Salmonella typhimurium virulence genes are induced upon bacterial invasioninto phagocytic and nonphagocytic cells

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella t yphimurium genes that are induced inside Salmonella-containing vacuoles wit hin macrophage and epithelial cells. A promoterless luciferase gene cassett e was inserted randomly into the Salmonella chromosome, and the resulting m utants were screened for genes upregulated in intracellular bacteria compar ed to extracellular bacteria. We identified four genes in S. typhimurium th at were upregulated upon bacterial invasion of both phagocytic and nonphago cytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time poi nts (2 to 4 h) postinvasion and continued to be upregulated within host cel ls at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished ba cterial virulence in a murine typhoid model. Two other mutants contained in sertions within SPI-5, one in the sopB/sigD gene and the other in a downstr eam gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, i icA (induced intracellularly A). We detected no effect on virulence as a re sult of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustra ting the requirement for specific gene expression inside mammalian cells an d indicating the key role that virulence factor regulation plays In Salmone lla pathogenesis.