Participation of cell-surface hydrophobins for hemin binding in Helicobacter pylori.

Treatment of Helicobacter pylori cells with several chaotropic agents resulted in different degrees of inhibition in the binding of the bacteria to hemin and Congo-red dye. Polyanions also yielded a >50% inhibitory effect. Furthermore, hydrophobic interaction chromatography was used to determine the relative surface hydrophobicity of cell-associated proteins extracted with 3 mol/L urea, revealing proteins with a significant hydrophobic profile.

Antibacterial and antifungal activities of some Mexican medicinal plants.

In Mexico about 4,000 plant species have some medicinal use. The aim of this work was to evaluate the antimicrobial activity of six Mexican medicinal plants against fungi and Gram-positive and Gram-negative bacteria. Methanolic extracts were prepared from the Mexican medicinal plants Amphypteringium adstrigens, Castella tortuosa, Coutarea latiflora, Ibervillea sonorae, Jatropha cuneata, and Selaginella lepidophylla. The antibacterial and antifungal activities of the plants were determined by the broth microdilution method and the radial growth inhibition assay, respectively. All Mexican plants tested showed antimicrobial activity. Among the six plant extracts analyzed, J. cuneata showed the highest growth-inhibitory activity against fungi, Gram-positive and Gram-negative bacteria (J. cuneata > A. adstrigens > C. latiflora > C. tortuosa > I. sonorae approximately S. lepidophylla). Shigella flexneri and Staphylococcus aureus were the most susceptible bacteria to plant extracts. Complete inhibition of S. flexneri growth was observed with J. cuneata methanolic extract at 90 microg/mL. This plant extract also showed the strongest antifungal activity against Fusarium verticillioides and Aspergillus niger. Our data suggest that the medicinal plants tested have important antimicrobial properties. This is the first report describing the antimicrobial activities of several of the Mexican medicinal plants used in this study.

Mucosal immune response of spotted sand bass Paralabrax maculatofasciatus (Steindachner, 1868) orally immunised with an extracellular lectin of Aeromonas veronii.

To assess the immunogenic and immunoprotective role of the extracellular lectin from Aeromonas veronii (MCBP), which has affinity for mucosal constituents such as mucin, lactoferrin, immunoglobulins and collagen, spotted sand bass (Paralabrax maculatofasciatus) were orally immunised either with soluble MCBP, adjuvant-conjugated MCBP or immobilised MCBP on latex microspheres. The results suggest that the MCBP is capable of eliciting protective immunity against A. veronii infections when administered orally. The highest mucosal immune response was elicited in fish immunised with MCBP covalently linked to cholera toxin B subunit (CTB) or to Escherichia coli heat-labile toxin (hLT). MCBP-CTB was found to elicit immunoprotection against a challenge with live Aeromonas cells with a relative percent survival of almost 70% and without the expression of the severe histopathological alterations induced by A. veronii.

Isolation and characterisation of putative adhesins from Helicobacter pylori with affinity for heparan sulphate proteoglycan.

A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylori culture supernates was obtained by sequential ammonium sulphate precipitation and affinity chromatography on heparin-Sepharose. The chromatographic procedure yielded one major fraction that contained proteins with heparan sulphate affinity as revealed by inhibition studies of heparan sulphate binding to H. pylori cells. Preparative iso-electric focusing, SDS-PAGE and blotting experiments, with peroxidase(POD)-labelled heparan sulphate as a probe, indicated the presence of two major extracellular proteins with POD-heparan sulphate affinity. One protein had a molecular mass of 66.2 kDa and a pI of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins of H. pylori, whereas the 66.2-kDa HSBP showed a high homology to an Escherichia coli chaperon protein and equine haemoglobin. A third HSBP was isolated from an outer-membrane protein (OMP) fraction of H. pylori cells with a molecular mass of 47.2 kDa. The amino acid sequence of an internal peptide of the OMP-HSBP did not show homology to the extracellular HSBP of H. pylori, or to another microbial HSBP.

Protection of BALB/c mice against experimental Helicobacter pylori infection by oral immunisation with H pylori heparan sulphate-binding proteins coupled to cholera toxin beta-subunit.

The presence of Helicobacter pylori in the gastroduodenal mucosae is associated with chronic active gastritis, peptic ulcers and gastric cancers such as adenocarcinoma and low-grade gastric B-cell lymphoma. In response to the presence of antibiotic-resistant strains, the use of vaccines to combat this infection has become an attractive alternative. The present study used a murine model of infection by a mouse-adapted H. pylori strain to determine whether infection in BALB/c mice can be successfully eradicated by intragastric vaccination with H. pylori heparan sulphate-binding proteins (HSBP) covalently coupled to the beta-subunit of cholera toxin (CTB). It was shown that vaccination confers protection against exposure of BALB/c mice to the pathogen, as revealed by microbiological, histopathological and molecular methods.

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori.

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin.

Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori. Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with alpha-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a kd 2.88 x 10(-6) M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or alpha-chymotrypsin after isolation from iron-restricted and iron-containing media.