Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns.

Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen.

Mycobacterial escape from macrophage phagosomes to the cytoplasm represents an alternate adaptation mechanism.

Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.

Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage.

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.