method to estimate the number of obligate intracellular bacteria for use in in vitro infection studies.

Cell culture is an important prerequisite for many of the basic and applied researches in life sciences and contamination is a major problem faced by the researchers in maintaining healthy cell lines. We noted a dot like contamination in our THP1 cell line, which does not make the culture medium turbid as fast as other common contaminants and remains in the culture for many days before it makes the culture unusable. The contaminant was identified as a member of the genus Brevibacterium and we could find that the antibiotic rifampicin effectively suppresses the growth of the bacterium.

Two dimensional gel electrophoresis (2DE) resolves a mixture of proteins based on both isoelectric point and molecular weight of the individual proteins. Even when we followed a standard protocol for 2DE we got lesser number of proteins focused especially in the basic region of the IPG strip. Since the common troubleshooting measures did not solve the problem we replaced the protease inhibitor cocktail in the lysis buffer with PMSF which resulted in an ideal protein map following 2DE. We also found that the presence of the cocktail results in skewing of the mass spectra of a purified protein which eventually resulted in incorrect identification of the protein by MASCOT search. Later we found that dimethyl sulfoxide, the solvent of protease inhibitor cocktail also resulted in focusing of lesser number of proteins. Addition of 1% dimethyl sulfoxide to bovine serum albumin resulted in lesser sequence coverage for the protein by LC-MS/MS. Also, dimethyl sulfoxide was found to decrease the intensity value of the dominant peptide in a fraction when MALDI-TOF-TOF was done. Even though we do not provide a reason behind our observations, we guess dimethyl sulfoxide might have changed the charge distribution of peptides or proteins resulting in the peculiar observations we had.

Mycobacterium tuberculosis when phagocytosed by macrophages is not cleared completely and many of the bacteria remain in phagosomes indefinitely. In this study we considered abnormal retention of filamentous actin on early phagosomes contributing to defective phagosome acidification. The actin depolymerizing protein cofilin1 was found downregulated in macrophages infected with virulent M. tuberculosis. Also, phosphocofilin1, the inactive form of cofilin1, which leads to retention of filamentous actin, and the total filamentous actin itself were found upregulated in macrophages infected with virulent M. tuberculosis . Over expression of constitutively active cofilin1 in macrophages was found to decrease the level of filamentous actin and increase phagosome acidification when infected with virulent M. tuberculosis . The anticancer drug sorafenib which activates cofilin1 in PI3K dependent manner was also found to decrease the filamentous actin level and increase phagosome acidification. Cofilin1, known to be positively regulated by superoxide was found to be downregulated by ESAT-6 of M. tuberculosis where the latter is known to reduce ROS in macrophages. Ectopic expression of ESAT-6 in macrophages was found to downregulate cofilin1, increase filamentous actin and to transform the macrophages more spindle shaped. ESAT-6 was also found to decrease phagosome acidification in macrophages infected with an avirulent M. tuberculosis strain. Finally, this study proposes a role for the amino acid methionine in resisting ROS by creating M93 mutants of ESAT-6.