Transcriptional Organization and Function of Invasion Genes within Salmonella enterica Serovar Typhimurium Pathogenicity Island 1, Including the prgH , prgI , prgJ , prgK , orgA , orgB , and orgC Genes

Abstract

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, ...

DNA-Binding Activities of the HilC and HilD Virulence Regulatory Proteins ofSalmonella entericaSerovar Typhimurium

“…The HilC and HilD proteins repressed the upstream promoter, which uses an extended Ϫ10 element at positions Ϫ88 to Ϫ80, and activate the silent downstream promoter. Reverse transcriptase PCR analysis suggested that hilC is also transcribed from even farther upstream (21). These results indicate that HilC and HilD can function as direct transcription activators at the hilC promoter, unlike their proposed role at the hilA promoter to relieve repression.…”

Salmonella Pathogenicity Island 2-Encoded Type III Secretion System Mediates Exclusion of NADPH Oxidase Assembly from the Phagosomal Membrane

“…The 2-kb DNA product was cloned into pGEM-T Easy (Promega, Madison, WI) and a blunt-ended aphT gene encoding kanamycin resistance was ligated into the middle of the sseD gene digested with EcoRV. The sseD-aphT construct was cloned into the pBDJ129 vector, followed by selection for allelic exchange of the mutated sseD into the SL1344 chromosome (18,19). This strain was designated S. typhimurium JK22.…”

The HilA Box and Sequences outside It Determine the Magnitude of HilA-Dependent Activation of P prgH from Salmonella Pathogenicity Island 1

“…The core of the complex, probably found embedded in the inner membrane and crossing the periplasmic space, is comprised of PrgH and PrgK, while the barrel of the syringe is predominantly PrgI, a protein that requires PrgJ for its stability (20). The genes encoding these proteins are in an operon (prgHIJK-orgABC) at one end of SPI1 (20)(21)(22)(23) (Fig. 1A).…”