SummaryThe Salmonella enterica serotype Typhimurium ( S. Typhimurium) genome contains 13 putative fimbrial operons termed agf ( csg ), fim , pef , lpf , bcf , saf , stb , stc , std , stf , sth , sti and stj . Evidence for in vitro expression of fimbrial proteins encoded by these operons is currently only available for agf , fim and pef . We raised antisera against putative major fimbrial subunits of S. Typhimurium, including AgfA, FimA, PefA, LpfA, BcfA, StbA, StcA, StdA, StfA, SthA and StiA. Elaboration of StcA on the bacterial surface could be detected by flow cytometry and immunoelectron microscopy after expression of the cloned stcABCD operon from a heterologous T7 promoter in Escherichia coli. To study the expression of fimbrial antigens in S. Typhimurium by flow cytometry, we constructed strains carrying deletions of agfAB , pef-BACDI , lpfABCDE , bcfABCDEFG , stbABCD , stcABC , stdAB , stfACDEFG , sthABCDE or stiABCDE . Using these deletion mutants for gating, expression of fimbrial antigens was measured by flow cytometry in cultures grown in vitro or in samples recovered 8 h after infection of bovine ligated ileal loops with S.

SummaryKnowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen-associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant ( csgBA ) was attenuated in its ability to elicit fluid accumulation and GRO α α α α mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL-8 production than the wild type in human macrophage-like cells. Purified thin curled fimbriae induced IL-8 expression in human embryonic kidney (HEK293) cells transfected with Toll-like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione-S-transferase (GST) elicited

Salmonella enterica serotype Typhimurium causes human infections that can frequently be traced back through the food chain to healthy livestock whose intestine is colonized by the pathogen. Little is known about the genes important for intestinal carriage of S. enterica serotype Typhimurium in vertebrate animals. Here we characterized the role of 10 fimbrial operons, agf, fim, lpf, pef, bcf, stb, stc, std, stf, and sth, using competitive infection experiments performed in genetically susceptible (BALB/c) and resistant (CBA) mice. Deletion of agfAB, fimAICDHF, lpfABCDE, pefABCDI, bcfABCDEFG, stbABCD, stcABCD, stdAB, stfACDEFG, or sthABCDE did not reduce the ability of S. enterica serotype Typhimurium to colonize the spleen and cecum of BALB/c mice 5 days after infection. Similarly, deletion of agfAB, fimAICDHF, pefABCDI, and stfACDEFG did not result in reduced recovery of S. enterica serotype Typhimurium from fecal samples collected from infected CBA mice over a 30-day time period. However, S. enterica serotype Typhimurium strains carrying deletions in lpfABCDE, bcfABCDEFG, stbABCD, stcABCD, stdAB, or sthABCDE were recovered at significantly reduced numbers from the feces of CBA mice. There was a good correlation (R 2 ‫؍‬ 0.9626) between competitive indices in the cecum and fecal samples of CBA mice at 30 days after infection, suggesting that the recovery of S. enterica serotype Typhimurium from fecal samples closely reflected its ability to colonize the cecum. Collectively, these data show that six fimbrial operons (lpf, bcf, stb, stc, std, and sth) contribute to long-term intestinal carriage of S. enterica serotype Typhimurium in genetically resistant mice.Nontyphoidal Salmonella serotypes are the leading cause of food-borne infections with a lethal outcome in the United States (30). S. enterica serotype Typhimurium is the serotype most frequently associated with this diarrheal disease (34). S. enterica serotype Typhimurium is commonly transmitted from animal to human through food products derived from livestock or domestic fowl (34). Intestinal carriage results in fecal contamination of equipment surfaces or workers hands with S. enterica serotype Typhimurium, thereby leading to contamination of carcasses and processed foods at processing plants. For this reason, the presence of S. enterica serotype Typhimurium in the intestine of healthy food animals arriving at slaughter is the main risk factor for introducing this pathogen into the human food supply. Although intestinal carriage of S. enterica serotype Typhimurium in livestock and domestic fowl is of considerable importance for food safety, the mechanisms that enable this pathogen to persist in the alimentary tract of vertebrate hosts remain poorly characterized.Mice of genetically susceptible lineages (e.g., BALB/c) commonly used to study S. enterica serotype Typhimurium pathogenesis succumb to infection within 6 to 10 days after inoculation and are thus not well suited for studying mechanisms of long-term intestinal persistence. As a result, reports on ge...

The shdA gene is carried on a 25-kb genetic island at centisome 54 (CS54 island) of the Salmonella enterica serotype Typhimurium chromosome. In addition to shdA, the CS54 island of Salmonella serotype Typhimurium strain LT2 contains four open reading frames designated ratA, ratB, sivI, and sivH. DNA hybridization analysis revealed that the CS54 island is comprised of two regions with distinct phylogenetic distribution within the genus Salmonella. Homologues of shdA and ratB were detected only in serotypes of Salmonella enterica subsp. I. In contrast, sequences hybridizing with ratA, sivI, and sivH were present in S. enterica subsp. II and S. bongori in addition to S. enterica subsp. I. Deletion of the ratA and sivI genes did not alter the ability of Salmonella serotype Typhimurium to colonize the organs of mice. Insertional inactivation of the sivH gene resulted in defective colonization of the Peyer's patches of the terminal ileum but normal colonization of the cecum, mesenteric lymph nodes, and spleen. Deletion of the shdA gene resulted in decreased colonization of the cecum and Peyer's patches of the terminal ileum and colonization to a lesser degree in the mesenteric lymph nodes and spleen 5 days post-oral inoculation of mice. A strain containing a deletion in the ratB gene exhibited a defect for the colonization of the cecum but not of the Peyer's patches, mesenteric lymph nodes, and spleen. The shdA and ratB deletion strains exhibited a shedding defect in mice, whereas the sivH deletion strain was shed at numbers similar to the wild type. These data suggest that colonization of the murine cecum is required for efficient fecal shedding in mice.

SummaryMisL is an autotransporter protein encoded by Salmonella pathogenicity island 3 (SPI3). To investigate the role of MisL in Salmonella enterica serotype Typhimurium ( S. Typhimurium) pathogenesis, we characterized its function during infection of mice and identified a host receptor for this adhesin. In a mouse model of S. Typhimurium intestinal persistence, a misL mutant was shed with the faeces in significantly lower numbers than the wild type and was impaired in its ability to colonize the cecum. Previous studies have implicated binding of extracellular matrix proteins as a possible mechanism for S. Typhimurium intestinal persistence. A gluthathione-S-transferase (GST) fusion protein to the MisL passenger domain (GST-MisL 29-281 ) was constructed to investigate binding to extracellular matrix proteins. In a solid-phase binding assay the purified GST-MisL 29-281 fusion protein bound to fibronectin and collagen IV, but not to collagen I. MisL expression was not detected by Western blot in S. Typhimurium grown under standard laboratory conditions. However, when expression of the cloned misL gene was driven by the Escherichia coli arabinose promoter, MisL could be detected in the S. Typhimurium outer membrane by Western blot and on the bacterial cell surface by flow cytometry. Expression of MisL enabled S. Typhimurium to bind fibronectin to its cell surface, resulting in attachment to fibronectin-coated glass slides and in increased invasiveness for human epithelial cells derived from colonic carcinoma (T84 cells). These data identify MisL as an extracellular matrix adhesin involved in intestinal colonization.