Linking the behavior of individual host cells to their transcriptional signatures in tuberculosis Tuberculosis is an infectious disease of enormous proportions with no effective vaccine and the emergence of extensively drug resistant strains. Major holes in our understanding of its pathogenesis present roadblocks to new therapeutic and preventive interventions. My laboratory has developed zebrafish infection by Mycobacterium marinum as a powerful surrogate model for exploring tuberculosis pathogenesis. The transparency of zebrafish larvae allows real-time examination of cellular movements and behaviors in unprecedented detail. We have made surprising discoveries about central aspects of tuberculosis pathogenesis, in particular regarding the organized aggregate of immune cells called the granuloma. This hallmark structure was long thought to be a key host protective element. However, detailed kinetic monitoring of granuloma formation revealed that granulomas are converted by mycobacteria into tools for expansion and dissemination. Phagocytes are recruited to the nascent granuloma, where they phagocytose the contents of dying infected cells. A subset of newly infected cells leaves the granuloma to disseminate infection to new foci. Here I propose to systematically link the behaviors of immune cells to their expression profiles to gain insight into the molecular blueprint of the host processes that promote pathogenesis. I propose to use laser capture microdissection techniques combined with multiple fluorescent bacterial and host reporters to isolate cells engaged in distinct movements, i.e. migrating towards newly infecting bacteria, returning to tissues from peripheral infection sites, migrating to forming granulomas, and departing granulomas. The gene expression profiles of these cells will then be deciphered using single cell gene expression techniques. We will then perturb these molecular signatures with genetic techniques to determine their impact on infection. These studies should yield a broad mechanistic understanding of basic processes such as cell migration and aggregation that are at the root of all inflammatory diseases and suggest new host-directed intervention strategies to fight them.
This work takes an innovative approach to understanding the basic mechanisms of humanpathogen interactions in tuberculosis that may identify new strategies for therapeutic and immunological intervention.
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