MECHANISMS OF SALMONELLA INVASION AND TRANSMIGRATION

沙门氏菌的发病机制是以侵入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，这在非极化细胞中由GTP酶的Rho家族的成员介导。 然而，上皮细胞的顶端肌动蛋白细胞骨架是高度专业化的，我们已经发现，ADP-核糖基化因子（ARF）家族的GTP酶，ARF 6的成员，也可能介导沙门氏菌内化极化上皮细胞。 在具体目标1中，我们将研究Rho GTPases和ARF 6在沙门氏菌内化和顶端质膜信号传导中的特定作用。 一旦在宿主细胞内，沙门氏菌就驻留在来源于宿主细胞膜的空泡体内，所述空泡体支持其细胞内复制，转运至基底外侧质膜并随后释放到固有层中。这些空泡基本上是由细菌从正常的膜运输途径转移的大的和专门的内体，并且它们的组成随着细菌从细胞的顶端向基底外侧极迁移而随时间变化。 使用MDCK细胞模型，其中上皮细胞在可渗透的过滤器支持物上培养，我们已经表明，细菌从细胞的基底外侧极的出现是一个矢量的，非随机的过程，表明沙门氏菌招募宿主细胞靶向机制的组件，以指导其运输到基底外侧膜并与基底外侧膜融合。 为了支持这一假设，我们已经确定了一个成员的rab家庭的小GTP酶，rab 5，这是目前对沙门氏菌的空泡在早期阶段的生物发生。 在具体目标2中，我们将确定rab 5在液泡成熟中的功能，并确定其他可能参与这一过程的rabs。普遍认为伊加通过防止细菌及其产物粘附到上皮细胞表面来保护粘膜表面免受细菌侵袭。 然而，还假设伊加可以在细胞内起作用，以抑制细胞内病原体的复制并增强细胞内病原体从细胞中的清除。 在具体目标3中，我们将确定细胞内伊加是否与迁移沙门氏菌相互作用，以及这种相互作用是否足以干扰液泡生物发生或运输。 这些研究的结果将提供显着的洞察沙门氏菌致病机制在肠上皮细胞。

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.