Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function.

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.

Isolation and whole genome sequencing of a Ruminococcus-like bacterium, associated with irritable bowel syndrome.

In our previous studies on the intestinal microbiota in irritable bowel syndrome (IBS), we identified a bacterial phylotype with higher abundance in patients suffering from diarrhea than in healthy controls. In the present work, we have isolated in pure culture strain RT94, belonging to this phylotype, determined its whole genome sequence and performed an extensive genomic analysis and phenotypical testing. This revealed strain RT94 to be a strict anaerobe apparently belonging to a novel species with only 94% similarity in the 16S rRNA gene sequence to the closest relatives Ruminococcus torques and Ruminococcus lactaris. The G + C content of strain RT94 is 45.2 mol% and the major long-chain cellular fatty acids are C16:0, C18:0 and C14:0. The isolate is metabolically versatile but not a mucus or cellulose utilizer. It produces acetate, ethanol, succinate, lactate and formate, but very little butyrate, as end products of glucose metabolism. The mechanisms underlying the association of strain RT94 with diarrhea-type IBS are discussed.

Genome Sequence of the Butyrate-Producing Anaerobic Bacterium Anaerostipes hadrus PEL 85.

Anaerostipes hadrus PEL 85, which was isolated from human feces, is a Gram-positive rod-shaped bacterium. The species may play an important role in gut health, as it was previously reported to produce butyric acid. Here, we present the genome assembly of PEL 85, a novel strain of A. hadrus.

Functional characterization of probiotic surface layer protein-carrying Lactobacillus amylovorus strains.

BACKGROUND: Adhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) -layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins. RESULTS: Three of the L. amylovorus strains studied adhered to IPEC-1 cells, while four strains inhibited the adherence of E. coli, indicating additional mechanisms other than competition for binding sites being involved in the inhibition. None of the strains bound to porcine mucus. The culture supernatants of all of the strains exerted inhibitory effects on the growth of E. coli, Salmonella, Listeria and Yersinia, and a variable, strain-dependent induction was observed of both pro- and anti-inflammatory cytokines in human DCs. L. amylovorus DSM 16698 was shown to carry two S-layer-like proteins on its surface in addition to the major S-layer protein SlpA. In contrast to expectations, none of the major S-layer proteins of the IPEC-1 -adhering strains mediated bacterial adherence. CONCLUSIONS: We demonstrated adhesive and significant pathogen inhibitory efficacies among the swine intestinal L. amylovorus strains studied, pointing to their potential use as probiotic feed supplements, but no independent role could be demonstrated for the major S-layer proteins in adherence to epithelial cells. The results indicate that many intestinal bacteria may coexist with and confer benefits to the host by mechanisms not attributable to adhesion to epithelial cells or mucus.

Effect of Lactobacillus brevis ATCC 8287 as a feeding supplement on the performance and immune function of piglets.

Lactobacillus brevis ATCC 8287, a surface (S-layer) strain, possesses a variety of functional properties that make it both a potential probiotic and a good vaccine vector candidate. With this in mind, our aim was to study the survival of L. brevis in the porcine gut and investigate the effect of this strain on the growth and immune function of recently weaned piglets during a feeding trial. For this, 20 piglets were divided evenly into a treatment and a control group. Piglets in the treatment group were fed L. brevis cells (1×10(10)) daily for three weeks, whereas those in the control group were provided an equivalent amount of probiotic-free placebo. For assessing the impact of L. brevis supplementation during the feeding trial, health status and weight gain of the piglets were monitored, pre- and post-trial samples of serum and feces were obtained, and specimens of the small and large intestinal mucosa and digesta were collected at slaughter. The results we obtained indicated that L. brevis-supplemented feeding induced a non-significant increase in piglet body weight and caused no change in the morphology of the intestinal mucosa. L. brevis cells were found to localize mainly in the large intestine, but they could not be isolated from feces. To a lesser extent, L. brevis was detected in the small intestine, although there was no specific attachment to the Peyer's patches. Changes in total serum IgG and IgA concentrations were not caused by supplemented L. brevis and no measurable rise in L. brevis-specific IgG was observed. However, analysis of cytokine gene expression in intestinal mucosa revealed downregulation of TGF-ß1 in the ileum and upregulation of IL-6 in the cecum in the L. brevis-supplemented group. Based on the results from this study, we conclude that whereas L. brevis appears to have some intestinal immunomodulatory effects, the ability of this strain to survive and colonize within the porcine gut appears to be limited.

Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG.

Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects.

Using recombinant Lactococci as an approach to dissect the immunomodulating capacity of surface piliation in probiotic Lactobacillus rhamnosus GG.

Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-α, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut.

Comparative genomic and functional analysis of Lactobacillus casei and Lactobacillus rhamnosus strains marketed as probiotics.

Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-κB response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities.

Nonpathogenic Lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages.

In this study, we have utilized global gene expression profiling to compare the responses of human primary macrophages to two closely related, well-characterized Lactobacillus rhamnosus strains GG and LC705, since our understanding of the responses elicited by nonpathogenic bacteria in human innate immune system is limited. Macrophages are phagocytic cells of the innate immune system that perform sentinel functions to initiate appropriate responses to surrounding stimuli. Macrophages that reside on gut mucosa encounter ingested and intestinal bacteria. Bacteria of Lactobacillus genus are nonpathogenic and used in food and as supplements with health-promoting probiotic potential. Our results demonstrate that live GG and LC705 induced quantitatively different gene expression profiles in macrophages. A gene ontology analysis revealed functional similarities and differences in responses to GG and LC705 that were reflected in host defense responses. Both GG and LC705 induced interleukin-1ß production in macrophages that required caspase-1 activity. LC705, but not GG, induced type I interferon -dependent gene activation that correlated with its ability to prevent influenza A virus replication and production of viral proteins in macrophages. Our results indicate that nonpathogenic bacteria are able to activate the inflammasome. In addition, our results suggest that L. rhamnosus may prime the antiviral potential of human macrophages.

Inhibition of dynamin-dependent endocytosis interferes with type III IFN expression in bacteria-infected human monocyte-derived DCs.

Type I IFNs (IFN-α/ßs) and type III IFNs (IFN-λ1-3) play an important role in host defense against viral infections. The induction of type I IFNs has recently been found to take place also in bacterial infections, and therefore, this study focuses on analyzing the regulation of type III IFNs in response to bacterial stimulation. We found by quantitative RT-PCR that the expression of IFN-λ1 and IFN-λ2/3 mRNAs, as well as that of IFN-ß, was similarly up-regulated in response to stimulation with live Salmonella typhimurium or TLR4 agonist LPS in human moDCs. The induction of IFN-λ mRNAs did not require ongoing protein synthesis, and only IFN-λ1 was detected at the protein level. The induction of IFN-λ mRNAs was sensitive to SB202190, Ly294002, and PDTC, which inhibit p38 MAPK, PI3K, and NF-κB activation, respectively. Furthermore, we observed that blocking dynamin-dependent endocytosis pathways with dynasore led to decreased cell surface expression of CD86 and HLA class II molecules and reduced production of IFN-λ1, CXCL10, and IL-6 when the cells were infected with S. typhimurium. Cytokine production was also impaired in dynasore-treated, Streptococcus thermophilus-stimulated cells. Further, inhibition of dynamin prevented S. typhimurium-induced phosphorylation of IRF3 and the internalization of the bacteria. In summary, induction of type III IFNs in bacteria-infected human moDCs requires multiple signaling pathways and involves bacterial phagocytosis.

Rab8 regulates ABCA1 cell surface expression and facilitates cholesterol efflux in primary human macrophages.

