Biogenesis Of The Salmonella-containing Vacuole

沙门氏菌属是兼性胞内细菌，其在空泡（SCV）内驻留和复制。SCV的生物发生由细菌决定，并涉及通过III型分泌系统易位到宿主细胞中的效应蛋白。由于肌动蛋白细胞骨架的局部重排，导致局部膜皱褶，从而发生侵袭。这一过程是由几个易位效应蛋白的协同效应诱导的。膜皱褶含有大量必须迅速输送的膜。由于在SCV中仅检测到内吞标志物，因此已经假设生物合成/分泌途径不参与该过程。与蒙特利尔大学的Michel Desjardins合作，我们已经证明SCV迅速获得内质网膜，并且确实与ER相邻。因此，可能是ER对质膜皱褶的膜内容物起作用。事实上，在吞噬细胞中，ER膜可以看到与含有乳胶珠或利什曼原虫的新生吞噬体邻接。 我们已经开发了一个活细胞系统，我们正在使用它来表征沙门氏菌-宿主细胞相互作用的3个阶段;入侵，SCV形成和Sif动态。SIF是从SCV延伸并与SCV邻接的小管。Sifs的形成依赖于几种沙门氏菌效应蛋白和未鉴定的宿主因子。我们已经发现，这些广泛的小管是非常动态的，并依赖于微管。对微管、马达和其他宿主细胞因子的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来消耗可能在SCV/sif形成中起作用的宿主细胞蛋白。

Salmonella sp are facultative intracellular bacteria that reside and replicate within a vacuole (SCV). The biogenesis of the SCV is determined by the bacteria and involves effector proteins that are translocated into the host cell via Type III secretion systems. Invasion occurs as a result of localized membrane ruffles that are due to local rearrangement of the actin cytoskeleton. This process is induced by the co-operative effectives of several translocated effector proteins. Membrane ruffles contain large amounts of membrane that must be rapidly delivered. It has been assumed, since only endocytic markers are detected in SCVs, that the biosynthetic/secretory pathway is not involved in this process. In collaboration with Michel Desjardins at the University of Montreal, we have shown that the SCV rapidly acquires Endoplasmic Reticulum membranes and indeed appears contiguous with the ER. Thus it is possible that the ER is contributing membrane content to ruffles at the plasma membrane. Indeed, in phagocytic cells, ER membrane can be seen to be contiguous with nascent phagosomes containing either latex beads or Leishmania. We have developed a live cell system that we are using to characterize 3 stages of Salmonella-host cell interactions; invasion, SCV formation and Sif dynamics. Sifs are tubules that extend from, and are contiguous with, SCVs. The formation of Sifs is dependent on several Salmonella effector proteins and unidentified host factors. We have found that these extensive tubules are extremely dynamic and are dependent on microtubules. 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.