Veera Kainulainen

Akkermansia muciniphila Adheres to Enterocytes and Strengthens the Integrity of the Epithelial Cell Layer

ABSTRACT Akkermansia muciniphila is a Gram-negative mucin-degrading bacterium that resides in the gastrointestinal tracts of humans and animals. A. muciniphila has been linked with intestinal health and improved metabolic status in obese and type 2 diabetic subjects. Specifically, A. muciniphila has been shown to reduce high-fat-diet-induced endotoxemia, which develops as a result of an impaired gut barrier. Despite the accumulating evidence of the health-promoting effects of A. muciniphila, the mechanisms of interaction of the bacterium with the host have received little attention. In this study, we used several in vitro models to investigate the adhesion of A. muciniphila to the intestinal epithelium and its interaction with the host mucosa. We found that A. muciniphila adheres strongly to the Caco-2 and HT-29 human colonic cell lines but not to human colonic mucus. In addition, A. muciniphila showed binding to the extracellular matrix protein laminin but not to collagen I or IV, fibronectin, or fetuin. Importantly, A. muciniphila improved enterocyte monolayer integrity, as shown by a significant increase in the transepithelial electrical resistance (TER) of cocultures of Caco-2 cells with the bacterium. Further, A. muciniphila induced interleukin 8 (IL-8) production by enterocytes at cell concentrations 100-fold higher than those for Escherichia coli, suggesting a very low level of proinflammatory activity in the epithelium. In conclusion, our results demonstrate that A. muciniphila adheres to the intestinal epithelium and strengthens enterocyte monolayer integrity in vitro, suggesting an ability to fortify an impaired gut barrier. These results support earlier associative in vivo studies and provide insights into the interaction of A. muciniphila with the host.

Simple faecal preparation and efficacy of frozen inoculum in faecal microbiota transplantation for recurrent Clostridium difficile infection – an observational cohort study

Faecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (rCDI). The finding of suitable donor, donor screening and preparation of faecal transplants are challenging in clinical work.

Dancing to another tune - adhesive moonlighting proteins in bacteria

Biological moonlighting refers to proteins which express more than one function. Moonlighting proteins occur in pathogenic and commensal as well as in Gram-positive and Gram-negative bacteria. The canonical functions of moonlighting proteins are in essential cellular processes, i.e., glycolysis, protein synthesis, chaperone activity, and nucleic acid stability, and their moonlighting functions include binding to host epithelial and phagocytic cells, subepithelia, cytoskeleton as well as to mucins and circulating proteins of the immune and hemostatic systems. Sequences of the moonlighting proteins do not contain known motifs for surface export or anchoring, and it has remained open whether bacterial moonlighting proteins are actively secreted to the cell wall or whether they are released from traumatized cells and then rebind onto the bacteria. In lactobacilli, ionic interactions with lipoteichoic acids and with cell division sites are important for surface localization of the proteins. Moonlighting proteins represent an abundant class of bacterial adhesins that are part of bacterial interactions with the environment and in responses to environmental changes. Multifunctionality in bacterial surface proteins appears common: the canonical adhesion proteins fimbriae express also nonadhesive functions, whereas the mobility organelles flagella as well as surface proteases express adhesive functions.

Glutamine Synthetase and Glucose-6-Phosphate Isomerase Are Adhesive Moonlighting Proteins of Lactobacillus crispatus Released by Epithelial Cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.

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.

Identification of a high-molecular-mass Lactobacillus epithelium adhesin (LEA) of Lactobacillus crispatus ST1 that binds to stratified squamous epithelium

Lactobacilli belong to the normal gastrointestinal and genital tract microbiota of human and animal hosts. Adhesion is important for bacterial colonization; however, only a few Lactobacillus adhesins have been identified so far. We studied extracted surface proteins from an adhesive Lactobacillus crispatus strain, ST1, which efficiently colonizes the chicken alimentary tract, for their binding to tissue sections of the chicken crop, and identified a novel high-molecular-mass repetitive surface protein that shows specific binding to stratified squamous epithelium. The adhesin binds to both crop epithelium and epithelial cells from human vagina, and was named Lactobacillus epithelium adhesin (LEA). Expression of LEA is strain-specific among L. crispatus strains and corresponds directly to in vitro bacterial adhesion ability. The partial sequence of the lea gene predicts that the LEA protein carries an N-terminal YSIRK signal sequence and a C-terminal LPxTG anchoring motif, as well as a highly repetitive region harbouring 82 aa long repeats with non-identical sequences that show similarity to Lactobacillus Rib/alpha-like repeats. LEA-mediated epithelial adherence may improve bacterial colonization in the chicken crop and the human vagina, which are the natural environments for L. crispatus.

