Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(8):e23278.
doi: 10.1371/journal.pone.0023278. Epub 2011 Aug 15.

Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway

Shuichi Segawa  1 , Mikihiro Fujiya, Hiroaki Konishi, Nobuhiro Ueno, Naoyuki Kobayashi, Tatsuro Shigyo, Yutaka Kohgo
Affiliations

Probiotic-derived polyphosphate enhances the epithelial barrier function and maintains intestinal homeostasis through integrin-p38 MAPK pathway

Shuichi Segawa et al. PLoS One. 2011.

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-µm 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 µg) 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 β1-p38 MAPK.

PubMed Disclaimer

Conflict of interest statement

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.

Figures

Figure 1
Figure 1. HSP27-inducible substance was separated from L. brevis SBC8803 culture supernatant.
(A) HSP27 induction from Caco2/BBE cells by culture supernatant obtained from the L. brevis 8803 culture broth at 12, 36 and 60 h. (B) HSP27 induction from Caco2/BBE cells by culture supernatant of L. brevis 8803, L. brevis 8013 and LGG obtained from the bacterial culture broth at 6 and 24 h. (C) HSP27 induction by filtrate of L. brevis SBC8803 culture supernatant passed through a 5-, 10-, 30- or 50-kDa membrane. (D) HSP27 induction by 65% or 90% ammonium sulfate precipitate of L. brevis SBC8803 culture supernatant. (E) HSP27 induction by fraction separated from 65% ammonium sulfate precipitate by DEAE anion-exchange chromatography. (F) HSP27 induction by fraction separated from 0.1 M NaCl-eluted fraction by size-exclusion chromatography. (G) HPLC chromatogram of the HSP-inducible fraction. Sample was separated on a Shodex KW800 column (300 mm×8 mm ID), eluted with 20 mM phosphate buffer (pH 6.5) at a flow rate of 0.1 mL/min. The eluent was monitored with an ultraviolet spectrophotometer at 220 nm. (H) HSP27 induction by 10 µg/mL of HSP27-inducible fraction (n = 3).
Figure 2
Figure 2. Anti-apoptopic property of HSP27-inducible fraction.
Time course of inactive full-length caspase-3 and -9 degradation in Caco2/BBE induced by 1 µM staurosporine treatment in the presence or absence of HSP27-inducible fraction.
Figure 3
Figure 3. HSP27-inducible fraction separated from culture supernatant of L. brevis 8803 was neither protein nor polysaccharide.
(A) Amino acid analysis after acid degradation of HSP27-inducible fraction by HPLC. (B) Neutral sugar and uronic acid content in the fraction. (C) Peptidoglycan content in the fraction. Concentration of peptidoglycan was measured by SLP test. (D) Western blot analysis of HSP27 induction from Caco2/BBE cells by LPS derived from E. coli or Salmonella enterica, or LTA derived from B. subtilis or Staphylococcus aureus (concentration: 0.1–100 µg/mL).
Figure 4
Figure 4. Enzymatically synthesized poly P induced HSP27 from Caco2/BBE cells through the activation of p38 MAPK pathway.
(A) HPLC chromatogram of poly P synthesized by PPK. (B) Western blot analysis of HSP27 induction in Caco2/BBE cells by poly P synthesized by PPK (0–100 µg/mL), (n = 3). (C) Poly P degradation by PPK treatment with 10 µM ADP led to an inability to induce HSP27 from Caco2/BBE cells (n = 3). (D) Poly P content in the culture supernatant of L. brevis 8803 at 8, 24 and 24 h from bacterial culture (n = 3). * Significantly different from corresponding control group at P<0.05.
Figure 5
Figure 5. Poly P prevented [3H]-mannitol flux induced by exposure to an oxidant, and integrin and p38 antagonist inhibited its protective action.
