Torkel Wadström

A new test based on ‘salting out’ to measure relative hydrophobicity of bacterial cells

A simple method for quantification of the hydrophobic surface properties of bacteria is described. The method is based on precipitation of cells by salts, for instance (NH4)2SO4. The order in which cells are precipitated is a measure of their surface hydrophobicities, the most hydrophobic cells being first precipitated at low salt concentration. Temperature, pH, time and the bacterial cell concentration were shown to affect the results. When these variables were kept constant the method was highly reproducible. This ‘salting out’ method was applied to enterotoxigenic Escherichia coli strains with different surface protein antigens (fimbriae, fibrillae and colonization factor antigen, CFA). These enterotoxigenic E. coli strain were found to have a surface hydrophobicity in the following order: CFA/I >CFA/II > K88∼K99 > type 1.

Glycosaminoglycan-binding microbial proteins in tissue adhesion and invasion: key events in microbial pathogenicity

Glycosaminoglycans such as heparin, heparan sulphate and dermatan sulphate, are distributed widely in the human body. Several glycosaminoglycans form part of the extracellular matrix and heparan sulphate is expressed on all eukaryotic surfaces. The identification of specific binding to different glycosaminoglycan molecules by bacteria (e.g., Helicobacter pylori, Bordetella pertussis and Chlamydia trachomatis), viruses (e.g., herpes simplex and dengue virus), and protozoa (e.g., Plasmodium and Leishmania), is therefore of great interest. Expression of glycosaminoglycan-binding proteins depends on growth and culture conditions in bacteria, and differs in various phases of parasite development. Glycosaminoglycan-binding microbial proteins may mediate adhesion of microbes to eukaryotic cells, which may be a primary mechanism in mucosal infections, and are also involved in secondary effects such as adhesion to cerebral endothelia in cerebral malaria or to synovial membranes in arthritis caused by Borrelia burgdorferi. It has been suggested that they may enhance intracellular survival in macrophages. Microbial binding of heparin may interfere with heparin-dependent growth factors. Whether or not glycosaminoglycan-binding proteins mediate invasion of epithelial cells is a matter of controversy. Heparin and other glycosaminoglycans may have potential uses as therapeutic agents in microbial infections and could form part of future vaccines against such infections.

SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated glycans

Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid–binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid–dependent in vitro agglutination of erythrocytes (i.e., sialic acid–dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid–dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcα2–3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.

Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR

Abstract A sample preparation method based on a two-phase aqueous polymer system, composed of 8% (w/w) polyethylene glycol 4000 and 11% (w/w) dextran 40, was employed to remove PCR-inhibitory substances from human faeces prior to PCR. The majority of the PCR-inhibitory substances, including bile salts, were shown to be distributed in the polyethylene glycol-rich top phase, whereas target bacteria were detected by PCR in the dextran-rich bottom phase. The efficiency of the aqueous two-phase system in removing PCR-inhibitory substances from faeces was evaluated with a standardised PCR assay containing different concentrations of pure Helicobacter pylori DNA. The detection level for a pure water solution of DNA was in the range 10 −12 to 10 −13 g DNA per reaction tube. Untreated faecal homogenate totally inhibited PCR even after 1000 times dilution in water. A positive PCR result was obtained when 10 −6 g H. pylori DNA was present in a 50 μl reaction mixture containing 10 μl homogenised faeces, which had been briefly centrifuged, boiled and diluted 100 times in water. After extraction of an undiluted heat-treated homogenate in the aqueous two-phase system, the detection level was lowered to the range 10 −10 to 10 −11 g DNA. When the aqueous two-phase system was evaluated on five faecal samples the detection level was decreased by three to five orders of magnitude. Finally, the aqueous two-phase system was applied to faecal samples inoculated with H. pylori cells. The sensitivity of the PCR assay after extraction was found to be 40 times above the detection level of H. pylori in a pure water solution. The target bacteria were detected directly by PCR in the dextran-rich bottom phase. Furthermore, most of the H. pylori cells were shown to be present in the dextran-rich bottom phase.

Treatment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes.

