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. 2014 Aug 13;9(8):e104258.
doi: 10.1371/journal.pone.0104258. eCollection 2014.

A Proteinaceous Fraction of Wheat Bran May Interfere in the Attachment of Enterotoxigenic E. Coli K88 (F4+) to Porcine Epithelial Cells

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A Proteinaceous Fraction of Wheat Bran May Interfere in the Attachment of Enterotoxigenic E. Coli K88 (F4+) to Porcine Epithelial Cells

Gemma González-Ortiz et al. PLoS One. .
Free PMC article

Abstract

Wheat bran (WB) from Triticum aestivum has many beneficial effects on human health. To the best of our knowledge, very little has been published about its ability to prevent pathogenic bacterial adhesion in the intestine. Here, a WB extract was fractionated using different strategies, and the obtained fractions were tested in different in vitro methodologies to evaluate their interference in the attachment of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal porcine epithelial cells (IPEC-J2) with the aim of identifying the putative anti-adhesive molecules. It was found that a proteinaceous compound in the >300-kDa fraction mediates the recognition of ETEC K88 to IPEC-J2. Further fractionation of the >300-kDa sample by size-exclusion chromatography showed several proteins below 90 kDa, suggesting that the target protein belongs to a high-molecular-weight (MW) multi-component protein complex. The identification of some relevant excised bands was performed by mass spectrometry (MS) and mostly revealed the presence of various protease inhibitors (PIs) of low MW: Serpin-Z2B, Class II chitinase, endogenous alpha-amylase/subtilisin inhibitor and alpha-amylase/trypsin inhibitor CM3. Furthermore, an incubation of the WB extract with ETEC K88 allowed for the identification of a 7S storage protein globulin of wheat, Globulin 3 of 66 kDa, which may be one of the most firmly attached WB proteins to ETEC K88 cells. Further studies should be performed to gain an understanding of the molecular recognition of the blocking process that takes place. All gathered information can eventually pave the way for the development of novel anti-adhesion therapeutic agents to prevent bacterial pathogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The adhesive (Figure A) and anti-adhesive (Figure B) abilities of fractions evaluated using the enterotoxigenic E. coli (ETEC) K88.
A) Number of bacteria (log CFU per well) attached to wells coated with the different molecular-weight (MW) fractions obtained from wheat bran (WB) in the in vitro adhesion test (AT). The higher the log CFU counts than those of PBS, the higher the adhesive ability. The samples tested were the WB extract (14 mg/ml), the >300-kDa fraction (2.7 mg/ml) and the <300-kDa fraction (17 mg/ml). B) Number of bacteria (log CFU per well) that attached to IPEC-J2 cells after being co-incubated for 30 min with the different fractions obtained from WB. The lower the log CFU counts than those of PBS, the higher the blocking-adhesion ability. Different letters mean significant differences (P<0.05) between fractions. Data result from the experiments performed in triplicate in two independent assays. Error bars represent the standard error of the mean.
Figure 2
Figure 2. Dose-response of WB extract (A) and the >300-kDa fraction (B) to block the attachment of ETEC K88 to IPEC-J2.
Log CFU: number of bacteria attached to the intestinal cells that were not blocked, when compared to those of PBS. The lower the log CFU counts, the higher the blocking-adhesion ability. Linear, quadratic and cubic contrasts were performed to analyze the dose-response. Data result from the experiments performed in triplicate in two independent assays. Error bars represent the standard error of the mean.
Figure 3
Figure 3. Recognition of ETEC K88 to the size-exclusion chromatography (SEC) fractions obtained.
A) SEC of the >300-kDa fraction. Distribution of the eight fractions obtained. B) Number of bacteria (log CFU per well) attached to wells coated with the different fractions in the in vitro adhesion test (AT). The higher the log CFU counts than those of PBS, the higher the adhesive ability. C) Number of bacteria (log CFU per well) that attached to IPEC-J2 cells in the blocking test (BT). The lower the log CFU counts than those of PBS, the higher the blocking-adhesion ability. In Figures B and C, different letters mean significant differences (P<0.05) between fractions. Data result from two independent assays performed in triplicate. Error bars represent the standard error of the mean. D) Dot-blot analysis with purified fimbriae on immobilized WB extract, >300-kDa and <300-kDa fractions, CGMP, BSA, fetuin and the eight fractions (F1 to F8). (i) Binding of purified K88ac fimbriae of ETEC strain FV12408. (ii) Binding of purified K88ac fimbriae of ETEC strain 5/95. E) One-dimension SDS-PAGE of the eight fractions obtained by SEC.
Figure 4
Figure 4. Recognition of ETEC K88 to the size-exclusion chromatography (SEC) fractions obtained with acetonitrile (ACN).
A) SEC of the >300-kDa fraction treated with solvent. Representation of the six fractions obtained. C) Number of bacteria (log CFU per well) attached to wells coated with the different fractions in the in vitro adhesion test (AT). The higher the log CFU counts than those of PBS, the higher adhesive ability. D) Number of bacteria (log CFU per well) that attached to IPEC-J2 cells in the blocking test (BT). The lower the log CFU counts than those of PBS, the higher the blocking-adhesion ability. In Figures C and D, different letters mean significant differences (P<0.05) between fractions. Data result from two independent assays performed in triplicate. Error bars represent the standard error of the mean. D) Dot-blot analysis with purified fimbriae on immobilized WB extract, >300-kDa, <300-kDa, CGMP, BSA, fetuin and the six fractions (FA1 to FA6). (i) Binding of purified K88ac fimbriae of ETEC strain FV12408. (ii) Binding of purified K88ac fimbriae of ETEC strain 5/95. E) One-dimension SDS-PAGE of the six fractions obtained by SEC treated with solvent. From B1 to B6 are indicated the excised bands which were identified by MS.
Figure 5
Figure 5. Isolation of wheat-bran proteins attaching to ETEC K88.
ETEC K88 cells were incubated with wheat-bran extract (1) and PBS (2). The proteins obtained after the shaving process were separated in 1D (A) and 2D (B) gels. The spots with a fold >2 are labeled as B7, B8 and B9.

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Grant support

This work was supported by a public research project of the Spanish Ministry of Education and Science (Project AGL 2009-07328). Gemma González-Ortiz was supported by a mobility scholarship from the Spanish Ministry of Education and Science, which she enjoyed in Utrecht Universiteit. The funders had no role in study study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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