(A) 10 or 100 µg/mL of poly P inhibited transmural [3H]-mannitol fluxes in the presence or absence of 0.3 mM NH2Cl (n = 5, each group). (B–F) Inhibition of 0.3 mM NH2Cl-induced transmural [3H]-mannitol fluxes by 100 µg/mL of poly P was abolished by 1 µM of p38 MAPK inhibitor SB203580 (B), peptide antagonist of integrin, 1 µM of echistatin or 10 µM of Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP) (C), 10 µM of reverse sequence of GRGDS, Ser-Thr-Asp-Gly-Arg-Gly (D), 1 µg/mL of human integrin α3β1 (E), 1 µM of cytochalasin D (F), 1 µM of EGFR inhibitor AG1478 or 1 µM of PDGFR inhibitor AG1296 treatment (n = 5, each group). *, ** Significantly different from corresponding control group at P<0.05 and P<0.01, respectively.
Figure 6
Figure 6. Poly P prevented F-actin and E-cadherin degradation induced by the oxidative stress.
(A–F) Caco2/BBE cells were treated with 10 µg/mL of poly P, 10 µM of Gly-Arg-Gly-Asp-Ser (GRGDS) or Ser-Asp-Gly-Arg-Gly (SDGRG) overnight, followed by stimulated with 30 mM of H2O2 for 1 h. (G, H) Caco2/BBE cells were treated with 10 µg/mL of poly P overnight, followed by stimulated with 0.3 mM of NH2Cl for 1 h. F-actin was stained with phalloidin (green) and E-cadherin (red). Nuclei were counterstained with 4′,6-Diamidino-2-Phenylindole (DAPI) (blue).
Figure 7
Figure 7. Poly P interacts with integrin β1 and activates p38 MAPK pathway.
(A) Western blot analysis of p38, ERK, JNK and Akt phosphorylation in Caco2/BBE by 10 µg/mL of poly P. (B) Expression and localization of integrin β1 and β3 in mouse small intestine. Immunohistochemistry for integrin β1 and β3 were performed by using anti-integrin β1, rabbit-poly antibody and anti-integrin β3, rabbit-poly antibody. (C) Inhibition of poly P-induced p38 MAPK phosphorylation by RGD peptide. C57Bl/6 mice small intestinal loops were filled with RPMI 1640 medium containing 10 µg/mL of poly P, 10 µM of Gly-Arg-Gly-Asp-Ser (GRGDS) or Ser-Asp-Gly-Arg-Gly (SDGRG) 2 h at 37°C in a 5% CO2 incubator. Their p38 MAPK phosphorylation was detected by western blot (n = 3, each group). (D) Poly P-integrin β1 or β3 interaction analysis. Reaction mixture of 32P-labeled poly P and integrin α3β1 or αVβ3 was immunoprecipitated with integrin β1 or β3 antibody and protein G beads. Immunoprecipitated 32P-labeled poly P was determined with liquid scintillation counting.
Figure 8
Figure 8. Anti-inflammatory property of HSP27-inducible fraction.
Time course of NF-κB pathway activation in Caco2/BBE cells induced by 10 ng/mL TNF-α treatment in the presence or absence of 10 µg/mL of poly P.
Figure 9
Figure 9. Poly P improved intestinal injury and survival rate of mice treated with a lethal dose of DSS.
(A) Survival of 4% DSS administered C57Bl/6 mice challenged intrarectally with 10 µg/100 µL of poly P or 100 µL of PBS once a day throughout the experimental period (n = 5, each group). (B–D) Representative picture of a colon and average colon length (B), representative colon histopathology and histological score (C) and cytokines, IL-1β, IL-6, IL-10, IL-12a and IL-4 expression in the colon (D) of C57Bl/6 mice, which were orally administered 0% (normal) or 3% DSS, challenged intrarectally with 10 µg/100 µL of poly P or 100 µL of PBS (vehicle) once a day for 7 days (n = 5, each group).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hooper LV, Gordon JI. Commensal host-bacterial relationships in the gut. Science. 2001;292:1115–1118. - PubMed
    1. De Keersmaecker SC, Verhoeven TL, Desair J, Marchal K, Vanderleyden J, et al. Strong antimicrobial activity of Lactobacillus rhamnosus GG against Salmonella typhimurium is due to accumulation of lactic acid. FEMS Microbiol Lett. 2006;259:89–96. - PubMed
    1. Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME, et al. Secretion of microbicidal α-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol. 2000;1:113–118. - PubMed
    1. Vaishnava S, Behrendt CL, Ismail AS, Eckmann L, Hooper LV. Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface. Proc Natl Acad Sci USA. 2008;105:20858–20863. - PMC - PubMed
    1. Belkaid Y, Oldenhove G. Tuning microenvironments: induction of regulatory T cells by dendritic cells. Immunity. 2008;29:362–371. - PMC - PubMed

Publication types

MeSH terms