InvB is a type III secretion chaperone specific for SspA.

A wide variety of gram-negative bacteria utilize a specialized apparatus called the type III secretion system (TTSS) to translocate virulence factors directly into the cytoplasm of eukaryotic cells. These translocated effectors contribute to the pathogen's ability to infect and replicate within plant and animal hosts. The amino terminus of effector proteins contains sequences that are necessary and sufficient for both secretion and translocation by TTSS. Portions of these sequences contain binding sites for type III chaperones, which facilitate efficient secretion and translocation of specific effectors through TTSS. In this study, we have utilized the yeast two-hybrid assay to identify protein-protein interactions between effector and chaperone proteins encoded within Salmonella pathogenicity island 1 (SPI-1). Several interactions were identified including a novel interaction between the effector protein, SspA (SipA), and a putative chaperone, InvB. InvB was demonstrated to bind to the amino terminus of SspA in the bacterial cytoplasm. Furthermore, InvB acts as a type III chaperone for the efficient secretion and translocation of SspA by SPI-1. InvB also permitted translocation of SspA through the SPI-2 TTSS, indicating that it is an important regulator in the recognition of SspA as a target of TTSS. Finally, it was determined that InvB does not alter the transcription of sspA but that its absence results in reduced SspA protein levels in Salmonella enterica serovar Typhimurium.

A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium.

Type III secretion systems (TTSS) are important virulence factors that Gram-negative bacteria use to translocate proteins into the cytoplasm of eukaryotic host cells. Salmonellae encode two virulence-associated TTSS. The Salmonella pathogenicity island 1 (SPI1)-encoded TTSS is active on contact with host cells, whereas the Salmonella pathogenicity island 2 (SPI2)-encoded TTSS is expressed after phagocytosis of bacteria by host cells. Previously, no consensus signal sequence for translocation has been identified among TTSS effector proteins. In this work, seven proteins, termed Salmonella-translocated effectors (STE), are described that contain conserved amino acid sequences that direct translocation by TTSS in Salmonella typhimurium. STE that are coordinately regulated with SPI2 gene expression contain translocation signals that are recognized by the SPI2 but not by the SPI1 TTSS. STE that are constitutively expressed contain signals that direct translocation through both SPI1 and SPI2 TTSS. Of the seven STE examined, SspH1 and SspH2 have been previously shown to be translocated and involved in virulence; SlrP and SifA were identified as virulence factors, but were not previously known to be associated with TTSS; and SseI, SseJ, and SifB were previously unidentified. Three STE genes (sspH1, sspH2, and sseI) are located within temperate bacteriophages, suggesting a common mechanism for the dissemination of more recently evolved STE.

Identification of a putative Salmonella enterica serotype typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis.

The genetic basis for the host adaptation of Salmonella serotypes is currently unknown. We have explored a new strategy to identify Salmonella enterica serotype Typhimurium (S. typhimurium) genes involved in host adaptation, by comparing the virulence of 260 randomly generated signature-tagged mutants during the oral infection of mice and calves. This screen identified four mutants, which were defective for colonization of only one of the two host species tested. One mutant, which only displayed a colonization defect during the infection of mice, was further characterized. During competitive infection experiments performed with the S. typhimurium wild type, the mutant was defective for colonization of murine Peyer's patches but colonized bovine Peyer's patches at the wild-type level. No difference in virulence between wild type and mutant was observed when calves were infected orally with 10(10) CFU/animal. In contrast, the mutant possessed a sixfold increase in 50% lethal morbidity dose when mice were infected orally. The transposon in this mutant was inserted in a 2.9-kb pathogenicity islet, which is located between uvrB and yphK on the S. typhimurium chromosome. This pathogenicity islet contained a single gene, termed slrP, with homology to ipaH of Shigella flexneri and yopM of Yersinia pestis. These data show that comparative screening of signature-tagged mutants in two animal species can be used for scanning the S. typhimurium genome for genes involved in host adaptation.

Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems.

Salmonellae encode two virulence-associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2). Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified. SspH1 and SspH2 are proteins containing leucine-rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS. sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2-encoded regulator ssrA/ssrB. In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells. Infection of eukaryotic cells with strains expressing a SspH2-CyaA fusion protein resulted in SPI2 TTSS-dependent cAMP increases. In contrast, SspH1-CyaA-mediated cAMP increases were both SPI1 and SPI2 TTSS dependent. sspH2-like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S. typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes. S. typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S. typhimurium virulence for animals.