[Abstract unavailable

The centisome 63 type III secretion system (T3SS-1) encoded by Salmonella pathogenicity island 1 (SPI1) mediates invasion of epithelial cells by Salmonella enterica serotype Typhimurium. Characterization of mutants lacking individual genes has revealed that T3SS-1 secreted proteins (effectors) SopE2 and SopB are required for invasion while the SipA protein accelerates entry into cells. Here we have revisited the question of which T3SS-1 effectors contribute to the invasion of epithelial cells by complementing a strain lacking all of the effector genes that are required to cause diarrhea in a calf (a sipA sopABDE2 mutant). Introduction of either the cloned sipA, the cloned sopB, or the cloned sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for nonpolarized HT-29 cells. However, a contribution of sopA or sopD to invasion was not apparent when invasion assays were performed with the nonpolarized colon carcinoma cell lines T84 and HT-29. In contrast, introduction of either the sopA, the sopB, the sopD, or the sopE2 gene increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells. Furthermore, introduction of a plasmid carrying sipA and sopB increased the invasiveness of the sipA sopABDE2 mutant for polarized T84 cells significantly compared to the introduction of plasmids carrying only sipA or sopB. We conclude that SipA, SopA, SopB, SopD, and SopE2 contribute to S. enterica serotype Typhimurium invasion of epithelial cells in vitro.

Silver nanoparticles (AgNP) are widely used for their antibacterial properties. Incorporation of AgNP into food-related products and health supplements represents a potential route for oral exposure to AgNP; however, the effects of such exposure on the gastrointestinal system are mostly unknown. This study evaluated changes in the populations of intestinal-microbiota and intestinal-mucosal gene expression in Sprague-Dawley rats (both male and female) that were gavaged orally with discrete sizes of AgNP (10, 75 and 110 nm) and silver acetate. Doses of AgNP (9, 18 and 36 mg/kg body weight/day) and silver acetate (100, 200 and 400 mg/kg body weight/day) were divided and administered to rats twice daily (∼10 h apart) for 13 weeks. The results indicate that AgNP prompted size- and dose-dependent changes to ileal-mucosal microbial populations, as well as, intestinal gene expression and induced an apparent shift in the gut microbiota toward greater proportions of Gram-negative bacteria. DNA-based analyses revealed that exposure to 10 nm AgNP and low-dose silver acetate caused a decrease in populations of Firmicutes phyla, along with a decrease in the Lactobacillus genus. Analysis of host gene expression demonstrated that smaller sizes and lower doses of AgNP exposure prompted the decreased expression of important immunomodulatory genes, including MUC3, TLR2, TLR4, GPR43 and FOXP3. Gender-specific effects to AgNP exposure were more prominent for the gut-associated immune responses. These results indicate that the oral exposure to AgNP alter mucosa-associated microbiota and modulate the gut-associated immune response and the overall homeostasis of the intestinal tract.

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.

SummaryModels of the outer epithelia of the human body -namely the skin, the intestine and the lung -have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.