Guignot Julie, Caron Emmanuelle, Beuzón Carmen, Bucci Cecilia, Kagan Jonathan, Roy Craig, Holden David W
Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, London, SW7 2AZ, UK.
J Cell Sci. 2004 Mar 1;117(Pt 7):1033-45. doi: 10.1242/jcs.00949. Epub 2004 Feb 17.
Infection of host cells by Salmonella enterica serovar Typhimurium (S. typhimurium) leads to the formation of specialised membrane-bound compartments called Salmonella-containing vacuoles (SCVs). Bacteria remain enclosed by the vacuolar membrane as they divide, and by translocating effector proteins across the vacuolar membrane through the SPI-2 type III secretion system, they interfere with host cell processes in ways that promote bacterial growth. One such effector is SifA, which is required to maintain the integrity of the vacuolar membrane and for the formation in epithelial cells of long tubular structures called Sifs that are connected to SCVs. Unknown effector(s) mediate the assembly of a meshwork of F-actin around SCVs. We report that intracellular bacteria also cause a dramatic accumulation of microtubules around S. typhimurium microcolonies in both epithelial cells and macrophages. Although this process appears to be independent of SPI-2-mediated F-actin assembly, it does require bacterial protein synthesis. In epithelial cells, microtubule accumulation is accompanied by the recruitment of both kinesin and dynein. Inhibition of the activity of either motor prevented both Sif formation and the loss of vacuolar membrane from sifA mutant bacteria. It also resulted in morphologically abnormal vacuoles enclosing wild-type bacteria, and impaired their replication. Our experiments indicate that recruitment of dynein to SCVs is dependent on Rab7 activity. We show that the recently described Rab7 effector RILP is also recruited to SCVs in a Rab7-dependent manner. However, overexpression of RILP did not restore dynein recruitment to SCVs in cells expressing dominant negative Rab7, suggesting that RILP requires a functional Rab7 to be activated at the SCV membrane, or that dynein recruitment is mediated by an effector other than RILP. Together, these experiments indicate that microtubule motors play important roles in regulating vacuolar membrane dynamics during intracellular replication of S. typhimurium.
鼠伤寒沙门氏菌（Salmonella enterica serovar Typhimurium，简称鼠伤寒沙门菌）感染宿主细胞会导致形成一种特殊的膜结合区室，称为含沙门氏菌液泡（Salmonella-containing vacuoles，简称SCV）。细菌在分裂时仍被液泡膜包围，并且通过SPI-2 III型分泌系统将效应蛋白转运穿过液泡膜，它们以促进细菌生长的方式干扰宿主细胞过程。其中一种效应蛋白是SifA，它对于维持液泡膜的完整性以及在上皮细胞中形成与SCV相连的称为Sifs的长管状结构是必需的。未知的效应蛋白介导了围绕SCV的F-肌动蛋白网络的组装。我们报告称，细胞内细菌还会在上皮细胞和巨噬细胞的鼠伤寒沙门菌微菌落周围引起微管的显著积累。尽管这个过程似乎独立于SPI-2介导的F-肌动蛋白组装，但它确实需要细菌蛋白质合成。在上皮细胞中，微管积累伴随着驱动蛋白和动力蛋白的募集。抑制任何一种马达的活性都会阻止Sif形成以及sifA突变细菌的液泡膜丢失。它还导致包围野生型细菌的液泡在形态上异常，并损害其复制。我们的实验表明，动力蛋白向SCV的募集依赖于Rab7活性。我们表明，最近描述的Rab7效应蛋白RILP也以Rab7依赖的方式被募集到SCV。然而，在表达显性负性Rab7的细胞中，RILP的过表达并没有恢复动力蛋白向SCV的募集，这表明RILP需要功能性的Rab7在SCV膜上被激活，或者动力蛋白的募集是由RILP以外的效应蛋白介导的。总之，这些实验表明微管马达在鼠伤寒沙门菌细胞内复制过程中调节液泡膜动力学方面发挥着重要作用。
