Pär Aleljung

Purification of collagen-binding proteins of Lactobacillus reuteri NCIB 11951

Collagen type-I-binding proteins ofLactobacillus reuteri NCIB 11951 were purified. The cell surface proteins were affinity purified on collagen Sepharose and eluted with an NaCl gradient. Two protein bands were eluted from the column (29 kDa and 31 kDa), and both bound radio-labeled collagen type I. Rabbit antisera raised against the 29 kDa and 31 kDa protein reacted with the affinity-purified proteins in a Western blot with whole-cell extract used as antigen. The N-terminal sequence of the 29-kDa and 31-kDa proteins demonstrated the closest homologies with internal sequences from anEscherichia coli trigger factor protein (TIG.ECOLI). Out of nine other lactobacilli, the antisera reacted only with theL. reuteri and not with the other species tested.

Immunomagnetic bead enrichment and PCR for detection of Helicobacter pylori in human stools

Abstract An immunomagnetic bead-based polymerase chain reaction assay (IMS-PCR) was developed for the detection of Helicobacter pylori in experimentally inoculated human stools and human clinical stool samples. Magnetic beads coated with anti- H. pylori rabbit antibodies were used for enrichment and concentration of H. pylori from faecal samples. Taq polymerase inhibitors, found in human faeces, are efficiently removed by the immunomagnetic separation (IMS) and subsequent washing of the magnetic beads. Conditions of the assay were developed and optimised with faeces from a healthy, H. pylori seronegative, individual. Faeces was inoculated with serial dilutions of either the spiral or the coccoid form of H. pylori . These two morphologic forms could be detected at similar concentrations when inoculated in faeces using an optimised IMS-PCR method. In 1 g of faeces less than 2.5×10 4 H. pylori cells were detected as measured with two separate sets of PCR-primers, based on a urease A subunit genesequence and a genesequence encoding a 26 kDa surface protein of H. pylori . Previously no report has shown a sensitivity below 10 6 H. pylori in faeces PCR. Preliminary analysis of stool samples from 17 patients with symptoms of gastritis and esophagitis by IMS-PCR showed a good correlation with EIA-analysis of H. pylori serum-antibodies from these patients. The results indicate that H. pylori cells are shed in faeces of infected patients and that immunomagnetic bead PCR might be an appropriate method for clinical diagnosis and studies involving immunoprophylaxis, antibiotic treatment as well as vaccine candidates.

Collagen Binding by Lactobacilli

Abstract125I-labeled type I collagen (Cn-I) binding of 92 fresh isolates and 18 type culture collection strains of lactobacilli was tested. More than 75% of the strains bound Cn-I. The binding was inhibited by excess of unlabeled Cn-I, gelatin, and was sensitive to proteinase K. Other proteins such as fibronectin and albumin and various carbohydrates such asD-galactose,D-fucose, andD-mannose did not inhibit the binding. Therefore, we propose binding of Cn-I to lactobacilli involving specific surface protein(s).

Growth conditions for the expression of fibronectin, collagen type I, vitronectin, and laminin binding toEscherichia coli strain NG7C

The ability ofEscherichia coli strains isolated from various human infections to bind fibronectin (Fn), collagen type I (Cn), laminin (Ln), and vitronectin (Vn) was studied. Binding of the proteins was shown to be specific and to be inhibited after protease treatment and heating of bacterial cells at 80°C. Binding of Fn, Cn type I, and Ln was not expressed in CFA broth and Voccanis medium with sodium chloride concentration above 0.5%. Fn binding was specifically enhanced for cells grown in CFA broth and Voccanis medium containing CaCl2 (up to 10 mM final concentration), whereas binding of Cn type I was decreased. Growth in liquid medium yielded cells with maximal binding of Fn, Cn type I, Vn, and Ln after 20–22 h of growth. The binding of Fn, Cn type I, and Vn to cells of strain NG7C was inhibited for cells preincubated with antisera raised to the homologous strain;E. coli NG7C cells seem to have specific binding sites for Fn, Cn type I, Vn, and possibly also Ln.

Sulfatides inhibit binding of Helicobacter pylori to the gastric cancer Kato III cell line.

Abstract.Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H. pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not α- or β-galactosidase, heparitinase, lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 μg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H. pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble 125I-labeled heparin and other sulfated carbohydrate compounds.

