Biogenesis Of The Salmonella-containing Vacuole

在人类和其他动物中引起各种严重疾病的沙门氏菌。致病的关键是这些兼性细胞内细菌入侵非吞噬细胞并在各种哺乳动物细胞内生存和复制的能力。沙门氏菌一旦进入宿主细胞，就会在一种被称为含有沙门氏菌的空泡(SCV)的修饰吞噬小体内居住和复制。SCV的侵袭和生物发生都是由效应蛋白介导的，效应蛋白通过III型分泌系统(TTSS)转运到宿主细胞中。沙门氏菌致病岛1(SPI1)编码的TTSS是侵袭所必需的，它是由几种效应蛋白协同作用诱导的，这些效应蛋白诱导肌动蛋白细胞骨架的局部重排导致膜褶皱。内质网参与了新生的SCV的形成，但SCV很快就具有早期内体的特征，然后经历一个成熟的过程，在这个过程中，早期的内体膜蛋白被溶酶体糖蛋白和其他通常存在于晚期内体/溶酶体中的蛋白质所取代。几个小时后，细胞内的细菌开始复制，巧合的是，广泛的膜小管开始从SCV延伸出来。SPI2编码的TTS是Sif形成和细胞内复制所必需的，然而，单个SPI2效应器的作用尚不清楚。尽管SPI1和SPI2 TTS先前分别被分配到明确定义和时间上不同的过程、侵袭和SCV的生物发生/细胞内生存，但我们的工作揭示了这两个系统的活动有相当大的重叠。 我们正在使用多方面的方法来表征沙门氏菌与宿主细胞的相互作用，特别是SCV的生物发生和SIF动力学以及SPI1和SPI2 TTS在这些过程中的作用。旋转圆盘共聚焦显微镜显示了迄今为止未检测到的内吞途径与SCV和SIFs之间的相互作用水平。此外，我们发现Sif小管是非常动态的，以微管依赖的方式在多轮中快速生长和回缩。对微管、马达和其他宿主细胞因子作用的进一步表征正在进行中。特别是，我们正在使用小的抑制性RNA来耗尽宿主细胞蛋白，这些蛋白可能在SCV/SIF的形成中发挥作用。

Salmonella species that cause a variety of serious diseases in man and other animals. Essential to pathogenesis is the ability of these facultative intracellular bacteria to invade nonphagocytic cells and to survive and replicate within a variety of mammalian cells. Once within the host cell Salmonella reside and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). Both invasion and SCV biogenesis are mediated by effector proteins, which are translocated into the host cell by Type III Secretion System (TTSS). The Salmonella Pathogenicity Island 1 (SPI1) encoded TTSS is required for invasion, which is induced by the co-operative effectives of several effector proteins that induce local rearrangement of the actin cytoskeleton leading to membrane ruffles. The Endoplasmic Reticulum is involved in nascent SCV formation but the SCV rapidly assumes characteristics of early endosomes and then undergoes a maturation process in which the early endosome membrane proteins are replaced by lysosomal glycoproteins and other proteins normally found in late endosomes/lysosomes. After several hours the intracellular bacteria begin to replicate and coincidently extensive membrane tubules, Sifs, begin to extend from the SCV. The SPI2 encoded TTSS is required for Sif formation and intracellular replication, however, the roles of individual SPI2 effectors are not well understood. Although the SPI1 and SPI2 TTSS have previously been assigned to well-defined and temporally distinct processes, invasion and SCV biogenesis/intracellular survival respectively, our work has revealed a considerable overlap of the activities of the two systems. We are using a multi-faceted approach to characterize Salmonella-host cell interactions, particularly SCV biogenesis and Sif dynamics and the roles of the SPI1 and SPI2 TTSS in these processes. Spinning disc confocal microscopy has revealed a hitherto undetected level of interaction between the endocytic pathway and SCVs and Sifs. In addition, we have found that Sif tubules are extremely dynamic, with rapid growth and retraction occurring in multiple rounds in a microtubule-dependent manner. Further characterization of the role of microtubules, motors and other host cell factors is ongoing. In particular we are using small inhibitory RNAs to deplete host cell proteins that may play a role in SCV/Sif formation.