MECHANISMS OF SALMONELLA INVASION AND TRANSMIGRATION

沙门氏菌的发病机制以侵袭和穿透肠上皮屏障为特征。进入宿主细胞需要肌动蛋白细胞骨架的剧烈重组，这在非极化细胞中是由GTPases的Rho家族成员介导的。然而，上皮细胞的顶端肌动蛋白细胞骨架是高度特化的，我们发现adp -核糖基化因子（ARF） gtpase家族的成员ARF6也可能介导沙门氏菌在极化上皮细胞中的内化。在Specific Aim 1中，我们将研究Rho GTPases和ARF6在沙门氏菌根尖质膜内化和信号传导中的具体作用。一旦进入宿主细胞，沙门氏菌就驻留在支持其细胞内复制的宿主细胞膜衍生的液泡体内，转移到基侧质膜，随后释放到固有层。这些液泡本质上是由细菌从正常的膜运输途径转移而来的大而特化的内体，它们的组成随着细菌从细胞的顶端迁移到细胞的基底外极而改变。在MDCK细胞模型中，上皮细胞被培养在可渗透的过滤器支架上，我们发现细菌从细胞的基底外侧极出现是一个载体的、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成部分，指导它们运输到基底外侧膜并与之融合。为了支持这一假设，我们已经确定了兔小gtp酶家族的一个成员，rab5，在生物发生的早期阶段存在于含沙门氏菌的液泡中。在Specific Aim 2中，我们将确定rab5在液泡成熟中的功能，并确定其他可能参与这一过程的rab5。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粘附来保护粘膜表面免受细菌的侵袭。然而，也有人假设IgA可能在细胞内起作用，抑制细胞内病原体的复制并增强其对细胞内病原体的清除。在Specific Aim 3中，我们将确定细胞内IgA是否与迁移的沙门氏菌相互作用，以及这种相互作用是否足以干扰液泡生物发生或运输。这些研究结果将为沙门氏菌在肠上皮内的发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.