Biogenesis Of The Salmonella-containing Vacuole

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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.