OBJECTIVE: ATP-binding cassette transporter A1 (ABCA1) is thought to lipidate apolipoprotein A-I (apoA-I) at the plasma membrane, with endosomal cholesterol contributing as substrate. The mechanisms of ABCA1 surface delivery are not well understood. We have shown that Rab8 regulates endosomal cholesterol removal to apoA-I in human fibroblasts. Here, we investigated whether Rab8 plays a role in ABCA1 plasma membrane expression and cholesterol removal in primary human macrophages. METHODS AND RESULTS: We found that Rab8 was abundantly expressed in human atherosclerotic lesional macrophages and upregulated on lipid loading of macrophages in vitro. Adenoviral overexpression of Rab8 increased ABCA1 protein levels and reduced cholesterol deposition in macrophage foam cells incubated with apoA-I. Depletion of Rab8 decreased the fraction of ABCA1 at the plasma membrane and inhibited the efflux of lipoprotein-derived endosomal cholesterol to apoA-I. In Rab8-depleted cells, ABCA1-GFP localized in beta1 integrin and transferrin receptor containing recycling organelles. CONCLUSIONS: Rab8 reduces foam cell formation by facilitating ABCA1 surface expression and stimulating endosomal cholesterol efflux to apoA-I in primary human macrophages.

Multiple signaling pathways contribute to synergistic TLR ligand-dependent cytokine gene expression in human monocyte-derived macrophages and dendritic cells.

TLRs are innate immune receptors that recognize pathogen-associated structures. Binding of ligands to different TLRs can induce the production of proinflammatory cytokines in a synergistic manner. We have analyzed the molecular mechanisms of synergy in TLR ligand-stimulated human monocyte-derived macrophages and dendritic cells (moDCs). Stimulation of moDCs with the TLR8 ligand together with the TLR3 or TLR4 ligand led to synergistic IL-6, IL-10, IL-12, and TNF-alpha mRNA expression and cytokine production. DNA-binding assays showed that TLR3 and TLR8 stimulation induced binding of multiple IFN regulatory factor (IRF) and STAT transcription factors to the IL-12p35 gene promoter IFN-stimulated response element in moDCs and macrophages but with different binding profiles and kinetics. We also demonstrate that NF-kappaB, MAPKs and PI-3K pathways have an important role in TLR-induced cytokine gene expression, as pharmacological inhibitors of these signaling pathways inhibited TLR3, TLR4, and TLR8 ligand-induced cytokine mRNA expression and protein production. Especially, synergistic IL-12p70 production was abolished completely in NF-kappaB, MAPK p38, and PI-3K inhibitor-treated moDCs. Our data suggest that TLR-dependent, synergistic cytokine gene expression results from enhanced activation and cooperation among NF-kappaB, IRF, MAPK, PI-3K, and STAT signaling pathways.

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells.

AIM: To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells (moDCs). METHODS: Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS), respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors. RESULTS: All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp. cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class II and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory (TNF-alpha, IL-12, IL-6, and CCL20) and Th1 type (IL-12 and IFN-gamma) cytokines, while B. breve and L. lactis were also potent inducers of anti-inflammatory IL-10. Mitogen-activated protein kinase (MAPK) p38, phosphatidylinositol 3 (PI3) kinase, and nuclear factor-kappa B (NF-kappaB) signaling pathways were shown to be involved in bacteria-induced cytokine production. CONCLUSION: Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another.

IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes.

Virus replication induces the expression of antiviral type I (IFN-alphabeta) and type III (IFN-lambda1-3 or IL-28A/B and IL-29) IFN genes via TLR-dependent and -independent pathways. Although type III IFNs differ genetically from type I IFNs, their similar biological antiviral functions suggest that their expression is regulated in a similar fashion. Structural and functional characterization of the IFN-lambda1 and IFN-lambda3 gene promoters revealed them to be similar to IFN-beta and IFN-alpha genes, respectively. Both of these promoters had functional IFN-stimulated response element and NF-kappaB binding sites. The binding of IFN regulatory factors (IRF) to type III IFN promoter IFN-stimulated response element sites was the most important event regulating the expression of these genes. Ectopic expression of the components of TLR7 (MyD88 plus IRF1/IRF7), TLR3 (Toll/IL-1R domain-containing adapter-inducing factor), or retinoic acid-inducible gene I (RIG-I) signal transduction pathways induced the activation of IFN-lambda1 promoter, whereas the IFN-lambda3 promoter was efficiently activated only by overexpression of MyD88 and IRF7. The ectopic expression of Pin1, a recently identified suppressor for IRF3-dependent antiviral response, decreased the IFN promoter activation induced by any of these three signal transduction pathways, including the MyD88-dependent one. To conclude, the data suggest that the IFN-lambda1 gene is regulated by virus-activated IRF3 and IRF7, thus resembling that of the IFN-beta gene, whereas IFN-lambda2/3 gene expression is mainly controlled by IRF7, thus resembling those of IFN-alpha genes.

