Genetic Dissection of Mycobacterial Pathogenesis During Eicosanoid-Mediated Immunity

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ABSTRACT Mycobacterium tuberculosis (Mtb) one of the most successful pathogen in the world due to its long-standing, ancient partnership with humans. Despite decades of effort put towards prevention and treatment, Mtb was the number one cause of death by any single infectious agent in 2019. An important regulator of mycobacterial infections are eicosanoids: host-produced, lipid signaling molecules that can drive both inflammatory and anti- inflammatory signaling cascades. Using a zebrafish-Mycobacterium marinum infection model that recapitulates important aspects of mycobacterial pathogenesis, the balance of specific pro- and anti-inflammatory eicosanoids was found to be critical to control of infection. Disruption of eicosanoid synthesis, through a mutation in leukotriene A4 hydrolase-deficient (lta4h-/-), results in altered granuloma structure. The granuloma is the central feature of Mtb infection and is an aggregation of immune cells coordinating to restrict bacterial growth. This is the principal site of host-pathogen interactions. Although the granuloma is a key part of tuberculosis pathogenesis, host-pathogen signaling programs that govern granuloma biology remain elusive. Our lack of understanding of granuloma formation and signaling is due to the difficulty in studying the granuloma because the complex cell organization can only be formed and observed in vivo. The zebrafish-Mycobacterium marinum model, with conserved virulence loci and host-pathogen genetic programs, is used to study the genetics of mycobacterial infection and granuloma structure. This proposal will use genetic approaches in both the zebrafish- M.marinum granuloma model and the mouse-Mtb model of infection to test the hypothesis that there are key differences in eicosanoid-mediated immunity and bacterial response that are integral to overall granuloma biology. To this end, the first single-cell map of mycobacterial granulomas, in an experimentally tractable system was assembled from wildtype and lta4h-/- zebrafish granuloma cells. The findings from this dataset informed the following approaches to interrogate the effects of host eicosanoid signaling on granuloma structure and mycobacterial response. Aim 1 will define the functional role of a previously undefined population of granuloma cells that are dependent on lta4h expression. Aim 2 will use Mtb TnSeq approaches to the examine the genetic requirements of Mtb in mice that lack key components of eicosanoid signaling. Together, these aims will enhance our understanding of granuloma biology and mycobacterial response and can help guide therapeutic approaches to treating tuberculosis.