Biogenesis of Salmonella enterica serovar typhimurium membrane vesicles provoked by induction of PagC.

Gram-negative bacteria ubiquitously release membrane vesicles (MVs) into the extracellular milieu. Although MVs are the product of growing bacteria, not of cell lysis or death, the regulatory mechanisms underlying MV formation remained unknown. We have found that MV biogenesis is provoked by the induction of PagC, a Salmonella-specific protein whose expression is activated by conditions that mimic acidified macrophage phagosomes. PagC is a major constituent of Salmonella MVs, and increased expression accelerates vesiculation. Expression of PagC is regulated at the posttranscriptional and/or posttranslational level in a sigmaS (RpoS)-dependent manner. Serial quantitative analysis has demonstrated that MV formation can accelerate when the quantity of the MV constituents, OmpX and PagC, rises. Overproduction of PagC dramatically impacts the difference in the relative amount of vesiculation, but the corresponding overproduction of OmpX was less pronounced. Quantitative examination of the ratios of PagC and OmpX in the periplasm, outer membrane, and MVs demonstrates that PagC is preferentially enriched in MVs released from Salmonella cells. This suggests that specific protein sorting mechanisms operate when MVs are formed. The possible role(s) of PagC-MV in host cells is discussed.

Coordinated regulation of expression of Salmonella pathogenicity island 1 and flagellar type III secretion systems by ATP-dependent ClpXP protease.

Salmonella enterica serovar Typhimurium delivers a variety of proteins via the Salmonella pathogenicity island 1 (SPI1)-encoded type III secretion system into host cells, where they elicit several physiological changes, including bacterial invasion, macrophage apoptosis, and enteropathogenesis. Once Salmonella has established a systemic infection, excess macrophage apoptosis would be detrimental to the pathogen, as it utilizes macrophages as vectors for systemic dissemination throughout the host. Therefore, SPI1 expression must be restricted to one or a few specific locations in the host. In the present study, we have demonstrated that the expression of this complex of genes is repressed by the ATP-dependent ClpXP protease, which therefore suppresses macrophage apoptosis. Depletion of ClpXP caused significant increases in the amounts of two SPI1-encoded transcriptional regulators, HilC and HilD, leading to the stimulation of hilA induction and therefore activation of SPI1 expression. Our evidence shows that ClpXP regulates cellular levels of HilC and HilD via the control of flagellar gene expression. Subsequent experiments demonstrated that the flagellum-related gene product FliZ controls HilD posttranscriptionally, and this in turn activates HilC. These findings suggest that the ClpXP protease coregulates SPI1-related virulence phenotypes and motility. ClpXP is a member of the stress protein family induced in bacteria exposed to hostile environments such as macrophages.