Septins in macrophage pyroptosis and Shigella infection

间隔蛋白是细胞骨架中的一种非常规成分，因其在细胞分裂和宿主防御中的作用而被广泛认识。利用组织培养细胞系，莫斯托维实验室发现，Septins可以将肌动蛋白聚合的志贺菌包裹在笼状结构中，实现细胞自主免疫。Mostowy实验室使用斑马鱼感染模型表明，毛壳素可以抑制炎症，保护幼虫免受志贺氏菌感染。尽管有这些见解，但Septins在宿主防御中的作用广度几乎是未知的。下睑下垂是一种已知的由志贺氏菌感染引起的炎性细胞死亡途径。Mostowy实验室最近的研究表明，隔膜在控制下垂中发挥了作用，但其潜在的机制和对整个动物的影响尚未被研究。在我的计划中，我将研究巨噬细胞(体外和体内)，以发现在进化上保守的作用，在下垂和志贺氏菌感染控制。在目标1，我将操纵SECTIN生物学，并研究脂多糖(LPS)和黑素治疗诱导的上睑下垂，脂多糖转染法和志贺氏菌感染。利用分子生物学方法，我将分析Septins在细胞死亡控制中的作用。这将确定Septin结合伙伴，并阐明焦下垂和/或志贺氏菌Septin笼化在宿主防御中的作用。在目标2中，我将用转座子定向插入测序(Tradis)随机突变志贺氏菌文库感染巨噬细胞，并发现促进Septin介导的巨噬细胞松弛控制的志贺氏菌效应物。目标1侧重于宿主细胞的反应，而目标2侧重于了解促进/限制细胞死亡的重要细菌因素。在目标3中，我将使用志贺氏菌-斑马鱼感染模型，从单个细胞水平到整个动物水平，研究间隔蛋白在巨噬细胞控制感染中的体内作用。我将使用斑马鱼品系(最近在Mostowy实验室开发)、药物治疗(使用诱导下垂的药物)或感染志贺氏菌来研究间隔蛋白和巨噬细胞死亡的作用。我将使用免疫印迹、流式细胞术、共聚焦和高分辨率显微镜技术分析细菌负荷、巨噬细胞死亡和嗜热症诱导。总之，我的研究提案有望为SECTIN生物学提供基本的见解，并发现控制下垂和志贺氏菌感染的新机制。我设想，在未来，基于Septin的药物可以被开发成治疗传染病和炎症性疾病的新疗法。

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.