Leptospiral-Phagocyte Dynamics in Leptospirosis

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Abstract: Project 1, University of California, Los Angeles Leptospirosis is a widespread and frequently fatal human health problem that disproportionately impacts individuals living in low resource settings. Research on phagocyte interactions in leptospirosis is significant because little is known about leptospiral virulence factors required for survival within phagocytes or the response of macrophages to leptospires. The proposed studies will involve collaborations between the PI and co- investigator Fayyaz Sutterwala, who is a leader in studies of inflammasome activation. Two related but non- interdependent Aims are proposed. Aim 1 is to examine leptospiral-phagocyte interactions in vitro. We will examine the murine vs human macrophage responses to L. interrogans in terms of type I interferon and inflammasome responses and determine the pathways involved in their activation. We will examine whether leptospiral virulence genes account for lower inflammasome activation in murine relative to human macrophages. We will examine the time course of leptospiral uptake and survival vs killing of leptospiral species and test the hypothesis that macrophage survival is a property of the pathogenic, but not the saprophytic, species. We will identify virulence genes required for survival after exposure to macrophages as a result of phagocytic uptake and examine the leptospiral response to macrophage uptake. Given the finding that LigB plays a role in macrophage infection, we are interested in examining macrophage uptake and killing of a ligAligB knock down mutant that lacks expression of LigA and LigB. Dual RNAseq will be performed with bone marrow-derived macrophages (BMDMs) and L. interrogans including mutants with impaired intracellular survival to identify genes potentially involved in leptospiral-phagocyte interactions. Aim 2 is to examine leptospiral-phagocyte interactions in vivo. We will examine the kinetics of local control vs dissemination after subcutaneous challenge of hamsters vs mice. We will test the hypothesis that L. interrogans survives within phagocytes during infection of the subcutaneous space, blood, and liver. We will identify virulence genes required for intracellular survival within phagocytes after subcutaneous challenge. We are particularly interested in defects in in vivo fitness for local and disseminated infection of our ligAligB knock-down mutant and other virulence gene candidates. We will screen our transposon mutant library for mutants with fitness defects in the subcutaneous space and for dissemination. Using mice with defects in type I interferon response involving cGAS/STING and inflammasome activation involving NLRP3, we will test the hypothesis that these macrophage responses improve local control and reduce dissemination after subcutaneous challenge.