Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual “Salmonella translocated effector.” Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS−/− mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.

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The type III secretion system (TTSS) encoded by Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI2) is employed by Salmonella enterica for interaction with host cells during the intracellular phase of pathogenesis. This TTSS secretes a set of SPI2-encoded proteins in vitro and translocates Salmonella serovar Typhimurium translocated effectors (STE) that are encoded by genes outside of SPI2 into host cells. Using an epitope-tagging approach, we analyzed secretion of proteins by the TTSS of SPI2 and identified SseF and SseG as further secreted substrate proteins. Three members of the STE family, SifA, SifB, and SseJ, were secreted under conditions that also induce secretion of SPI2-encoded substrate proteins.

Plasmid vectors and fluorescent protein reporter systems are commonly used in the study of bacterial pathogenesis. Here we show that they can impair the ability of Salmonella enterica serovar Typhimurium to productively infect either cultured mammalian cells or mice. This has significant implications for studies that rely on these systems.

Acquisition of genomic elements by horizontal gene transfer represents an important mechanism in the evolution of bacterial species. Pathogenicity islands are a subset of horizontally acquired elements present in various pathogens. These elements are frequently located adjacent to tRNA genes. We performed a comparative genome analysis of Salmonella enterica serovars Typhi and Typhimurium and Escherichia coli and scanned tRNA loci for the presence of species-specific, horizontally acquired genomic elements. A large number of species-specific elements were identified. Here, we describe the characteristics of four large chromosomal insertions at tRNA genes of Salmonella spp. The tRNA-associated elements harbor various genes previously identified as single virulence genes, indicating that these genes have been acquired with large chromosomal insertions. Southern blot analyses confirmed that the tRNA-associated elements are specific to Salmonella and also indicated a heterogeneous distribution within the salmonellae. Systematic scanning for insertions at tRNA genes thus represents a tool for the identification of novel pathogenicity islands.