Helicobacter pylori is a gram-negative bacterium affecting about half of the world population, causing chronic gastritis type B dominated by activated phagocytes. In some patients the disease evolves into gastric ulcer, duodenal ulcer, gastric cancer or MALT lymphoma. The pathogenesis is in part caused by the immunological response. In mouse models and in human disease, the mucosal immune response is characterized by activated phagocytes. Mucosal T-lymphocytes are producing IFN-gamma thus increasing mucosal inflammation and mucosal damage. A low dietary intake of antioxidants such as carotenoids and vitamin C may be an important factor for acquisition of H. pylori by humans. Dietary antioxidants may also affect both acquisition of the infection and the bacterial load of H. pylori infected mice. Antioxidants, including carotenoids, have anti-inflammatory effects. The aim of the present study was to investigate whether dietary antoxidant induced modulation of H. pylori in mice affected the cytokines produced by H. pylori specific T-cells. We found that treatment of H. pylori infected mice with an algal cell extract containing the antioxidant astaxanthin reduces bacterial load and gastric inflammation. These changes are associated with a shift of the T-lymphocyte response from a predominant Th1-response dominated by IFN-gamma to a Th1/Th2-response with IFN-gamma and IL-4. To our knowledge, a switch from a Th1-response to a mixed Th1/Th2-response during an ongoing infection has not been reported previously.

Astaxanthin-rich algal meal and vitamin C inhibit Helicobacter pylori infection in BALB/cA mice

ABSTRACT Helicobacter pylori infection in humans is associated with chronic type B gastritis, peptic ulcer disease, and gastric carcinoma. A high intake of carotenoids and vitamin C has been proposed to prevent development of gastric malignancies. The aim of this study was to explore if the microalga Haematococcus pluvialisrich in the carotenoid astaxanthin and vitamin C can inhibit experimental H. pylori infection in a BALB/cA mouse model. Six-week-old BALB/cA mice were infected with the mouse-passagedH. pylori strain 119/95. At 2 weeks postinoculation mice were treated orally once daily for 10 days (i) with different doses of algal meal rich in astaxanthin (0.4, 2, and 4 g/kg of body weight, with the astaxanthin content at 10, 50, and 100 mg/kg, respectively), (ii) with a control meal (algal meal without astaxanthin, 4 g/kg), or (iii) with vitamin C (400 mg/kg). Five mice from each group were sacrificed 1 day after the cessation of treatment, and the other five animals were sacrificed 10 days after the cessation of treatment. Culture of H. pylori and determination of the inflammation score of the gastric mucosae were used to determine the outcome of the treatment. Mice treated with astaxanthin-rich algal meal or vitamin C showed significantly lower colonization levels and lower inflammation scores than those of untreated or control-meal-treated animals at 1 day and 10 days after the cessation of treatment. Lipid peroxidation was significantly decreased in mice treated with the astaxanthin-rich algal meal and vitamin C compared with that of animals not treated or treated with the control meal. Both astaxanthin-rich algal meal and vitamin C showed an inhibitory effect on H. pylori growth in vitro. In conclusion, antioxidants may be a new strategy for treating H. pylori infection in humans.

Lactobacillus acidophilus autolysins inhibit Helicobacter pylori in vitro.

Antibacterial activity of 17 strains of lactobacilli was tested against 10 strains of H. pylori. The inhibition observed was related to the acid production and the low pH attained. No relationship between CagA phenotype of H. pylori strains and tolerance to lactic acid was observed. In mixed cultures, L. acidophilus CRL 639 showed an autolytic behavior after 24 h of culture. At this moment, H. pylori CCUG17874 showed a decrease of 2 log-cycle, and no viable count was detected after 48 h. The bactericidal effect of L. acidophilus CRL 639 in mixed cultures is related to a proteinaceous compound released after cell lysis.

