The reverse transmigration of Salmonella-infected cells into the bloodstream

 描述(申请人提供)：沙门氏菌是一个主要的公共卫生问题。每年有超过10亿例新的沙门氏菌感染人类，导致300多万人死亡。多重耐药菌株的出现大大加剧了这一问题。除了公共卫生问题外，研究鼠伤寒沙门氏菌也是因为它 是解剖基本致病过程的无与伦比的模式病原体，因为它结合了优秀遗传学的优势和易于处理的动物感染模型。沙门氏菌被认为是利用迁徙宿主细胞作为特洛伊木马从胃肠道传播到内脏的。树突状细胞就是这样一种细胞类型，它可以在正常的宿主过程中，通过从底部向心尖方向穿过内皮重新进入血流，称为反向迁移。反向轮回很可能与许多感染过程有关，包括病原微生物从胃肠道、肺组织和口腔粘膜传播到体循环。不足为奇的是，受感染的细胞通常不会逆转移行，可能是因为这可能会造成严重的血液感染。宿主可能有机制来检测保守的微生物成分，并适当地平衡消炎和抑制微生物传播的需要。鼠伤寒沙门氏菌利用反向迁移途径进入更深的组织，操纵感染细胞的迁移特性，作为其发病机制的一个未被认识的组成部分，直接从胃肠道进入它们内部的血流。沙门氏菌通过将III型效应器SrfH分泌到感染细胞中来颠覆宿主蛋白TRIP6来刺激反向转运，从而部分实现了这一点。在目标1中，我们将使用各种生化和遗传工具来机械地准确描述SrfH/TRIP6相互作用如何通过反向轮回途径促进旅行。由于III型分泌缺陷的突变体在触发感染细胞反向迁移方面通常比srfH突变体有更大的缺陷，因此必须涉及其他微生物和宿主因素。在目标2中，我们将利用体外逆转移行试验和小鼠体内传播实验来确定9种已知调节未感染细胞反向移行的宿主因素对感染沙门氏菌的细胞反向移行的影响。此外，我们还将确定这些分子在感染期间是否受到干扰。最后，我们将利用体外实验来鉴定额外的III型效应器(S)，该效应器参与了对血流的反向迁移途径的开发。

 DESCRIPTION (provided by applicant): Salmonella is a major public health problem. There are more than one billion new Salmonella infections of humans each year that lead to more than three million deaths. The problem is greatly exacerbated by the emergence of multi-drug resistant strains. In addition to public health concerns, S. Typhimurium is also studied because it is a model pathogen without parallel for dissecting basic pathogenic processes as it combines the advantages of excellent genetics with tractable animal models of infection. Salmonella is believed to exploit migratory host cells as Trojan horses to spread from the gastrointestinal (GI) tract to internal organs. Dendritic cells are one such cell type, which can reenter the bloodstream by traversing endothelium in the basal to apical direction in a normal host process referred to as reverse transmigration. Reverse transmigration is likely relevant to numerous infectious processes including the spread of pathogenic microbes from the GI tract, lung tissue and the oral mucosa to the systemic circulation. Not surprisingly, infected cells do not normally reverse transmigrate, presumably because this could create a serious bloodstream infection. The host likely has mechanisms to detect conserved microbial components and balances the need to resolve inflammation with inhibiting the spread of microbes appropriately. Salmonella Typhimurium exploits the reverse transmigration pathway to deeper tissue, manipulating the migratory properties of infected cells to enter the bloodstream within them directly from the GI tract as an unappreciated component of its pathogenesis. Salmonella achieves this in part by secreting the type III effector SrfH into infected cells to subvert the host protein TRIP6 to stimulate reverse transmigration. In Aim 1, we will employ a variety of biochemical and genetic tools to delineate mechanistically exactly how the SrfH/TRIP6 interaction promotes travel through the reverse transmigration pathway. As a mutant deficient in type III secretion generally has a larger defect in triggering the reverse transmigration of infected cells than a srfH mutant, additional microbial and host factors must be involved. In Aim 2, we will utilize an in vitro revere transmigration assay along with murine intra-host dissemination experiments to determine the impact of the 9 host factors that are known to regulate the reverse transmigration of uninfected cells on the reverse transmigration of ones infected with Salmonella. Also, we will determine if these molecules are perturbed during infections. Finally, we will utilize the in vitro assay to identify the additional type III effector(s) involved in exploiting the reverse transmigration pathway to the bloodstream.