Dietary factors influence the recovery rates of Helicobacter pylori in a BALB/cA mouse model

The aim of this study was to assess the ability of different mouse diets to sustain an H. pylori infection in BALB/cA mice. Four commercially available mouse diets were compared. Experiment 1: Mice were fed the four diets for seven days before infection, infected three times at two-day intervals with 0.1 ml of 10(9) colony-forming units/ml H. pylori cells. H. pylori strains (n = 4) were cultured on GAB-Camp agar for 2 days, harvested and suspended in PBS. All animals were sacrificed at 2 and 4 weeks post inoculation. Experiment 2: Mice infected for 8 weeks were fed RM2, changed to the different diets for 10 days and sacrificed. Stomachs were collected, cultured on GAB-Camp agar to estimate H. pylori growth and stomach biopsies were analyzed by PCR. There were significant differences between diets in their ability to sustain growth of H. pylori. The range was from a few hundred colonies to no growth at all on the GAB-Camp agar. PCR signals showed good correlation with the culture results. All H. pylori-infected mice gave a significantly higher inflammation score compared to non-infected mice. The diet RM2, having the highest number of culturable H. pylori in the mouse stomach, also showed the highest inflammation. These results suggest that the dietary factors affect the amounts of H. pylori in an infection of BALB/cA mice.

Helicobacter pylori Hemagglutinins — Possible Gut Mucosa Adhesins

A number of recent studies indicate that Helicobacter pylori has unique cellular properties which allow this pathogen to colonize the human stomach mucosa [1,2,4,7]. Despite this unique habitat for this microbe, very few studies have tried to identify surface proteins and other possible structures determining an association with the stomach mucosa. Emody et al. [2] reported on surface hemagglutinins in H. pylori followed by reports from the USA, Ireland, and Japan. Studies by Emody et al. [2] and Carlsson et al. [1] have defined a sialic acid specific hemagglutinin. More recently, a Canadian group of investigators defined a surface component of H. pylori interacting with a glycolipid from human erythrocyte membranes [5]. Furthermore, Huang et al. [4] described a hemagglutinin which caused neuraminidase and protease resistant hemagglutination. In view of these findings we decided to carry out a systematic study on H. pylori hemagglutinin profiles by screening clinical isolates and type culture collection strains for hemagglutination with a variety of erythrocytes.

Type I and IV collagen and fibrinogen binding to Aeromonas species isolated from various infections.

Collagen binding is a common property of strains of Aeromonas species. However, agglutination of latex beads coated with types I and IV collagen and fibrinogen with Aeromonas cells varied among strains of Aeromonas species and their source of isolation. Culture media and growth conditions greatly influenced expression of Aeromonas cell surface receptors to bind collagen (types I and IV) and fibrinogen immobilized on the latex particles as suggested by the particle agglutination assay (PAA). Aeromonas cells aggregated with the differentially coated latex beads in a specified manner. Furthermore, the PAA method was found to be rapid, easy to perform and sensitive for routine screening of a large number of strains for serum and connective-tissue protein cell surface receptors.

Crystal violet binding, cell surface properties and extracellular enzyme profiles of Staphylococcus aureus producing toxic shock syndrome toxin-1

Staphylococcus aureus isolated from clinically diagnosed cases of toxic shock syndrome (TSS) showed susceptibility to phage types belonging to both I and III groups (90.5%). Phage typing patterns showed a wide diversity among 87 toxic shock syndrome toxin-1 (TSST-1) positive strains isolated from different non TSS clinical sources. Toxin producing strains isolated from both TSS and non TSS showed a remarkable ability to bind to crystal violet (pattern C/D, 97.2%) incorporated into brain heart infusion agar media at subinhibitory concentrations and these isolates were traced to biotype var. hominis. The cellular fatty acid compositions of TSS and non-TSS strains belonging to the three biotypes S. aureus var. hominis, S. aureus var. bovis and S. aureus var. canis did not differ. TSST-1 producing strains demonstrated a high salt aggregation test value (above 1.5) indicating a low cell surface hydrophobicity. Both TSS and non TSS strains demonstrated a high lipolytic activity. TSST-1 positive strains in general, showed significantly higher lipase activity than strains isolated from septicemia (p less than 0.0001) and superficial (p less than 0.0001) infections. The proteolytic activity is higher among TSS (median value 0.075 U/ml) than to non TSS (median value 0.045 U/ml) strains. There was no correlation with the quantity of toxin production in vitro and to the properties described.

Adherence of haemagglutinating Helicobacter pylori to five cell lines

Helicobacter pylori causes gastritis and is an important factor for the development of peptic ulcer disease in man. We used two different methods to examine the adhesion of nine H. pylori strains, with different haemagglutinating properties, to five cell lines, HeLa S3, HFI, Vero, SW1222 and WEHI cells. The adhesion studies were performed a) as bacterial adhesion to a monolayer of tissue culture cells, visualizing bacteria with fluorescein-isothiocyanate-labelled antibodies, b) as cell agglutination with bacteria and eucaryotic cells mixed in a suspension. The H. pylori strains were divided into three groups according to their cell adhesion properties. In general, H. pylori strains which showed the best adhesion to the five cell lines were strains which showed the best capability of agglutinating erythrocytes of several animal species. It is likely that the same adhesins are involved in cell adhesion and in haemagglutination. The two methods gave similar results.