Biogenesis Of The Salmonella-containing Vacuole

沙门氏菌是一种兼性胞内细菌，在空泡(SCV)内寄生和复制。SCV的生物发生由细菌决定，涉及通过III型分泌系统转运到宿主细胞的效应蛋白。侵袭是由于肌动蛋白细胞骨架的局部重排引起的局部膜褶皱的结果。这一过程是由几种易位效应蛋白的协同效应诱导的。薄膜折边包含大量必须快速输送的薄膜。由于在SCV中只检测到内吞标记，因此一直认为生物合成/分泌途径不参与这一过程。在与蒙特利尔大学的Michel Desjardins的合作中，我们已经证明了SCV迅速获得内质网膜，并且确实看起来与内质网相邻。因此，内质网可能正在为质膜上的褶皱贡献膜内容物。事实上，在吞噬细胞中，可以看到内质网膜与含有乳胶珠或利什曼原虫的新生吞噬小体相连。 我们开发了一个活细胞系统，用来描述沙门氏菌与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延伸而来并与SCV相邻的小管。Sifs的形成依赖于几种沙门氏菌效应蛋白和未知的宿主因素。我们发现，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 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.