The role of Rv1860 from M. tuberculosis in modulating the host immune response

Abstract

Tuberculosis continues to prevail as the major cause of mortality from any single pathogen around the world, despite implementation of control programs and the availability of effective drugs and the vaccine BCG. Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only vaccine available for clinical use and protects children against disseminated disease; however, it has shown variable levels of efficacy against pulmonary TB in adults. The drawbacks of anti-TB chemotherapy such as the inability of currently used drugs to kill bacteria that are “dormant”, long term treatment required for the current drugs, and emergence of multidrug resistant M. tuberculosis (Mtb) pose an urgent need for a more effective vaccine against TB. Thus, one of the challenges is to develop effective TB vaccines which can confer long term protection against pulmonary TB and also better understanding of protective immune response against TB is essential to develop better vaccines. The general aim of the thesis was to understand role of Mtb Rv1860 in modulating the host macrophage immune response. Macrophages are the first line of defense against Mtb, but Mtb continues to reside in them for years. Macrophages also function as APC’s during the course of infection and they process Mtb antigens and present them to T cells by MHC molecules to trigger adaptive immunity. Glycoconjugate structures present on the surface of invading pathogens are of importance in the first contact with the host innate immune system. This interaction has a decisive role in the establishment of the initial immune response and in the outcome of the infection. M. tuberculosis is able to subvert the host innate immune response and survive within macrophages and modulate the immune response using its unique outer cell wall components like lipoarabinomannan (LAM), lipomannan (LM) and phosphatidyl-myo inositol mannosides (PIM’s). Previous studies form our laboratory showed human CD8+ T cells in healthy latently infected individuals correlated with protection from TB disease. Conversely, we also reported that the glycosylated form of Apa abrogated the activation of and T cell polarization by dendritic cells infected with BCG expressing Rv1860 from Mtb. In M. tuberculosis Rv1860 codes for an alanine-proline-rich antigen, also known as the 45/47-kDa protein complex. Its expression is restricted to members of Mtb complex; based on initial binding to concanavalin-A and later mass spectrometry analysis, Rv1860 was characterized as a mannosylated protein. In the present study, in chapter 1, we have investigated the effect of Rv1860 on the functions of mouse bone marrow derived primary macrophages (BMDM) which are innate immune cells and also identified the interacting partners of Rv1860 in BCG. Infection of BMDM with recombinant Mycobacterium bovis BCG expressing Mtb Rv1860 (BCG-Rv1860) increased the production of pro- inflammatory cytokines over the levels produced by infection with wild-type parent strain whereas expression of Rv1860 in M. smegmatis inhibited the production of pro- inflammatory cytokines by macrophages. BCG-Rv1860 enhances inflammation, evidenced by increase in cytokines like TNF-α, IL-6 and Il-12p40 which is transcriptionally mediated. In chapter 2, we have shown that BCG-Rv1860 induces enhanced phosphorylation of JNK and induce ROS production. BCG-Rv1860 induces early autophagy evidenced by conversion of LC3-I to LC3-II and enhanced by stability of p62. On early stage phagophore, p62 can contribute to the assembly of the necrosome complex on these membranes and induces necroptotic death. BCG-Rv1860 inhibits caspase activation, thereby inhibiting apoptosis. Death pathway induced by BCG-Rv1860 was unequivocally shown to be necroptosis confirmed by phosphorylation of MLKL, necrosome assembly, inhibition of cell death and inflammation by necrostatin-1.