The human intestinal epithelium is formed by a single layer of epithelial cells that separates the intestinal lumen from the underlying lamina propria. The space between these cells is sealed by tight junctions (TJ), which regulate the permeability of the intestinal barrier. TJ are complex protein structures comprised of transmembrane proteins, which interact with the actin cytoskeleton via plaque proteins. Signaling pathways involved in the assembly, disassembly, and maintenance of TJ are controlled by a number of signaling molecules, such as protein kinase C, mitogen-activated protein kinases, myosin light chain kinase, and Rho GTPases. The intestinal barrier is a complex environment exposed to many dietary components and many commensal bacteria. Studies have shown that the intestinal bacteria target various intracellular pathways, change the expression and distribution of TJ proteins, and thereby regulate intestinal barrier function. The presence of some commensal and probiotic strains leads to an increase in TJ proteins at the cell boundaries and in some cases prevents or reverses the adverse effects of pathogens. Various dietary components are also known to regulate epithelial permeability by modifying expression and localization of TJ proteins.

BackgroundIntestinal barrier function is important for preserving health, as a compromised barrier allows antigen entry and can induce inflammatory diseases. Probiotic bacteria can play a role in enhancing intestinal barrier function; however, the mechanisms are not fully understood. Existing studies have focused on the ability of probiotics to prevent alterations to tight junctions in disease models, and have been restricted to a few tight junction bridging proteins. No studies have previously investigated the effect of probiotic bacteria on healthy intestinal epithelial cell genes involved in the whole tight junction signalling pathway, including those encoding for bridging, plaque and dual location tight junction proteins. Alteration of tight junction signalling in healthy humans is a potential mechanism that could lead to the strengthening of the intestinal barrier, resulting in limiting the ability of antigens to enter the body and potentially triggering undesirable immune responses.ResultsThe effect of Lactobacillus plantarum MB452 on tight junction integrity was determined by measuring trans-epithelial electrical resistance (TEER) across Caco-2 cell layers. L. plantarum MB452 caused a dose-dependent TEER increase across Caco-2 cell monolayers compared to control medium. Gene expression was compared in Caco-2 cells untreated or treated with L. plantarum MB452 for 10 hours. Caco-2 cell RNA was hybridised to human oligonucleotide arrays. Data was analysed using linear models and differently expressed genes were examined using pathway analysis tools. Nineteen tight junction-related genes had altered expression levels in response to L. plantarum MB452 (modified-P < 0.05, fold-change > 1.2), including those encoding occludin and its associated plaque proteins that anchor it to the cytoskeleton. L. plantarum MB452 also caused changes in tubulin and proteasome gene expression levels which may be linked to intestinal barrier function. Caco-2 tight junctions were visualised by fluorescent microscopy of immuno-stained occludin, zona occludens (ZO)-1, ZO-2 and cingulin. Caco-2 cells treated with L. plantarum MB452 had higher intensity fluorescence of each of the four tight junction proteins compared to untreated controls.ConclusionsThis research indicates that enhancing the expression of genes involved in tight junction signalling is a possible mechanism by which L. plantarum MB452 improves intestinal barrier function.

Lactobacillus species can exert health promoting effects in the gastrointestinal tract (GIT) through many mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation, and enhancement of the epithelial barrier function. Different species of the genus Lactobacillus can evoke different responses in the host, and not all strains of the same species can be considered beneficial. Strain variations may be related to diversity of the cell surface architecture of lactobacilli and the bacteria's ability to express certain surface components or secrete specific compounds in response to the host environment. Lactobacilli are known to modify their surface structures in response to stress factors such as bile and low pH, and these adaptations may help their survival in the face of harsh environmental conditions encountered in the GIT. In recent years, multiple cell surface-associated molecules have been implicated in the adherence of lactobacilli to the GIT lining, immunomodulation, and protective effects on intestinal epithelial barrier function. Identification of the relevant bacterial ligands and their host receptors is imperative for a better understanding of the mechanisms through which lactobacilli exert their beneficial effects on human health.

The aim of this research was to identify bacterial isolates having the potential to improve intestinal barrier function. Lactobacillus plantarum strains and human oral isolates were screened for their ability to enhance tight junction integrity as measured by the transepithelial electrical resistance (TEER) assay. Eight commercially used probiotics were compared to determine which had the greatest positive effect on TEER, and the best-performing probiotic strain, Lactobacillus rhamnosus HN001, was used as a benchmark to evaluate the isolates. One isolate, L. plantarum DSM 2648, was selected for further study because it increased TEER 135% more than L. rhamnosus HN001. The ability of L. plantarum DSM 2648 to tolerate gastrointestinal conditions and adhere to intestinal cells was determined, and L. plantarum DSM 2648 performed better than L. rhamnosus HN001 in all the assays. Lactobacillus plantarum DSM 2648 was able to reduce the negative effect of Escherichia coli [enteropathogenic E. coli (EPEC)] O127:H6 (E2348/69) on TEER and adherence by as much as 98.75% and 80.18%, respectively, during simultaneous or prior coculture compared with EPEC incubation alone. As yet, the precise mechanism associated with the positive effects exerted by L. plantarum DSM 2648 are unknown, and may influence its use to improve human health and wellness.

Three ovine-derived cathelicidins, SMAP29, OaBac5mini, and OaBac7.5mini, were compared with respect to their antibacterial activities and interactions with membranes. SMAP29 was confirmed to be ␣-helical, broad spectrum, and able to disrupt both the outer and the cytoplasmic membranes at relatively low concentrations. In contrast, the two proline-and arginine-rich OaBac peptides had more-modest antibacterial activities, reduced levels of lipopolysaccharide binding, and a lesser ability to depolarize the cytoplasmic membrane, consistent with a cytoplasmic target.

Summary Faecalibacterium prausnitzii, an abundant member of the human commensal microbiota, has been proposed to have a protective role in the intestine. However, it is an obligate anaerobe, difficult to co‐culture in viable form with oxygen‐requiring intestinal cells. To overcome this limitation, a unique apical anaerobic model of the intestinal barrier, which enabled co‐culture of live obligate anaerobes with the human intestinal cell line Caco‐2, was developed. Caco‐2 cells remained viable and maintained an intact barrier for at least 12 h, consistent with gene expression data, which suggested Caco‐2 cells had adapted to survive in an oxygen‐reduced atmosphere. Live F. prausnitzii cells, but not ultraviolet (UV)‐killed F. prausnitzii, increased the permeability of mannitol across the epithelial barrier. Gene expression analysis showed inflammatory mediators to be expressed at lower amounts in Caco‐2 cells exposed to live F. prausnitzii than UV‐killed F. prausnitzii, This, consistent with previous reports, implies that live F. prausnitzii produces an anti‐inflammatory compound in the culture supernatant, demonstrating the value of a physiologically relevant co‐culture system that allows obligate anaerobic bacteria to remain viable.

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