Abstract: BACKGROUND: Mycobacterium tuberculosis (Mtb) causes death of 2-3 million people every year. The persistence of the pathogenic mycobacteria inside the macrophage occurs through modulation of host cell signaling which allows them, unlike the other non-pathogenic species, to survive inside the host. The secretory proteins of M. tuberculosis have gained attention in recent years both as vaccine candidates and diagnostic tools; they target the immune system and trigger a putatively protective response; however, they may also be involved in the clinical symptoms of the disease. RESULTS: Our studies showed that RD-1-encoded secretory protein ESAT-6 is involved in modulation of the mitogen-activated protein (MAP) kinase-signaling pathway inside the macrophage. ESAT-6 induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the cytoplasm but not in the nucleus, which normally is the case for MAP kinases. ESAT-6 also antagonized LPS-induced ERK1/2 phosphorylation in the nucleus. Stimulation of cells by ESAT-6 along with sodium orthovanadate (a tyrosine phosphatase inhibitor) restored phosphorylation of ERK1/2 in the nucleus, suggesting active dephosphorylation of ERK1/2 by some putative phosphatase(s) in the nucleus. Further, ESAT-6 was found to down regulate the expression of LPS-inducible gene c-myc in an ERK1/2-dependent manner. CONCLUSION: This study showed the effect of secretory proteins of M. tuberculosis in the modulation of macrophage signaling pathways particularly ERK1/2 MAP kinase pathway. This modulation appears to be achieved by limiting the ERK1/2 activation in the nucleus which ultimately affects the macrophage gene expression. This could be a mechanism by which secretory proteins of Mtb might modulate gene expression inside the macrophages.

Abstract: Secreted proteins encoded by different regions of difference (RDs) from the genome of Mycobacterium tuberculosis have been considered as attractive candidates for vaccination against tuberculosis owing to their absence in most BCG strains. In this study, the structural gene for the RD2 locus encoding protein CFP21 was PCR amplified and expressed as a fusion protein with hexahistidine residues in Escherichia coli. Expression of CFP21 in E. coli under transcriptional regulation of the T7 promoter yielded a protein located within inclusion bodies. The inclusion bodies were solubilized in the presence of 8M urea and the protein was purified to homogeneity under denaturing conditions at low pH using nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradient dialysis against a decreasing concentration of urea. The purified protein was shown to have esterase activity. CFP21 protein was evaluated for immunogenicity in C57BL/6J mice. We observed an elevated T cell proliferative response and production of IFN-gamma and IL-12 (p40). CFP21 also induced an optimum level of cytotoxic T cell activity and induced a strong humoral response as indicated by higher levels of specific IgG1 and IgG2a antibody isotypes. In addition, a moderate level of protection was observed against experimental tuberculosis. This is the first report describing esterase activity of the M. tuberculosis complex-restricted protein CFP21 and its protective potential against experimental tuberculosis.

Abstract: Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.