Septins in macrophage pyroptosis and Shigella infection

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Septins are an unconventional component of the cytoskeleton widely recognized for their role in cell division and host defense. Using tissue culture cell lines, the Mostowy Lab discovered that septins can entrap actin-polymerizing Shigella in cage-like structures for cell-autonomous immunity. Using a zebrafish infection model, the Mostowy Lab showed that septins can restrict inflammation and protect larvae from Shigella infection. Despite these insights, the breadth of roles for septins in host defense is mostly unknown. Pyroptosis is an inflammatory cell death pathway known to be induced by Shigella infection. Recent studies in the Mostowy Lab have suggested a role for septins in pyroptosis control, yet the underlying mechanisms and whole animal impact has not yet been studied. For my proposal, I will investigate macrophages (in vitro and in vivo) to discover evolutionarily conserved roles for septins in pyroptosis and infection control using Shigella.In Objective 1, I will manipulate septin biology and investigate pyroptosis induced by lipopolysaccharide (LPS) and nigericin treatment, LPS transfection and Shigella infection. Using molecular biology approaches, I will analyze the role of septins in cell death control. This will identify septin binding partners and elucidate the role of pyroptosis and/or Shigella septin caging in host defense. In Objective 2, I will infect macrophages with a Transposon Directed Insertion Sequencing (TraDis) randomized mutation Shigella library and discover Shigella effectors that promote septin-mediated control of macrophage pyroptosis. While Objective 1 is focused on the host cell response, Objective 2 is focused on understanding bacterial factors important for the promotion / restriction of cell death. In Objective 3, I will use a Shigella-zebrafish infection model to study the in vivo role of septins in infection control by macrophages from the level of the single cell to the whole animal. I will investigate the role of septins and macrophage cell death using zebrafish lines (recently developed in the Mostowy Lab) following drug treatment (using pyroptosis inducing drugs) or infection with Shigella. I will analyze bacterial burden, macrophage cell death and pyroptosis induction using immunoblotting, flow cytometry, confocal and high resolution microscopy techniques. Together, my research proposal is expected to provide fundamental insights into septin biology and discover new mechanisms which control pyroptosis and Shigella infection. I envision that, in the future, septin-based medicines can be developed into novel therapies for infectious and inflammatory diseases.