SspA is required for lethal Salmonella enterica serovar Typhimurium infections in calves but is not essential for diarrhea.

Salmonella pathogenicity island 1 (SPI-1) encodes virulence determinants, which are important for enteropathogenicity in calves. To determine whether the Salmonella enterica serovar Typhimurium SPI-1 effector proteins SspA and SptP are important for enteropathogenicity, strains lacking these proteins were tested during oral infection of calves. Calves infected with a sptP mutant or its isogenic parent developed diarrhea and lethal morbidity. In contrast, calves infected with an sspA mutant developed diarrhea, which resolved within 10 days but did not result in mortality. The sspA mutant was recovered from bovine intestinal tissues at numbers similar to those obtained for its isogenic parent and caused marked intestinal lesions. Thus, the severity of pathological changes caused by serovar Typhimurium strains or their ability to cause diarrhea were not predictive of their ability to cause lethal morbidity in calves. We conclude that factors other than or in addition to bacterial colonization, intestinal lesions, or electrolyte loss contribute to lethal morbidity in calves infected with serovar Typhimurium.

Physical and genetic map of Streptococcus mutans GS-5 and localization of five rRNA operons.

The physical map of the 2.1 megabase chromosome of Streptococcus mutans GS-5 has been refined by including all ApaI and SmaI fragments of 5 kbp or greater, and by positioning the fragments generated by the endonuclease I-CeuI. Sixty-three new genetic loci have been added to the map, so that it now contains 90 loci. The new loci include those for 35 cloned streptococcal genes of established function and for 23 S. mutans genes of putative function. In addition, five rrn operons were identified and placed on the map of the chromosome. The presence of a SmaI site in each of the rrn operons allowed the direction of transcription of each operon to be deduced. The orientation of the rrn loci indicates that their transcription is directed away from a small region of the chromosome, identifying a possible region for the initiation of chromosome replication.

Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins.

Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion. Here we describe four genes (SspB, sspC, sspD, and sspA), located between spaT and prgH, which encode proteins of 63, 42, 36, and 87 kDa, respectively. These Ssp are homologous to Shigella flexneri secreted proteins IpaB, IpaC, IpaD and IpaA. A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87, 42 and 36 kDa. Complementation and analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA. sspC and sspD, but not sspA, are required for invasion. Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing. We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK, homologous to the Shigella Ipa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant. A precipitate containing abundant SspC and three other major Ssp of 63, 59 and 22 kDa was isolated from culture supernatants of wild-type bacteria. These data indicate that major secreted invasion determinants of S. typhimurium are structurally and functionally homolgous to S. flexneri Ipa proteins.

PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion.

Previously, the PhoP-repressed locus prgH was identified as important for signalling epithelial cells to endocytose Salmonella typhimurium. Characterization of prgH revealed that it is an operon of four genes encoding polypeptides of 392 (prgH), 80 (prgI), 101 (prgJ) and 252 amino acid residues (prgK). Synthesis of the 2.6 kb prgHIJK transcript was repressed in bacteria that activate PhoP/PhoQ, indicating that PhoP/PhoQ regulates prgHIJK by transcriptional repression. The prgI, prgJ and prgK predicted gene products were similar to Shigella flexneri and Yersinia enterocolitica proteins required for secretion of Ipa and Yop virulence factors. Analysis of the culture supernatants from wild-type S. typhimurium demonstrated that at least 25 polypeptides larger than 14 kDa could be detected. In contrast, prgH1::TnphoA, phoP-constitutive and hil-deletion mutants had significant defects in their supernatant protein profiles. The invasion and supernatant protein profile defects of the prgH1::TnphoA mutant were both complemented by a 5.1 kb plasmid that included prgHIJK. These results suggest that PhoP/PhoQ regulates extracellular transport of proteins by transcriptional repression of secretion determinants and that secreted proteins may be involved in signalling epithelial cells to endocytose bacteria.

Chromosome organization of Streptococcus mutans GS-5.

Twenty-eight genetic loci have been physically mapped to specific large restriction fragments of the Streptococcus mutans GS-5 chromosome by hybridization with probes of cloned genes or, for transposon-generated amino acid auxotrophs, with probes for Tn916. In addition, restriction fragments generated by one low-frequency-cleavage enzyme were used as probes to identify overlapping fragments generated by other restriction enzymes. The approach allowed construction of a low resolution physical map of the S. mutans GS-5 genome using restriction enzymes ApaI (5'-GGGCC/C), SmaI (5'-CCC/GGG), and NotI (5'-GC/GGCCGC).