Acetylated Rhamnogalacturonans from Immature Fruits of Abelmoschus esculentus Inhibit the Adhesion of Helicobacter pylori to Human Gastric Cells by Interaction with Outer Membrane Proteins.

Polysaccharide containing extracts from immature fruits of okra (Abelmoschus esculentus) are known to exhibit antiadhesive effects against bacterial adhesion of Helicobacter pylori (H. pylori) to stomach tissue. The present study investigates structural and functional features of polymers responsible for this inhibition of bacterial attachment to host cells. Ammonium sulfate precipitation of an aqueous extract yielded two fractions at 60% and 90% saturation with significant antiadhesive effects against H. pylori, strain J99, (FE60% 68% ± 15%; FE90% 75% ± 11% inhibition rates) after preincubation of the bacteria at 1 mg/mL. Sequential extraction of okra fruits yielded hot buffer soluble solids (HBSS) with dose dependent antiadhesive effects against strain J99 and three clinical isolates. Preincubation of H. pylori with HBSS (1 mg/mL) led to reduced binding to 3'-sialyl lactose, sialylated Le(a) and Le(x). A reduction of bacterial binding to ligands complementary to BabA and SabA was observed when bacteria were pretreated with FE90%. Structural analysis of the antiadhesive polysaccharides (molecular weight, monomer composition, linkage analysis, stereochemistry, and acetylation) indicated the presence of acetylated rhamnogalacturonan-I polymers, decorated with short galactose side chains. Deacetylation of HBSS and FE90% resulted in loss of the antiadhesive activity, indicating esterification being a prerequisite for antiadhesive activity.

Low-Molecular-Weight Dextran Sulfate Nanocapsules Inhibit the Adhesion of Helicobacter pylori to Gastric Cells.

The Gram-negative bacterium Helicobacter pylori is the most common bacterial pathogen in humans, infecting 24-79% of the population at any time. Standard eradication protocols involve multi-target therapy including combinations of antibiotics, which has promoted the emergence of resistant strains. To address this challenge, we prepared antibiotic-free colloidal nanoparticles designed to interfere with the adhesion mechanisms of H. pylori and thus prevent both the onset and recurrence of infection. Our colloidal particles comprised a nanocapsule (NC) formulation based on an oil-core nanoemulsion co-stabilized with lysozyme and lecithin, coated with negatively charged low-molecular-weight (DexS40-NC) or high-molecular-weight (DexS500-NC) dextran sulfate, or positively charged chitosan (CSHMC+30-NC). The oil core of all NC formulations was also loaded with curcumin, a model lipophilic phytochemical substance with well-documented anti-inflammatory and anti-tumor activities. Our proof-of-principle experiments showed that the DexS40-NC formulation inhibited the adhesion of H. pylori to AGS stomach cells in a dose-dependent manner. DexS40-NC achieved more potent inhibition than DexS500-NC or uncoated control nanoemulsions, whereas the effect of CSHMC+30-NC was not clear-cut given the ability of this formulation to aggregate bacteria. DexS40-NC, unlike DexS500-NC, showed no cytotoxic effects against AGS, Caco-2, or MDCK cell lines. DexS40-NC is, therefore, a promising candidate for further development as an alternative or complementary therapy against H. pylori infections.

A NADH-accepting imine reductase variant: Immobilization and cofactor regeneration by oxidative deamination.

Engineering cofactor specificity of enzymes is a promising approach that can expand the application of enzymes for biocatalytic production of industrially relevant chemicals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This limits their applications to reactions employing whole cells as a cost-efficient cofactor regeneration system. For applications of IREDs as cell-free catalysts, (i) we created an IRED variant showing an improved activity for NADH. With rational design we were able to identify four residues in the (R)-selective IRED from Streptomyces GF3587 (IR-Sgf3587), which coordinate the 2'-phosphate moiety of the NADPH cofactor. From a set of 15 variants, the highest NADH activity was caused by the single amino acid exchange K40A resulting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability using an immobilisate obtained either from purified enzyme or from lysate using the EziG(™) carriers. Applying the variant and NADH, we reached 88% conversion in a preparative scale biotransformation when employing 4% (w/v) 2-methylpyrroline. (iii) We demonstrated a one-enzyme cofactor regeneration approach using the achiral amine N-methyl-3-aminopentanone as a hydrogen donor co-substrate.

Inhibition of Helicobacter pylori adhesion to human gastric adenocarcinoma epithelial cells by aqueous extracts and pectic polysaccharides from the roots of Cochlospermum tinctorium A. Rich. and Vernonia kotschyana Sch. Bip. ex Walp.

In Malian traditional medicine infusions of the roots of Vernonia kotschyana or Cochlospermum tinctorium in water are used for treating gastric ulcer. Helicobacter pylori is known to play a major role in gastric ulcer development, and it was of interest to evaluate a potential anti-adhesive activity towards H. pylori by crude water extracts and isolated polysaccharide fractions from the roots of V. kotschyana and C. tinctorium. The inhibitory effects were examined by an in vitro flow cytometric assay using human gastric adenocarcinoma epithelial cells, where fluorescent-labeled H. pylori were pre-treated with the test fractions. The crude extract Ctw50 from C. tinctorium, containing a mixture of inulin, pectic polysaccharides, phenols and protein, led to a 43% reduction of bacterial attachment. The isolated pectic type fractions CtwA1 and CtwA2 from C. tinctorium, and Vko-I from V. kotschyana resulted in approximately 30% inhibition of H. pylori adhesion. These fractions consist of rhamnogalacturonan backbones with side chains of arabinogalactans and/or arabinans. The low degree of uronic acids in the fractions compared to anti-adhesive polysaccharides reported previously, suggests that the neutral side chains might play a role in the binding of bacterial adhesins. The fraction Vko-III.1 from V. kotschyana consisting mainly of galacturonic acid resulted only in a 19% inhibition of H. pylori adhesion. The anti-adhesive properties shown by the crude water extracts and isolated polysaccharide fractions in the present study might partly explain the anti-ulcer activities by the roots of V. kotschyana and C. tinctorium.

Chemical characterization and complement fixation of pectins from Cola cordifolia leaves.

Defatted leaves from the medicinal tree Cola cordifolia were extracted with 50% EtOH, 50 °C and 100 °C water. The polysaccharide rich extracts were fractionated and the structure of the polysaccharides elucidated. Linkage analysis of the polysaccharides indicates a rhamnogalacturonan type I backbone where both Rha and parts of GalA are substituted in position 3, indicating a highly branched polymer with short side chains. The purified fractions were tested for complement fixation, macrophage stimulating activity and anti-adhesion activity towards Helicobacter pylori. Here we report on complex and polydisperse types of pectins (Mw: 3-1300 kDa) as well as the presence of low Mw (<3 kDa) acidic oligosaccharides. The fractions showed a moderate complement fixing activity and no macrophage activating effects after LPS removal. Anti-adhesion activity towards H. pylori was not found.

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

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.