Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS + ) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS + B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium . Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial–host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

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Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus . Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444 T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli . These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.

Listeria monocytogenes is a Gram-positive bacterium that can cause septicemia and meningitis. TLRs are central receptors of the innate immune system that drive inflammatory responses to invading microbes such as L. monocytogenes. Although intestinal epithelial cells (IECs) represent the initial point of entry used by L. monocytogenes for infection, the innate immune response to L. monocytogenes in these cells has been poorly characterized to date. The aim of this study was to determine which TLRs are involved in mediating the immune response to L. monocytogenes in IECs. We performed an RNA interference screen of TLRs 1–10 in the HT-29 IEC cell line and observed the most significant reduction in chemokine output following silencing of TLR10. This effect was also observed in the macrophage cell line THP-1. The chemokines CCL20, CCL1, and IL-8 were reduced following knockdown of TLR10. Silencing of TLR10 resulted in increased viability of L. monocytogenes in both HT-29 and THP-1 cells. TLR10 was found to be predominantly expressed intracellularly in epithelia, and activation required viable L. monocytogenes. NF-κB activation was seen to require TLR2 in addition to TLR10. Taken together, these data indicate novel roles for TLR10 in sensing pathogenic infection in both the epithelium and macrophages and have identified L. monocytogenes as a source of ligand for the orphan receptor TLR10.