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Salmonella enterica relies on a type III secretion system encoded in Salmonella pathogenicity island-2 (SPI-2) to survive and replicate within macrophages at systemic sites during typhoid. SPI-2 virulence is induced upon entry into macrophages, but the mechanisms of SPI-2 gene control in vivo remain unclear, particularly with regard to negative regulators that control the contextual activation of SPI-2. Here, we identified and characterized YdgT as a negative modulator of the SPI-2 pathogenicity island and established that this negative regulation is central to systemic pathogenesis because ydgT mutants overexpressing typhoid virulence genes were ultimately attenuated during infection. ydgT mutants displayed a biphasic virulence phenotype during in vivo competitive infections that consisted of an early "gain-of-virulence" dependent on SPI-2 activation, followed by attenuation later in infection indicating that proper contextual regulation of SPI-2 by YdgT is necessary for full virulence during systemic colonization. These data suggest that overexpression of virulence-associated type III secretion genes can have an adverse effect on bacterial pathogenesis in vivo. [ABSTRACT FROM AUTHOR]

The Salmonella pathogenicity island-2 (SPI2) is a virulence locus on the bacterial chromosome required for intracellular proliferation and systemic infection in mice. Cell culture models and a murine model of systemic infection were used to address the role of an uncharacterized SPI2 open reading frame, designated as sseA, in Salmonella virulence. A Salmonella strain with an unmarked internal deletion of sseA displayed a phenotype that was similar to an SPI2-encoded type III secretion system apparatus mutant. Moreover, SseA was required for survival and replication within epithelial cells and macrophages. Murine infection studies confirmed that the ΔsseA strain was severely attenuated for virulence. Using immunofluorescence microscopy, the virulence defect in the ΔsseA strain was attributed to an inability to translocate SPI2 effector proteins into host cells. These data demonstrate that SseA is essential for SPI2-mediated translocation of effector proteins. [Copyright &y& Elsevier]

Salmonella typhimurium is a facultative intracellular pathogen that utilizes two type III secretion systems to deliver virulence proteins into host cells. These proteins, termed effectors, alter host cell function to allow invasion into and intracellular survival/replication within a vacuolar compartment. Here we describe SopD2, a novel member of the Salmonella translocated effector (STE) family, which share a conserved N-terminal type III secretion signal. Disruption of the sopD2 gene prolonged the survival of mice infected with a lethal dose of Salmonella typhimurium , demonstrating a significant role for this effector in pathogenesis. Expression of sopD2 was induced inside host cells and was dependent on functional ssrA/B and phoP/Q, two component regulatory systems. HA-tagged SopD2 was delivered into HeLa cells in a SPI-2-dependent manner and associated with both the Salmonella -containing vacuole and with swollen endosomes elsewhere in the cell. Subcellular fractionation confirmed that SopD2 was membrane associated in host cells, while the closely related effector SopD was localized to the cytosol. A SopD2 fusion to GFP associated with small tubular structures and large vesicles containing late endocytic markers, including Rab7. Surprisingly, expression of N-terminal amino acids 1–150 of SopD2 fused to GFP was sufficient to mediate both binding to late endosomes/lysosomes and swelling of these compartments. These findings demonstrate that the N-terminus of SopD2 is a bifunctional domain required for both type III secretion out of Salmonella as well as late endosome/lysosome targeting following translocation into host cells . [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium is lysogenized by several temperate bacteriophages that encode lysogenic conversion genes, which can act as virulence factors during infection and contribute to the genetic diversity and pathogenic potential of the lysogen. We have investigated the temperate bacteriophage called Gifsy-1 in S.enterica serovar Typhimurium and show here that the product of the gogB gene encoded within this phage shares similarity with proteins from other Gram-negative pathogens. The amino-terminal portion of GogB shares similarity with leucine-rich repeat-containing virulence-associated proteins from other Gram-negative pathogens, whereas the carboxyl-terminal portion of GogB shares similarity with uncharacterized proteins in other pathogens. We show that GogB is secreted by both type III secretion systems encoded in Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 but translocation into host cells is a SPI-2-mediated process. Once translocated, GogB localizes to the cytoplasm of infected host cells. The genetic regulation of gogB in Salmonella is influenced by the transcriptional activator, SsrB, under SPI-2-inducing conditions, but the modular nature of the gogB gene allows for autonomous expression and type III secretion following horizontal gene transfer into a heterologous pathogen. These data define the first autonomously expressed lysogenic conversion gene within Gifsy-1 that acts as a modular and promiscuous type III-secreted substrate of the infection process. [Copyright &y& Elsevier]