Multiple NF-kappaB and IFN regulatory factor family transcription factors regulate CCL19 gene expression in human monocyte-derived dendritic cells.

CCL19 chemokine has a central role in dendritic cell (DC) biology regulating DC traffic and recruitment of naive T cells to the vicinity of activated DCs. In this study, we have analyzed the regulation of CCL19 gene expression in human monocyte-derived DCs. DCs infected with Salmonella enterica or Sendai virus produced CCL19 at late times of infection. The CCL19 promoter was identified as having two putative NF-kappaB binding sites and one IFN-stimulated response element (ISRE). Transcription factor binding experiments demonstrated that Salmonella or Sendai virus infection increased the binding of classical p50+p65 and alternative p52+RelB NF-kappaB proteins to both of the CCL19 promoter NF-kappaB elements. Interestingly, Salmonella or Sendai virus infection also increased the binding of multiple IFN regulatory factors (IRFs), STAT1, and STAT2, to the ISRE element. Enhanced binding of IRF1, IRF3, IRF7, and IRF9 to the CCL19 promoter ISRE site was detected in Salmonella or Sendai virus-infected cell extracts. The CCL19 promoter in a luciferase reporter construct was activated by the expression of NF-kappaB p50+p65 or p52+RelB dimers. IRF1, IRF3, and IRF7 proteins also activated CCL19 promoter in the presence of Sendai virus infection. CCL19 promoter constructs mutated at NF-kappaB and/or ISRE sites were only weakly activated. Ectopic expression of RIG-I (DeltaRIG-I, CARDIF) or TLR3/4 (TRIF, MyD88, IKKepsilon, or TBK1) signaling pathway components induced CCL19 promoter activity, suggesting that these pathways are important in CCL19 gene expression. Our experiments reveal that expression of the CCL19 gene is regulated by a combined action of several members of the NF-kappaB, IRF, and STAT family transcription factors.

Activation, cytokine production, and intracellular survival of bacteria in Salmonella-infected human monocyte-derived macrophages and dendritic cells.

Salmonella enterica serovar typhimurium (S. typhimurium) is an intracellular pathogen causing localized gastroenteritis in humans. Macrophages (Mphis) and dendritic cells (DCs) play an important role in innate immunity against Salmonella. In this report, we have compared the consequences of infection of human Mphis and DCs with wild-type S. typhimurium and an isogenic PgtE-defective strain. PgtE is an outer membrane protein hypothesized to have a role in intracellular survival of Salmonella. We observed that DCs undergo full maturation in response to Salmonella infection, as indicated by up-regulation of cell-surface marker proteins CD80, CD83, CD86, and human leukocyte antigen class II. CC chemokine ligand 5 (CCL5), CXC chemokine ligand 10, tumor necrosis factor alpha, interleukin (IL)-12, and IL-18 gene expression and protein production were readily induced by Salmonella-infected Mphis and DCs. CCL20 was preferentially produced by Mphis, whereas DCs secreted higher levels of CCL19 as compared with Mphis. DCs and Mphis infected with S. typhimurium also produced high levels of interferon-gamma (IFN-gamma). Cytokine neutralization and stimulation experiments suggest that the production was partly regulated by Salmonella-induced type I IFNs, IL-12, and IL-18. DC cytokine production induced by Salmonella was much higher as compared with the responses induced by Salmonella lipopolysaccharide or flagellin. Mphis and DCs were capable of internalizing and harboring Salmonella for several days. S. enterica PgtE provided no survival advantage for the bacteria in human Mphis or DCs. Our results demonstrate that although Mphis and DCs share similar functions, they may have different roles during Salmonella infection as a result of differential production of certain chemokines and cytokines.