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Bacterial lipoproteins are well-recognized microorganism-associated molecular patterns, which interact with Toll-like receptor (TLR) 2, an important pattern recognition receptor of the host innate immune system. Lipoproteins are conjugated with two- or three-acyl chains (di- or tri-acyl), which is essential for appropriate anchoring in the cell membrane as well as for the interaction with TLR2. Lipoproteins have mostly been studied in pathogens and have established roles in various biological processes, such as nutrient import, cell wall cross-linking and remodeling, and host-cell interaction. By contrast, information on the role of lipoproteins in the physiology and host interaction of probiotic bacteria is scarce. By deletion of lgt, encoding prolipoprotein diacylglyceryl transferase, responsible for lipidation of lipoprotein precursors, we investigated the roles of the collective group of lipoproteins in the physiology of the probiotic model strain Lactobacillus plantarum WCFS1 using proteomic analysis of secreted proteins. To investigate the consequences of the lgt mutation in host-cell interaction, the capacity of mutant and wild-type bacteria to stimulate TLR2 signaling and inflammatory responses was compared using (reporter-) cell-based models. These experiments exemplified the critical contribution of the acyl chains of lipoproteins in immunomodulation. To the best of our knowledge, this is the first study that investigated collective lipoprotein functions in a model strain for probiotic lactobacilli, and we show that the lipoproteins in L. plantarum WCFS1 are critical drivers of anti-inflammatory host responses toward this strain.

There is much interest in the immunomodulatory properties of dietary fibers but their activity may be influenced by contamination with microbial-Associated molecular patterns (MAMPs) such as lipopolysaccharide (LPS) and lipoteichoic acids, which are difficult to remove completely from biological samples. Bone marrow-derived dendritic cells (BMDCs) from TLR2x4 double-KO mice were shown to be a reliable approach to analyse the immunomodulatory properties of a diverse range of dietary fibers, by avoiding immune cell activation due to contaminating MAMPs. Several of the 44 tested dietary fiber preparations induced cytokine responses in BMDCs from TLR2x4 double-KO mice. The particulate fractions of linear arabinan (LA) and branched arabinan (BA) from sugar beet pectin were shown to be strongly immune stimulatory with LA being more immune stimulatory than BA. Enzymatic debranching of BA increased its immune stimulatory activity, possibly due to increased particle formation by the alignment of debranched linear arabinan. Mechanistic studies showed that the immunostimulatory activity of LA and BA was independent of the Dectin-1 recognition but Syk kinase-dependent.

Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc 1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc-1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function.

Orally ingested bacteria interact with intestinal mucosa and may impact immunity. However, insights in mechanisms involved are limited. In this randomized placebo-controlled cross-over trial, healthy human subjects were given Lactobacillus plantarum supplementation (strain TIFN101, CIP104448, or WCFS1) or placebo for 7 days. To determine whether L. plantarum can enhance immune response, we compared the effects of three stains on systemic and gut mucosal immunity, by among others assessing memory responses against tetanus toxoid (TT)-antigen, and mucosal gene transcription, in human volunteers during induction of mild immune stressor in the intestine, by giving a commonly used enteropathic drug, indomethacin [non-steroidal anti-inflammatory drug (NSAID)]. Systemic effects of the interventions were studies in peripheral blood samples. NSAID was found to induce a reduction in serum CD4+ /Foxp3 regulatory cells, which was prevented by L. plantarum TIFN101. T-cell polarization experiments showed L. plantarum TIFN101 to enhance responses against TT-antigen, which indicates stimulation of memory responses by this strain. Cell extracts of the specific L. plantarum strains provoked responses after WCFS1 and TIFN101 consumption, indicating stimulation of immune responses against the specific bacteria. Mucosal immunomodulatory effects were studied in duodenal biopsies. In small intestinal mucosa, TIFN101 upregulated genes associated with maintenance of T- and B-cell function and antigen presentation. Furthermore, L. plantarum TIFN101 and WCFS1 downregulated immunological pathways involved in antigen presentation and shared downregulation of snoRNAs, which may suggest cellular destabilization, but may also be an indicator of tissue repair. Full sequencing of the L. plantarum strains revealed possible gene clusters that mightbe responsible for the differential biological effects of the bacteria on host immunity. In conclusion, the impact of oral consumption L. plantarum on host

Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure-function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization.

Lactobacilli are thought to be beneficial for human health, with lactobacilli-associated infections being confined to immune-compromised individuals. However, Lactobacillus fermentum AGR1487 negatively affects barrier integrity in vitro so we hypothesized that it caused a pro-inflammatory response in the host. We compared germ-free rats inoculated with AGR1487 to those inoculated with another L. fermentum strain, AGR1485, which does not affect in vitro barrier integrity. We showed that rats inoculated with AGR1487 had more inflammatory cells in their colon, higher levels of inflammatory biomarkers, and increased colonic gene expression of pro-inflammatory pathways. In addition, our in vitro studies showed that AGR1487 had a greater capacity to activate TLR signaling and induce pro-inflammatory cytokines in immune cells. This study indicates the potential of strains of the same species to differentially elicit inflammatory responses in the host and highlights the importance of strain characterization in probiotic approaches to treat inflammatory disorders.

Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strainspecific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction.

Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen β-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.

BACKGROUND: In the intestinal mucosa, several adaptations of TLR signalling have evolved to avoid chronic inflammatory responses to the presence of commensal microbes. Here we investigated whether polarized monolayers of intestinal epithelial cells might regulate inflammatory responses by secreting IL-8 in a vectorial fashion (i.e. apical versus basolateral) depending on the location of the TLR stimulus. RESULTS: In the Caco-2 BBE model of polarized villus-like epithelium, apical stimulation with TLR2 and TLR5 ligands resulted in the apical secretion of IL-8. The CXCR1 receptor for IL-8 was expressed only on the apical membrane of Caco-2 BBE cells and differentiated epithelial cells in the human small intestine and colon. Transcriptome analyses revealed that Caco-2 BBE cells respond to stimulation with IL-8 supporting the hypothesis that IL-8 induces G protein-coupled receptor signalling. CONCLUSIONS: These results show that IL-8 induces autocrine signalling via an apical CXCR1 in Caco-2 BBE intestinal epithelial cells and that this receptor is also expressed on the apical surface of differentiated human intestinal epithelial cells in vivo, suggesting an autocrine function for IL-8 secreted in the lumen.