BopA Does Not Have a Major Role in the Adhesion of Bifidobacterium bifidum to Intestinal Epithelial Cells, Extracellular Matrix Proteins, and Mucus

ABSTRACT The ability of bifidobacteria to adhere to the intestine of the human host is considered to be important for efficient colonization and achieving probiotic effects. Bifidobacterium bifidum strains DSM20456 and MIMBb75 adhere well to the human intestinal cell lines Caco-2 and HT-29. The surface lipoprotein BopA was previously described to be involved in mediating adherence of B. bifidum to epithelial cells, but thioacylated, purified BopA inhibited the adhesion of B. bifidum to epithelial cells in competitive adhesion assays only at very high concentrations, indicating an unspecific effect. In this study, the role of BopA in the adhesion of B. bifidum was readdressed. The gene encoding BopA was cloned and expressed without its lipobox and hydrophobic signal peptide in Escherichia coli, and an antiserum against the recombinant BopA was produced. The antiserum was used to demonstrate the abundant localization of BopA on the cell surface of B. bifidum. However, blocking of B. bifidum BopA with specific antiserum did not reduce adhesion of bacteria to epithelial cell lines, arguing that BopA is not an adhesin. Also, adhesion of B. bifidum to human colonic mucin and fibronectin was found to be BopA independent. The recombinant BopA bound only moderately to human epithelial cells and colonic mucus, and it failed to bind to fibronectin. Thus, our results contrast the earlier findings on the major role of BopA in adhesion, indicating that the strong adhesion of B. bifidum to epithelial cell lines is BopA independent.

Mucosal Prevalence and Interactions with the Epithelium Indicate Commensalism of Sutterella spp.

Sutterella species have been frequently associated with human diseases, such as autism, Down syndrome, and inflammatory bowel disease (IBD), but the impact of these bacteria on health still remains unclear. Especially the interactions of Sutterella spp. with the host are largely unknown, despite of the species being highly prevalent. In this study, we addressed the interaction of three known species of Sutterella with the intestinal epithelium and examined their adhesion properties, the effect on intestinal barrier function and the pro-inflammatory capacity in vitro. We also studied the relative abundance and prevalence of the genus Sutterella and Sutterella wadsworthensis in intestinal biopsies of healthy individuals and patients with celiac disease (CeD) or IBD. Our results show that Sutterella spp. are abundant in the duodenum of healthy adults with a decreasing gradient toward the colon. No difference was detected in the prevalence of Sutterella between the pediatric IBD or CeD patients and the healthy controls. Sutterella parvirubra adhered better than the two other Sutterella spp. to differentiated Caco-2 cells and was capable of decreasing the adherence of S. wadsworthensis, which preferably bound to mucus and human extracellular matrix proteins. Furthermore, only S. wadsworthensis induced an interleukin-8 production in enterocytes, which could be due to different lipopolysaccharide structures between the species. However, its pro-inflammatory activity was modest as compared to non-pathogenic Escherichia coli. Sutterella spp. had no effect on the enterocyte monolayer integrity in vitro. Our findings indicate that the members of genus Sutterella are widely prevalent commensals with mild pro-inflammatory capacity in the human gastrointestinal tract and do not contribute significantly to the disrupted epithelial homeostasis associated with microbiota dysbiosis and increase of Proteobacteria. The ability of Sutterella spp. to adhere to intestinal epithelial cells indicate that they may have an immunomodulatory role.

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

BackgroundFor a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.ResultsWe found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.ConclusionIn conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

The Potential of Gut Commensals in Reinforcing Intestinal Barrier Function and Alleviating Inflammation

The intestinal microbiota, composed of pro- and anti-inflammatory microbes, has an essential role in maintaining gut homeostasis and functionality. An overly hygienic lifestyle, consumption of processed and fiber-poor foods, or antibiotics are major factors modulating the microbiota and possibly leading to longstanding dysbiosis. Dysbiotic microbiota is characterized to have altered composition, reduced diversity and stability, as well as increased levels of lipopolysaccharide-containing, proinflammatory bacteria. Specific commensal species as novel probiotics, so-called next-generation probiotics, could restore the intestinal health by means of attenuating inflammation and strengthening the epithelial barrier. In this review we summarize the latest findings considering the beneficial effects of the promising commensals across all major intestinal phyla. These include the already well-known bifidobacteria, which use extracellular structures or secreted substances to promote intestinal health. Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium hallii metabolize dietary fibers as major short-chain fatty acid producers providing energy sources for enterocytes and achieving anti-inflammatory effects in the gut. Akkermansia muciniphila exerts beneficial action in metabolic diseases and fortifies the barrier function. The health-promoting effects of Bacteroides species are relatively recently discovered with the findings of excreted immunomodulatory molecules. These promising, unconventional probiotics could be a part of biotherapeutic strategies in the future.