An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice. This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S. typhimurium.

SummaryThe type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for systemic infection of this pathogen in mice. Cloning and sequencing of a central region of SPI-2 revealed the presence of genes encoding putative chaperones and effector proteins of the secretion system. The predicted products of the sseB, sseC and sseD genes display weak but significant similarity to amino acid sequences of EspA, EspD and EspB, which are secreted by the type III secretion system encoded by the locus of enterocyte effacement of enteropathogenic Escherichia coli. The transcriptional activity of an sseA::luc fusion gene was shown to be dependent on ssrA, which is required for the expression of genes encoding components of the secretion system apparatus. Strains carrying nonpolar mutations in sseA, sseB or sseC were severely attenuated in virulence, strains carrying mutations in sseF or sseG were weakly attenuated, and a strain with a mutation in sseE had no detectable virulence defect. These phenotypes were reflected in the ability of mutant strains to grow within a variety of macrophage cell types: strains carrying mutations in sseA, sseB or sseC failed to accumulate, whereas the growth rates of strains carrying mutations in sseE, sseF or sseG were only modestly reduced. These data suggest that, in vivo, one of the functions of the SPI-2 secretion system is to enable intracellular bacterial proliferation.

Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type Ill secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90%o identity with those of the E. coli ydhE and pykF genes. Thus SP12 was probably acquired horizontally by insertion into a region corresponding to that between theydhE and pykF genes of E. coli. Virulence studies of SP12 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.Salmonella typhimurium is a principal agent of gastroenteritis in humans and produces a systemic illness in mice that serves as a model for human typhoid fever (1). After oral inoculation of mice with S. typhimurium, the bacteria pass from the lumen of the small intestine through the intestinal mucosa via enterocytes or M cells of the Peyer's patch follicles (2). The bacteria then invade macrophages and neutrophils, enter the reticuloendothelial system, and disseminate to other organs, including the spleen and liver, where further reproduction results in an overwhelming and fatal bacteremia (3). To invade host cells, to survive and replicate in a variety of physiologically stressful intracellular and extracellular environments, and to circumvent the specific antibacterial activities of the immune system, S. typhimurium employs a sophisticated repertoire of virulence factors (4).To gain a more comprehensive understanding of virulence mechanisms of S. typhimurium and other pathogens, we recently developed a transposon mutagenesis system called signature-tagged mutagenesis, which combines the strength of mutational analysis with the ability to follow simultaneously the fate of a large number of different mutants within a single animal (5). By using this approach, we identified 43 mutants with attenuated virulence from a total of 1152 mutants that were screened. The nucleotide sequences of DNA flanking the insertion points of transposons in 5 of these mutants showed that they were related to genes encoding type III secretion systems of a variety of bacterial pathogens (6, 7). The products of the inv/spa gene cluster of S. typhimurium (8,9) are proteins that form a type III secretion system required for the assembly

SummaryWe have investigated the structure and transcriptional organization of 13 genes of Salmonella Pathogenicity Island 2 (SPI2) that encode components of the second type III secretion apparatus of Salmonella typhimurium. ssaK, L, M, V, N, O, P, Q, R, S, T, U constitute one operon of 10 kb. ssaJ lies upstream of ssaK and is the terminal gene of another operon. The deduced products of ssaJ, ssaK, ssaV, ssaN, ssaO, ssaQ, ssaR, ssaS, ssaT, and ssaU show greatest similarity to the Yersinia spp. genes yscJ, yscL, lcrD, yscN, yscO, yscQ, yscR, yscS, yscT, and yscU, respectively. The products of the ssaL, ssaM and ssaP genes do not have significant similarity to products of other type III secretion systems, and might be important for the specific function of the SPI2 type III secretion system. Bacterial strains carrying different ssa mutations display minor alterations in terms of serum sensitivity when compared with the wild-type strain, but none are defective in replication within macrophage-like RAW 264.7 cells. However, some of the ssa mutant strains invade HEp2 cells less efficiently and are less cytotoxic to RAW 264.7 macrophages than the wild-type strain. We show that the invasion defect is correlated with a lack of SipC in culture supernatants of these mutant strains.SipC is a product of the SPI1 type III secretion system of S. typhimurium, and is important for epithelial cell invasion. Therefore, mutations in SPI2 can affect the SPI1 secretion system, which raises the possibility of an interaction between the two type III secretion systems.

We recently identified a pathogenicity island (SPI2) located at 30.7 centisomes on the Salmonella typhimurium chromosome. SPI2 contains genes encoding a type III secretion system whose function is distinct from that of the type III secretion system encoded by a pathogenicity island (SPI1) at 63 centisomes which is involved in epithelial cell entry. An analysis of the boundaries of SPI2 and comparison with the corresponding region of the Escherichia coli chromosome revealed that SPI2 inserted adjacent to the tRNA Val gene. The E. coli chromosome contains 9 kb of DNA at the region corresponding to the SPI2 insertion point which appears to be absent in S. typhimurium. The distribution of SPI1 and SPI2 was examined in various Salmonella isolates. In contrast to type III secretion system genes of SPI1, those of SPI2 are not present in Salmonella bongori, which diverged at the first branch point in the Salmonella lineage. These and other data indicate that SPI2 was acquired by a Salmonella strain already harboring SPI1 by horizontal transfer from an unknown source.Diseases caused by Salmonella spp. range from self-limiting gastroenteritis to typhoid fever, which can be fatal (28). A useful tool for investigation of the systemic form of salmonellosis is the murine model of typhoid-like illness caused by Salmonella typhimurium (full taxonomical name, Salmonella enterica subsp. enterica serotype Typhimurium). Many virulence factors required at different stages of S. typhimurium infection have been characterized at the molecular level. The genes encoding these virulence factors are distributed on the Salmonella chromosome and the 92-kb virulence plasmid (20).An important stage in S. typhimurium pathogenesis is invasion of the gut epithelium. A large number of genes is required for epithelial cell invasion, and it has been shown that these encode the structural components of a type III (contact-dependent) secretion system, the secreted effector proteins, and associated regulatory proteins (17). These genes are clustered at 63 centisomes (cs) on the S. typhimurium chromosome, and recent analysis of this locus revealed that it constitutes a pathogenicity island (PAI) (29). PAIs comprise large, sometimes unstable chromosomal regions harboring clusters of virulence genes and are often either flanked by insertion sequence elements (16) or appear to have inserted in or adjacent to tRNA genes (6, 12). The locus for enterocyte effacement of enteropathogenic Escherichia coli is a PAI; like the invasion locus of S. typhimurium, it contains genes for a type III secretion system. Homologs of these genes are also found on the virulence plasmids of Yersinia spp. (for a review, see reference 13) and Shigella spp. (for a review, see reference 32). The DNA base composition of PAIs often differs from those of the bacterial chromosomes in which they are located, indicating that they have probably been acquired by horizontal gene transfer (for a review, see reference 11).During a search for new virulence genes of S. typhimurium by signature-t...