The impact of two-pore channel calcium signaling on MR1 antigen presentation of Mycobacterium tuberculosis metabolites

Oregon Health & Science University, Portland OR

Investigators

Abstract

PROJECT SUMMARY Tuberculosis (TB) caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb) is the leading cause of infectious disease morbidity and mortality worldwide. Immune system sampling of the intracellular environment for Mtb antigens is crucial to control infection and prevent active tuberculosis. Major Histocompatibility Complex, Class I-Related molecule (MR1) is a conserved nonclassical antigen presenting molecule expressed in several cell types that presents Mtb derived riboflavin metabolites to MR1-restricted T cells (MR1Ts). MR1 antigen presentation to this subset of CD8+ T cells is poised to play a crucial role in controlling Mtb infection; MR1Ts are enriched in the lung and rapidly release pro-inflammatory cytokines and apoptotic factors to kill infected cells. Our laboratory has found that endosomal trafficking facilitates MR1 antigen presentation, yet the exact mechanisms by which MR1 loading and trafficking to the cell surface occurs are not known. The specific purpose of this project is to decipher molecular mechanisms of endosomal trafficking to ultimately inform our understanding of MR1 antigen presentation. We have found that endosomal calcium signaling mediates MR1 antigen presentation during Mtb infection. Interference of two-pore calcium channels (TPCs) via siRNA-mediated knockdown specifically decreases MR1 presentation of Mtb antigens. Nicotinic acid adenine dinucleotide phosphate (NAADP) facilitates endosomal calcium release by activating TPCs; treatment with a selective, membrane-permeant NAADP antagonist or knockdown of an NAADP binding protein important for TPC activation also decrease MR1 presentation of Mtb antigens. NAADP is synthesized from NADP, and this reaction requires catalysis by ADP-ribosyl cyclase. Cluster of Differentiation 157 (CD157) is one of few proteins that possesses this enzymatic activity. The central hypothesis of this proposal is that Mtb infection triggers CD157-mediated NAADP generation to facilitate MR1 antigen presentation of the Mtb-infected cell via the TPC. To test this hypothesis, I will infect antigen presenting cells with Mtb and assess MR1 antigen presentation using a human MR1T cell clone. Experiments in Aim 1 will further elucidate the role of TPC calcium release in MR1 antigen presentation via T cell IFN-γ release ELISpot assays, and characterize interactions of TPCs with MR1 and Mtb via live-cell imaging. Aim 2 will identify the source of NAADP and establish where NAADP generation occurs in relation to MR1 antigen presentation of Mtb-infected cells. This proposed work will have broad implications for how endosomal calcium sensing facilitates recognition of Mtb-infected cells. The results from this research will elucidate key, novel pathways that underpin the immune response to Mtb, which have potential to inform tuberculosis vaccine development.