Affinity of the gastric pathogen Helicobacter pylori for the N-sulphated glycosaminoglycan heparan sulphate

Binding of 125I-heparan sulphate was a common property of Helicobacter pylori strains isolated from patients with gastroduodenal ulcer diseases. Binding was (i) saturable; (ii) reversible by the addition of unlabelled heparan sulphate and heparin; (iii) inhibited by unlabelled heparan sulphate, heparin, and heparin oligosaccharides but not by other glycosaminoglycans of comparable size (chondroitin sulphate and dermatan sulphate) or by highly glycosylated glycoproteins (hog gastric mucin and fetuin); (iv) reduced by heat treatment (80 degrees C, 10 min) and exposure of the bacteria to pronase E, proteinase K, trypsin and chymotrypsin, but unaffected by treatment with pepsin and neuraminidase; and (v) time-, pH-, and ionic strength-dependent. Scatchard plot analysis of the binding data indicated the presence of one class of high-affinity receptor (Kd = 9 x 10(-9) M) for heparan sulphate.

Endothelial Dysfunction After Repeated Chlamydia pneumoniae Infection in Apolipoprotein E–Knockout Mice

BackgroundArterial relaxation is largely regulated by endothelial nitric oxide (NO). Its diminished activity has been associated with incipient atherosclerosis. We investigated the endothelium-dependent relaxation of aorta in apolipoprotein E–knockout (apoE-KO) mice exposed to single or repeated Chlamydia pneumoniae inoculation. Methods and ResultsForty-eight apoE-KO mice, 8 weeks old, were inoculated intranasally with C pneumoniae (n=24) or saline (n=24) every 2 weeks over a 6-week period. Twenty mice (10 infected and 10 controls) were killed at 2 weeks and 6 weeks, respectively, after the first inoculation. The smooth muscle tone of aortic rings was measured in vitro at both time points. The norepinephrine-precontracted thoracic aortic rings were successively exposed to methacholine in the absence and presence of NG-nitro-l-arginine methyl ester (L-NAME) and diclofenac. The methacholine-induced relaxation was attenuated in the infected mice at 6 weeks in both the absence and presence of L-NAME (P <0.05 and P <0.01, respectively). When administered together with L-NAME, diclofenac enhanced the relaxation of the L-NAME–pretreated aortas in infected mice at 2 weeks (P <0.05) but not in noninfected mice. The relaxation response from infected mice tended to differ in the same manner at 6 weeks (P <0.1). No intimal thickening was detected at either time point. ConclusionsC pneumoniae impairs arterial endothelial function, and the NO pathway is principally involved. Cyclooxygenase-dependent vasoconstricting products may also account for the infection-induced impaired relaxation. These findings further support the role of C pneumoniae infection in atherosclerosis development.

Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a BAlb/cA mouse model.

The attachment of Helicobacter pylori to the human gastric mucosa is a complex process involving several specific structures recognised by the cell surface receptors. Sialylated multivalent high mol. wt glycoproteins have been shown to inhibit H. pylori sialic acid-specific haemagglutination. This study explored whether sialylated glycoconjugates from bovine milk could inhibit an experimental H. pylori infection in a mouse model. BALB/cA mice (6-8 weeks old) were inoculated with a mouse-passaged H. pylori strain 317p. Four weeks after infection the mice were given lactoferrin (iron-free LF or 20% iron-saturated LF) or bovine milk fat globule membrane fractions (MFGM or defatted MFGM) orally (400 mg/kg body weight) once daily for 10 days and then killed to examine for bacterial colonisation and gastritis. Mice treated with iron-free LF, 20% iron-saturated LF, MFGM or defatted MFGM showed 30%, 10%, 20% or 20% healing rates, respectively, when compared with the H. pylori-infected control. Gastric colonisation by H. pylori was remarkably decreased in all mice treated with bovine milk glycoconjugates and the inflammation score was also significantly lower in treated mice than in infected control animals. The fact that there was no significant difference between iron-free LF and iron-saturated LF or MFGM and defatted MFGM suggested that iron is not crucial for inhibition of H. pylori by lactoferrin and that the lipid part of MFGM is not important for anti-H. pylori activity. In conclusion, bovine milk glycoconjugates showed potencies to inhibit H. pylori infection in this mouse model and, therefore, could be considered as candidates for non-antibiotic strategies against H. pylori infection in man.