SlyA Regulates Function of Salmonella Pathogenicity Island 2 (SPI-2) and Expression of SPI-2-Associated Genes

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Abstract
During the systemic phase of murine infection with Salmonella enterica serovar Typhimurium, bacterial virulence is correlated with the ability to grow and survive within host macrophages. Salmonella pathogenicity island 2 (SPI-2), encoding a type three secretion system, has emerged as an important contributor to Salmonella intracellular growth. SPI-2 mutants have been proposed to be more accessible than wild-type Salmonella to oxyradicals generated by the NADPH phagocyte oxidase. We performed mixed infections of mice to investigate the relationship between SPI-2 and SlyA, a transcriptional regulator that confers resistance to oxyradicals. In mixed-infection experiments, the SPI-2 null mutant was severely attenuated in virulence, whereas slyA mutants were only mildly attenuated. Surprisingly, further experiments indicated that the function of SPI-2 was partially dependent on slyA . The intracellular behavior of a slyA mutant in infected cells was consistent with inefficient SPI-2 expression, as formation of Salmonella -induced filaments and the intracellular F-actin meshwork, features that depend on SPI-2, were present at abnormally low frequencies. Furthermore, the translocated levels of the SPI-2 effector SseJ were severely reduced in a strain carrying a mutation in slyA . We used flow cytometry to investigate the role of SlyA in expression of green fluorescent protein (GFP) from transcriptional fusions with promoters of either of two other SPI-2 effector genes, sifB and sifA . The slyA mutant exhibited reduced GFP expression from both promoters. Combining mutations in slyA and other regulators of SPI-2 indicated that SlyA acts through the SsrAB two-component regulatory system. SlyA exhibits partial functional redundancy with OmpR-EnvZ and contributes to the transcriptional response to low osmolarity and the absence of calcium, two environmental stimuli that promote SPI-2 gene expression.