Salmonella typhimurium initiates infection of a host by invading M cells of Peyer's patches within the small intestine. The ability of the bacteria to invade mammalian cells has been shown to be regulated by environmental conditions, including oxygen concentrations, osmolarity, and growth phase. We have previously created oxygen-regulated Tn5lacZY S. typhimurium mutants that are defective in invasion. We have now identified the invasion genes disrupted by eight of the transposon insertions. These genes encode transcriptional regulators (hilA and invF), type III secretory components (orgA, invG and spaR) and secreted proteins (invC and invD). Examination of the protein-secretion profiles of the non-invasive mutants indicated that each of the mutants was defective in secretion of between one and six proteins. We have also demonstrated that the loss of tissue culture cell invasiveness corresponds to an inability to invade and destroy M cells of Peyer's patches in a murine ligated loop model. Virulence studies, performed in mice, demonstrated that these defects significantly reduced the ability of the mutants to cause murine typhoid fever by an oral route of infection. Virulence by an intraperitoneal route of infection was unaffected. The data indicate that in vitro invasiveness, invasion-protein secretion, and M-cell invasion are critical indicators of S. typhimurium virulence.

The ability of Salmonella enterica serovar Typhimurium to traverse the intestinal mucosa of a host is an important step in its ability to initiate gastrointestinal disease. The majority of the genes required for this invasive characteristic are encoded on Salmonella pathogenicity island 1 (SPI1), and their expression is controlled by the transcriptional activator HilA, a member of the OmpR/ToxR family of proteins. A variety of genes (hilC, hilD, fis, sirA/barA, csrAB, phoB, fadD, envZ/ompR, fliZ, hilE, ams, lon, pag, and hha) have been identified that exert positive or negative effects on hilA expression, although the mechanisms by which these gene products function remain relatively unclear. Recent work indicates that the small DNA-binding protein, Hha, has a significant role in repressing hilA transcription and the invasive phenotype, particularly in response to osmolarity signals. We have characterized the Salmonella-specific gene, hilE, and found that it plays an important regulatory role in hilA transcription and invasion gene expression. Mutation of hilE causes derepression of hilA transcription, and overexpression of hilE superrepresses hilA expression and the invasive phenotype. Bacterial two-hybrid experiments indicate that the HilE protein interacts with HilD, suggesting a possible mechanism for HilE negative regulation of hilA gene expression and the Salmonella invasive phenotype. Finally, we have found that the hilE gene resides on a region of the serovar Typhimurium chromosome that has many characteristics of a pathogenicity island.

An early step in the establishment of Salmonella enterica serovar Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine. The majority of the genes responsible for the Salmonella invasive phenotype are encoded on Salmonella pathogenicity island 1, and their transcription is controlled by the hilA transcriptional activator. The expression of hilA is regulated by environmental signals including oxygen, osmolarity, pH, and growth phase such that the presence of any one suboptimal condition results in repression of hilA expression and the invasive phenotype. We have conducted a search for negative regulators of hilA by introduction of a Salmonella enterica serovar Typhimurium chromosomal DNA gene bank into a Salmonella enterica serovar Typhimurium hilA::Tn5lacZY reporter strain. This screen has identified the hha gene as a regulator that exerts a negative influence on hilA expression. Plasmid-encoded hha significantly reduces hilA::Tn5lacZY chromosomal expression, as well as expression of the invasion genes invF, prgH, and sipC. An hha null mutation results in substantial derepression of both chromosomally encoded and plasmidencoded hilA::Tn5lacZY expression. Introduction of plasmid-encoded hha into strain SL1344 results in attenuation of invasion using in vitro and in vivo assays. Importantly, purified Hha protein was found to bind to a hilA DNA promoter fragment, suggesting that the regulatory activity of the Hha protein occurs at the hilA promoter. These data add detail to the developing model of the regulation of Salmonella invasion genes.

Salmonella enterica serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of Salmonella into intestinal epithelial cells is dependent upon invasion genes that are clustered together in Salmonella pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the prgH, -I, -J, and -K and orgA genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published orgA DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated orgA and orgB. Additionally, an opening reading frame downstream of orgB, which we have designated orgC, has been identified and partially characterized. Previously published work has indicated that the prgH, -I, -J, and -K genes are transcribed from a promoter distinct from that used by the gene immediately downstream, orgA. Here, we present experiments indicating that orgA expression is driven by the prgH promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of prgH to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the prgH, prgI, prgJ, prgK, orgA, and orgB genes are each required for invasion and secretion, while orgC is not essential for the invasive phenotype.Salmonella infections are an important health problem in both the developing and developed world (9,32,34). Pathogenic Salmonella species cause infections that range in severity from self-limiting gastroenteritis to life-threatening systemic dissemination (45). After entry into a host, the bacteria establish infection by attaching to and invading specialized M cells associated with Peyer's patches in the small intestine (5,23,26). Following M-cell invasion and destruction, host-restricted Salmonella species cause localized destruction of the intestinal epithelium (gastroenteritis). In contrast, passage of hostadapted Salmonella species through M cells allows rapid dissemination to the mesenteric lymph nodes and then to the liver and spleen, where unchecked growth causes death (25).A critical determinant in the development of Salmonella disease is the ability of the bacteria to invade cells. Salmonella enterica serovar Typhimurium mutants that are unable to invade tissue culture cells are defective in their ability to invade and destroy M cells (26,43). This defect severely limits the ability of the bacteria to initiate infection and reduces their virulence in mice (14,24,43). Genes required for Salmonella internalization into mammalian cells have been identified (4, 7, ...

The ability of Salmonella enterica serovar Typhimurium to cause disease depends upon the co‐ordinated expression of many genes located around the Salmonella chromosome. Specific pathogenicity loci, termed Salmonella pathogenicity islands, have been shown to be crucial for the invasion and survival of Salmonella within host cells. Salmonella pathogenicity island 1 (SPI‐1) harbours the genes required for the stimulation of Salmonella uptake across the intestinal epithelia of the infected host. Regulation of SPI‐1 genes is complex, as invasion gene expression responds to a number of different signals, presumably signals similar to those found within the environment of the intestinal tract. As a result of our continued studies of SPI‐1 gene regulation, we have discovered that the nucleoid‐binding protein Fis plays a pivotal role in the expression of HilA and InvF, two activators of SPI‐1 genes. A S. typhimurium fis mutant demonstrates a two‐ to threefold reduction in hilA::Tn5lacZY and a 10‐fold reduction in invF::Tn5lacZY expression, as well as a 50‐fold decreased ability to invade HEp‐2 tissue culture cells. This decreased expression of hilA and invF resulted in an altered secreted invasion protein profile in the fis mutant. Furthermore, the virulence of a S. typhimurium fis mutant is attenuated 100‐fold when administered orally, but has wild‐type virulence when administered intraperitoneally. Expression of hilA::Tn5lacZY and invF::Tn5lacZY in the fis mutant could be restored by introducing a plasmid containing the S. typhimurium fis gene or a plasmid containing hilD, a gene encoding an AraC‐like regulator of Salmonella invasion genes.