Salmonella enterica serovar Typhimurium can cross the epithelial barrier using either the invasion-associated type III secretion system (T3SS-1) or a T3SS-1-independent mechanism that remains poorly characterized. Here we show that flagellum-mediated motility supported a T3SS-1-independent pathway for entering ileal Peyer’s patches in the mouse model. Flagellum-dependent invasion of Peyer’s patches required energy taxis toward nitrate, which was mediated by the methyl-accepting chemotaxis protein (MCP) Tsr. Generation of nitrate in the intestinal lumen required inducible nitric oxide synthase (iNOS), which was synthesized constitutively in the mucosa of the terminal ileum but not in the jejunum, duodenum, or cecum. Tsr-mediated invasion of ileal Peyer’s patches was abrogated in mice deficient for Nos2, the gene encoding iNOS. We conclude that Tsr-mediated energy taxis enables S. Typhimurium to migrate toward the intestinal epithelium by sensing host-derived nitrate, thereby contributing to invasion of Peyer’s patches.

Expression of ABO and Lewis histo-blood group antigens by the gastrointestinal epithelium is governed by an α-1,2-fucosyltransferase enzyme encoded by the Fut2 gene. Alterations in mucin glycosylation have been associated with susceptibility to various bacterial and viral infections. Salmonella enterica serovar Typhimurium is a food-borne pathogen and a major cause of gastroenteritis. In order to determine the role of Fut2 -dependent glycans in Salmonella -triggered intestinal inflammation, Fut2 +/+ and Fut2 -/- mice were orally infected with S . Typhimurium and bacterial colonization and intestinal inflammation were analyzed. Bacterial load in the intestine of Fut2 -/- mice was significantly lower compared to Fut2 +/+ mice. Analysis of histopathological changes revealed significantly lower levels of intestinal inflammation in Fut2 -/- mice compared to Fut2 +/+ mice and measurement of lipocalin-2 level in feces corroborated histopathological findings. Salmonella express fimbriae that assist in adherence of bacteria to host cells thereby facilitating their invasion. The std fimbrial operon of S . Typhimurium encodes the π-class Std fimbriae which bind terminal α(1,2)-fucose residues. An isogenic mutant of S . Typhimurium lacking Std fimbriae colonized Fut2 +/+ and Fut2 -/- mice to similar levels and resulted in similar intestinal inflammation. In vitro adhesion assays revealed that bacteria possessing Std fimbriae adhered significantly more to fucosylated cell lines or primary epithelial cells in comparison to cells lacking α(1,2)-fucose. Overall, these results indicate that Salmonella -triggered intestinal inflammation and colonization are dependent on Std-fucose interaction.

The std locus of Salmonella enterica, an operon acquired by horizontal transfer, encodes fimbriae that permit adhesion to epithelial cells in the large intestine. Expression of the std operon is bistable, yielding a major subpopulation of StdOFF cells (99.7%) and a minor subpopulation of StdON cells (0.3%). In addition to fimbrial proteins, the std operon encodes two proteins, StdE and StdF, that have DNA binding capacity and control transcription of loci involved in flagellar synthesis, chemotaxis, virulence, conjugal transfer, biofilm formation, and other cellular functions. As a consequence of StdEF pleiotropic transcriptional control, StdON and StdOFF subpopulations may differ not only in the presence or absence of Std fimbriae but also in additional phenotypic traits. Separation of StdOFF and StdON lineages by cell sorting confirms the occurrence of lineage-specific features. Formation of StdOFF and StdON lineages may thus be viewed as a rudimentary bacterial differentiation program.

Bistable expression of the Salmonella enterica std operon is controlled by an AND logic gate involving three transcriptional activators: the LysR-type factor HdfR and the StdE and StdF regulators encoded by the std operon itself. StdE activates transcription of the hdfR gene, and StdF activates std transcription together with HdfR. Binding of HdfR upstream of the std promoter is hindered by methylation of GATC sites located within the upstream activating sequence (UAS). Epigenetic control by Dam methylation thus antagonizes formation of the StdE-StdF-HdfR loop and tilts the std switch toward the StdOFF state. In turn, HdfR binding hinders methylation of the UAS, permitting activation of the StdE-StdF-HdfR loop and concomitant formation of StdON cells. Bistability is thus the outcome of competition between DNA adenine methylation and the StdE-StdF-HdfR activator loop.

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