Antiadhesive properties of Abelmoschus esculentus (Okra) immature fruit extract against Helicobacter pylori adhesion - PubMed (original) (raw)

Antiadhesive properties of Abelmoschus esculentus (Okra) immature fruit extract against Helicobacter pylori adhesion

Jutta Messing et al. PLoS One. 2014.

Abstract

Background: Traditional Asian and African medicine use immature okra fruits (Abelmoschus esculentus) as mucilaginous food to combat gastritis. Its effectiveness is due to polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach tissue. The present study investigates the antiadhesive effect in mechanistic detail.

Methodology: A standardized aqueous fresh extract (Okra FE) from immature okra fruits was used for a quantitative in vitro adhesion assay with FITC-labled H. pylori J99, 2 clinical isolates, AGS cells, and fluorescence-activated cell sorting. Bacterial adhesins affected by FE were pinpointed using a dot-blot overlay assay with immobilized Lewis(b), sialyl-Lewis(a), H-1, laminin, and fibronectin. (125)I-radiolabeled Okra FE polymer served for binding studies to different H. pylori strains and interaction experiments with BabA and SabA. Iron nanoparticles with different coatings were used to investigate the influence of the charge-dependence of an interaction on the H. pylori surface.

Principal findings: Okra FE dose-dependently (0.2 to 2 mg/mL) inhibited H. pylori binding to AGS cells. FE inhibited the adhesive binding of membrane proteins BabA, SabA, and HpA to its specific ligands. Radiolabeled compounds from FE bound non-specifically to different strains of H. pylori, as well as to BabA/SabA deficient mutants, indicating an interaction with a still-unknown membrane structure in the vicinity of the adhesins. The binding depended on the charge of the inhibitors. Okra FE did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors.

Conclusion: Non-specific interactions between high molecular compounds from okra fruits and the H. pylori surface lead to strong antiadhesive effects.

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Conflict of interest statement

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

Figures

Figure 1

Figure 1. Relative adhesion (%, related to the untreated control UC) of FITC-labeled H. pylori to AGS cells after pretreatment of the bacteria with different concentrations of Okra FE.

UC: untreated control; Values are mean ± SD; n = 3 independent experiments with 3 replicates each, ** p<0.01.

Figure 2

Figure 2. Representative images of the adhesion of FITC-labeled H. pylori wild-type strain J99 to immobilized ligands on PVDF membranes.

(I) untreated control and pretreated bacteria with (II) 3′-sialyllactose and (III) Okra FE. (Neo)glycoproteins spotted on PVDF membranes (1 µg per spot) were overlaid with FITC-labeled H. pylori and adherent bacteria were detected by fluorescence imaging. The respective locations of spotted (neo)glycoproteins are indicated below.

Figure 3

Figure 3. Binding of radiolabeled fractions Okra FE subfractions FE3, 4 and 5 to different H. pylori bacterial strains and E. coli reference strain.

Figure 4

Figure 4. Influence of Okra FE on H. pylori strain 17875/Leb binding to Leb-HSA.

Data are related to the Leb-HSA binding of the control (only 17875/Leb together with Leb, = 100% Leb binding).

Figure 5

Figure 5. Influence of Okra FE on H. pylori strain 17875_babA1A2_ binding to sialyl-Lex-HSA.

Data are related to the control (only 17875_babA1A2_ with sLex-HAS = 100%).

Figure 6

Figure 6. Adhesion assay with MNPs.

Amount of bacteria in the untreated control was calculated as 100% and is indicated as broken line. Results of the samples incubated with MNPs are related to the values of the untreated control. Samples are named according to the type of MNPs added. Results are mean values with ± SD from six independent experiments with *: p<0.05 and **: p<0.01. Significance values refer to the comparison with the fluidMAG-DX® values, as indicated by the brackets.

Figure 7

Figure 7. Differential gene expression of H. pylori pretreated with Okra FE.

Endogenous control: 23S rRNA. Data are related to untreated control (UC) H. pylori in liquid growth medium (RQ = 1), with n = 2 replicates from three independent experiments (mean ± SD). * _p_≤0.05

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Grants and funding

Financial support by the German Research Foundation, DFG (International Research Training Group 1549 Molecular and Cellular Glyco-Sciences MCGS, Münster Hyderabad), to AH, JM, and CT and from Vetenskapsrådet/VR(M), Cancerfonden to TB is acknowledged, and was in part performed within the Umeå Centre for Microbial Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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