Project 1: Listeria metabolites and innate immunity

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Project Summary/Abstract (Project 1, Portnoy) This proposal is Project 1 within a P01 renewal, entitled “The intersection of innate and adaptive immunity to intracellular pathogens.” A central problem that we address is how intracellular pathogens are recognized by the host innate immune system and how multiple signals are integrated to induce an appropriate response, and conversely, how pathogens avoid and/or manipulate host responses to promote their pathogenesis. In Project 1, we have chosen to approach this problem by continuing a detailed analysis of Listeria monocytogenes, a facultative intracellular, food-borne pathogen that has been studied for decades as a model system with which to dissect basic aspects of infection & immunity. Previously, we discovered that L. monocytogenes secretes c-di-AMP which binds to and activates host STING leading to a type I interferon response. In Aim 1, we exploit a newly developed oral model of listeriosis where we find that bacterially secreted c-di-AMP induces a STING-dependent host protective response. We propose to analyze how STING provides protection and test the hypothesis that STING also promotes dissemination to the placenta and brain. In Aims 2 and 3, we begin to explore the idea that c-di-AMP might be just one example of the principle that small bacterial metabolites play important roles in shaping host immunity. L. monocytogenes is a riboflavin requiring microorganism, and we explore the hypothesis that that this nutritional requirement allows the bacteria to avoid activation of mucosal-associated invariant T-cells (MAIT cells), which are innate T-cells stimulated by a modified intermediate of riboflavin biosynthesis. In preliminary data, we introduced five Bacillus subtilis genes (ribDEAHT) into L. monocytogenes converting it into a riboflavin-synthesizing bacterium and are now poised to test our hypothesis and learn about the role of MAIT cells during bacterial infection and immunity. We also propose to explore the activation of the dominant gamma delta T-cell in humans (Vg9Vd2 T-cells). These cells are activated by an intermediate of bacterial isoprenoid biosynthesis (HMBPP). Our ultimate goal is to determine the importance of this innate immune pathway during infection of primates (human and non-human). Here we propose to construct bacterial mutants that either fail to make HMBPP or make more of it to examine how bacterial HMBPP is detected during infection in tissue culture models. We further propose to collaborate with the Cox lab (Project 2) to explore the importance of HMBPP during M. tuberculosis infection and immunity.