MECHANISMS OF SALMONELLA INVASION AND TRANSMIGRATION

沙门氏菌的致病机制以肠上皮屏障的侵袭和穿透为特征。进入宿主细胞需要肌动蛋白细胞骨架的戏剧性重组，在非极化细胞中，肌动蛋白骨架是由Rho家族的GTP酶家族成员介导的。然而，上皮细胞的顶端肌动蛋白细胞骨架高度专门化，我们发现GTP酶中ADP-核糖化因子(ARF)家族的一个成员ARF6也可能介导沙门氏菌在极化上皮细胞中的内化。在特定的目标1中，我们将研究Rho GTP酶和ARF6在沙门氏菌内化和顶端质膜信号转导中的特定作用。一旦进入宿主细胞，沙门氏菌就居住在来源于宿主细胞膜的空泡体中，这些液泡体支持沙门氏菌在细胞内复制，转移到基侧质膜，然后释放到固有层。这些液泡本质上是由细菌从正常的膜运输途径转移过来的大而专门的内体，当细菌从细胞的顶端迁移到基端时，它们的组成随着时间的变化而变化。使用MDCK细胞模型，在该模型中，上皮细胞培养在可渗透的过滤器支架上，我们已经证明了细菌从细胞的基侧极出现是一个矢量的、非随机的过程，这表明沙门氏菌招募宿主细胞靶向机制的组件，以指导它们向基侧膜运输并与基侧膜融合。为了支持这一假说，我们已经确定了Rab家族中的一个成员，Rab5，它存在于生物发生的早期阶段的含有沙门氏菌的液泡上。在特定的目标2中，我们将确定Rab5在空泡成熟中的功能，并寻找可能参与这一过程的其他RAB。人们普遍认为，IgA通过阻止细菌及其产物与上皮细胞表面的黏附来保护粘膜表面免受细菌入侵。然而，也有人假设，IgA可能在细胞内发挥作用，抑制细胞内病原体的复制，并增强细胞内病原体的清除。在具体目标3中，我们将确定细胞内IgA是否与移行的沙门氏菌相互作用，以及这种相互作用是否足以扰乱空泡的生物发生或运输。这些研究的结果将为沙门氏菌在肠上皮内的致病机制提供重要的见解。

Salmonella pathogenesis is characterized by the invasion and penetration of the intestinal epithelial barrier. Entry into host host cells requires dramatic reorganization of the actin cytoskeleton, which in non-polarized cells is mediated by members of the Rho family of GTPases. However, the apical actin cytoskeleton of epithelial cells is highly specialized, and we have found that a member of the ADP-ribosylation factor (ARF) family of GTPases, ARF6, may also mediate Salmonella internalization in polarized epithelial cells. In Specific Aim 1, we will examine the specific roles of Rho GTPases and ARF6 in Salmonella internalization and signaling at the apical plasma membrane. Once inside the host cell, Salmonella reside within vacuolar bodies derived from host cell membranes that support their intracellular replication, transit to the basolateral plasma membrane and subsequent release into the lamina propria. These vacuoles are essentially large and specialized endosomes diverted by the bacteria from normal membrane trafficking pathways, and their composition changes with time as the bacteria migrate from the apical to basolateral pole of the cell. Using an MDCK cell model, in which epithelial cells are cultured on permeable filter supports, we have shown that the emergence of bacteria from the basolateral pole of the cell is a vectorial, non-random process, suggesting that Salmonella recruit components of the host cell targeting machinery to direct their transport to and fusion with the basolateral membrane. In support of this hypothesis, we have identified one member of the rab family of small GTPases, rab5, that is present on Salmonella-containing vacuoles at an early stage of biogenesis. In Specific Aim 2, we will determine the function of rab5 in vacuolar maturation, and identify other rabs that may participate in this process. It is widely accepted that IgA protects mucosal surfaces from bacterial invasion by preventing the adhesion of bacteria and their products to the epithelial cell surface. However, it has also been hypothesized that IgA may act intracellularly, to inhibit the replication of and enhance the clearance of intracellular pathogens from the cell. In Specific Aim 3, we will determine whether intracellular IgA interacts with transmigrating Salmonella, and whether such interaction is sufficient to perturb vacuolar biogenesis or transport. The results of these studies will provide significant insight into the mechanisms of Salmonella pathogenesis within the intestinal epithelium.