Probiotic-Derived Polyphosphate Enhances the Epithelial Barrier Function and Maintains Intestinal Homeostasis through Integrin–p38 MAPK Pathway Shuichi Segawa 1 , Mikihiro Fujiya2*, Hiroaki Konishi2, Nobuhiro Ueno 2 , Naoyuki Kobayashi1, Tatsuro Shigyo1, Yutaka Kohgo2 1 Frontier Laboratories of Value Creation, Sapporo Breweries Ltd., Yaizu, Japan, 2 Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Asahikawa, Japan Abstract Probiotics exhibit beneficial effects on human health, particularly in the maintenance of intestinal homeostasis in a complex manner notwithstanding the diversity of an intestinal flora between individuals. Thus, it is highly probable that some common molecules secreted by probiotic and/or commensal bacteria contribute to the maintenance of intestinal homeostasis and protect the intestinal epithelium from injurious stimuli. To address this question, we aimed to isolate the cytoprotective compound from a lactobacillus strain, Lactobacillus brevis SBC8803 which possess the ability to induce cytoprotective heat shock proteins in mouse small intestine. L. brevis was incubated in MRS broth and the supernatant was passed through with a 0.2-mm filter. Caco2/bbe cells were treated with the culture supernatant, and HSP27 expression was evaluated by Western blotting. HSP27-inducible components were separated by ammonium sulfate precipitation, DEAE anion exchange chromatography, gel filtration, and HPLC. Finally, we identified that the HSP27-inducible fraction was polyphosphate (poly P), a simple repeated structure of phosphates, which is a common product of lactobacilli and other bacteria associated with intestinal microflora without any definitive physiological functions. Then, poly P was synthesized by poly P-synthesizing enzyme polyphosphate kinase. The synthesized poly P significantly induced HSP27 from Caco2/BBE cells. In addition, Poly P suppressed the oxidant-induced intestinal permeability in the mouse small intestine and pharmacological inhibitors of p38 MAPK and integrins counteract its protective effect. Daily intrarectal administration of poly P (10 mg) improved the inflammation grade and survival rate in 4% sodium dextran sulfate-administered mice. This study, for the first time, demonstrated that poly P is the molecule responsible for maintaining intestinal barrier actions which are mediated through the intestinal integrin b1-p38 MAPK. Citation: Segawa S, Fujiya M, Konishi H, Ueno N, Kobayashi N, et al. (2011) Probiotic-Derived Polyphosphate Enhances the Epithelial Barrier Function and Maintains Intestinal Homeostasis through Integrin–p38 MAPK Pathway. PLoS ONE 6(8): e23278. doi:10.1371/journal.pone.0023278 Editor: Stefan Bereswill, Charite´-University Medicine Berlin, Germany Received June 17, 2011; Accepted July 11, 2011; Published August 15, 2011 Copyright: 2011 Segawa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Grant-in Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology in Japan to M.F. (#20590734), Sapporo Bio Cluster ‘‘Bio-S’’, the Knowledge Cluster Initiative of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) in Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sapporo Breweries Ltd also funded this study and their role was to provide the lactic acid bacteira strain. Through the employment of Shuichi Segawa, Naoyuki Kobayashi and Tatsuro Shigyo they were also involved in conceiving, designing and performing the experiments and also with analyzing the data. Competing Interests: S. Segawa, N. Kobayashi and T. Shigyo are employed by Sapporo Breweries Ltd. Sapporo Breweries Ltd and Asahikawa Medical University have a patent application on this study. The patent name is Intestinal Tract Protective Agent. The patent number is PCT/JP2011/057689. There is no product in development and marketed product related to this research. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: fjym@asahikawa-med.ac.jp Introduction The human gastrointestinal tract is colonized by a vast community of bacteria. Well-balanced interactions between the host and gut microbiota provides health benefits for the body [1]. Probiotics in particular deliver beneficial effects including competitive exclusion of pathogenic bacteria [2], induction of defensin production [3,4], modulation of host immune functions [5], and improvement of the intestinal barrier function [6]. Although these actions appear to be mediated by soluble factors secreted from probiotics [7–12], the molecules responsible for the probiotic effects have rarely been identified [7,8]. We previously found that competence and sporulation factor, a pentapeptide produced by Bacillus subtilis, exerts a cytoprotective effect by inducing heat shock proteins and activating the p38 mitogen- activated protein kinase (MAPK) and phosphatidylinositol 3- kinase (Akt) pathways [7]. Subsequently, soluble proteins p75 and p40 produced by Lactobacillus rhamnosus GG (LGG) were identified as functional molecules possessing inhibitory effects on inflamma- tory cytokine-induced cell apoptosis by activating the Akt pathway [8]. The identification of these three molecules, which are specifically produced by B. subtilis and LGG helped us to elucidate the probiotic mechanisms of actions. Intestinal homeostasis, on the other hand, is maintained in a complex manner notwithstanding the diversity of an intestinal flora between individuals. Thus, it is highly probable that some common molecules secreted by probiotic and/or commensal bacteria contribute to the maintenance of intestinal homeostasis and protect the intestinal epithelium from injurious stimuli. To address this question, we selected a lactobacillus strain, Lactobacillus brevis SBC8803 which possess the ability to induce cytoprotective heat shock proteins in mouse small intestine, protect intestinal PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23278