Summary: The Salmonella pathogenicity island 2 (SPI‐2) type III secretion system (TTSS) of Salmonella typhimurium is required for bacterial replication within host cells. It acts by translocating effector proteins across the membrane of the Salmonella‐containing vacuole (SCV). The SifA effector is required to maintain the integrity of the SCV membrane, and for the formation in epithelial cells of Salmonella‐induced filaments (Sifs), which are tubular extensions of SCVs. We have investigated the role in S. typhimurium virulence of the putative SPI‐2 effector genes sifB, srfJ, sseJ and sseI. An S. typhimurium strain carrying a mutation in sseJ was mildly attenuated for systemic virulence in mice, but strains carrying mutations in either srfJ, sseI or sifB had very little or no detectable virulence defect after intraperitoneal inoculation. Expression of SseJ in HeLa cells resulted in the formation of globular membranous compartments (GMCs), the composition of which appears to be similar to that of SCV membranes and Sifs. The formation of GMCs was dependent on the serine residue of the predicted acyltransferase/lipase active site of SseJ. Transiently expressed SseJ also inhibited Sif formation by wild‐type bacteria, and was found to associate with Sifs, SCV membranes and simultaneously expressed SifA. Intracellular vacuoles containing sseJ mutant bacteria appeared normal but, in contrast to a sifA mutant, a sifA sseJ double mutant strain did not lose its vacuolar membrane, indicating that loss of vacuolar membrane around sifA mutant bacteria requires the action of SseJ. Collectively, these results suggest that the combined action of SseJ and SifA regulate dynamics of the SCV membrane in infected cells.

The type III secretion system of Salmonella pathogenicity island 2 (SPI‐2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI‐2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI‐2 type III secretion system. As the pH of the Salmonella‐containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI‐2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI‐2‐mediated secretion in vivo.

The type III secretion system (TTSS) encoded bySalmonella typhimuriumpathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA–SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA–B is in turn regulated by the OmpR–EnvZ two-component system, by direct binding of OmpR to thessrABpromoter. Several environmental signals have been shown to inducein vitroexpression of genes regulated by the SsrA–B or OmpR–EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA–B and OmpR–EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifAandsifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca²⁺was determined. SsrA–B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR–EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA–B system to mediate their effect on SPI-2 TTSS gene expression.

Summary: Many stimuli such as hormones and elicitors induce changes in intracellular calcium levels to integrate information and activate appropriate responses. The Ca²⁺signals are perceived by various Ca²⁺sensors, and calmodulin (CaM) is one of the best characterized in eukaryotes. Calmodulin‐like (CML) proteins extend the Ca²⁺toolkit in plants; they share sequence similarity with the ubiquitous and highly conserved CaM but their roles at physiological and molecular levels are largely unknown. Knowledge of the contribution of Ca²⁺decoding proteins to plant immunity is emerging, and we report here data onArabidopsis thaliana CML9, whose expression is rapidly induced by phytopathogenic bacteria, flagellin and salicylic acid. Using a reverse genetic approach, we present evidence that CML9 is involved in plant defence by modulating responses to bacterial strains ofPseudomonas syringae. Compared to wild‐type plants, the later responses normally observed upon flagellin application are altered in knockout mutants and over‐expressing transgenic lines. Collectively, using PAMP treatment andP. syringaestrains, we have established that CML9 participates in plant innate immunity.