Åsa Ljungh

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.

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.

Adhesion of staphylococci to chemically modified and native polymers, and the influence of preadsorbed fibronectin, vitronectin and fibrinogen.

A commercially available poly(ether urethane), polyethylene, and modifications of these polymers have been compared with respect to adsorption of fibronectin, fibrinogen and vitronectin. The adhesion of staphylococcal strains (characterized for ability to bind immobilized proteins, cell surface hydrophobicity and charge) was studied by bioluminescence with and without preadsorption of proteins to the surfaces. The least amount of proteins and the fewest bacteria adhered to the amphiphilic surfaces. When polymers were preincubated with plasma or albumin, lower numbers of bacteria adhered, except to Pellethane grafted with PEG 20,000, to which coagulase-negative staphylococci adhered to a higher extent.

Effect of Cold Starvation, Acid Stress, and Nutrients on Metabolic Activity of Helicobacter pylori

ABSTRACT Helicobacter pylori can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoids, with intermediate forms called U forms. The importance of nonculturable coccoid forms of H. pylori in disease transmission and antibiotic treatment failures is unclear. Metabolic activities of actively growing as well as nonculturable H. pylori were investigated by comparing the concentrations of cellular ATP and total RNA, gene expression, presence of cytoplasmic polyphosphate granules and iron inclusions, and cellular morphology during extended broth culture and nutritional cold starvation. In addition, the effect of exposing broth-cultured or cold-starved cells to a nutrient-rich or acidic environment on the metabolic activities was investigated. ATP was detectable up to 14 days and for at least 25 days after transformation from the spiral form to the coccoid form or U form in broth-cultured and cold-starved cells, respectively. mRNAs of VacA, a 26-kDa protein, and urease A were detected by using reverse transcription-PCR in cells cultured for 2 months in broth or cold starved for at least 28 months. The ATP concentration was not affected during exposure to fresh or acidified broth, while 4- to 12-h exposures of nonculturable cells to lysed human erythrocytes increased cellular ATP 12- to 150-fold. Incubation of nonculturable cold-starved cells with an erythrocyte lysate increased total RNA expression and ureA mRNA transcription as measured by quantitative real-time reverse transcription-PCR. Furthermore, the number of structurally intact starved coccoids containing polyphosphate granules increased almost fourfold (P = 0.0022) under the same conditions. In conclusion, a specific environmental stimulus can induce ATP, polyphosphate, and RNA metabolism in nonculturable H. pylori, indicating viability of such morphological forms.

Enterobacteriaceae found in high numbers in fish, minced meat and pasteurised milk or cream and the presence of toxin encoding genes

Enterobacteriaceae were found in high numbers after storage at 7 degrees C in 6% of consumers packs of pasteurised milk or cream, in 31% of retailed fish and in 100% of retail packs of minced meat. Seventy two fresh-water fishes, 40 packs of minced meat and 430 milk packs were sampled. One hundred and eighty four isolates were randomly picked from Tryptone glucose extract (TGE) agar (30 degrees C for 3d) or Violet red bile glucose (VRBG) agar (37 degrees C for 1d). In minced meat, Serratia liquefaciens, Hafnia alvei, Rahnella aquatilis were frequently encountered. On fish, the most frequently found species were R. aquatilis, and in milk, the dominating species were S. liquefaciens, H. alvei and R. aquatilis. One to three isolates of Citrobacter freundii were found in all three food categories. Using a polymerase chain reaction (PCR) technique, the gene of Escherichia coli heat-labile toxin (lt) was indicated in one fish isolate of R. aquatilis whereas heat-stable toxin genes (s.t.) were indicated in four H. alvei isolates, two originating from fish and two from minced meat. Positive PCR-reaction for vero cytotoxin genes were found in one H. alvei strain originating from fish (vt1), in two S. liquefaciens strains from minced meat (vt2), and in a C. freundii reference strain. One of the st-positive H. alvei strains from meat harboured the eaeA gene involved in the attaching phenotype of enteropathogenic E. coli.

A novel von Willebrand factor binding protein expressed by Staphylococcus aureus

When a shotgun phage-display library of Staphylococcus aureus Newman was affinity selected (panned) against recombinant von Willebrand factor (vWf), a novel von Willebrand factor binding protein (vWbp) was found. Experimental data indicate that the interaction between vWbp and vWf is very specific and mediated by a region of 26 aa residues in the C-terminal part of vWbp. vWbp has an N-terminal secretory signal sequence but no cell wall anchoring motif, suggesting a soluble extracellular location. Mature vWbp could be purified from the culture supernatant and the identity of the protein was confirmed by N-terminal sequencing. vWbp migrates with an apparent molecular mass of 66 kDa and the deduced protein consists of 482 aa. The gene encoding vWbp, named vwb, was present in all S. aureus strains investigated.

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.

Detection of Antibodies to Helicobacter pylori Cell Surface Antigens

Serum IgG antibodies of Helicobacter pylori were detected in single-dilution ELISA using glycine extracted material. Among 148 endoscopy patients 59% displayed antibodies; as expected, a higher occurrence (90%) was found in patients with positive gastric culture for H. pylori than in culture negative patients (37%). Among 68 blood donors the frequency of H. pylori antibodies was 28%. In 73 children less than 15 years of age examined for unrelated disorders the occurrence was 4%. By immunoblotting using the same extract, 3 prominent bands, 29K, 54K and 60K and several weak bands were identified. These were formed by 57%, 92%, and 65%, respectively, of the ELISA positive patient sera. Comparing culture positive and negative patients, the 3 bands occurred more often among the culture positive subjects though between 18 and 61% of the sera from culture negative patients gave either of the bands. When comparing the glycine extracts of 4 different H. pylori strains with separate haemagglutinating patterns no differences in the position of the major bands emerged. By absorption experiments no immunological cross-reactivity with components of Escherichia coli, Klebsiella pneumoniae, Campylobacter jejuni or C. fetus was found. Thus, the glycine extract seemed specific for the detection of antibodies to H. pylori.

Cell Surface Proteins of Helicobacter pylori as Antigens in an ELISA and a Comparison with Three Commercial ELISA

Cell surface proteins of Helicobacter pylori were solubilized by extraction with acidic glycine buffer, N-octyl-glucoside, lithium chloride, and distilled water, and by sonication. The preparations were evaluated as antigens in ELISA to detect serum IgG responses in patients and healthy subjects. SDS-PAGE analyses of the preparations from a type strain (NCTC 11637) and of acidic glycine extracts of 4 clinical isolates showed multiple protein bands. The sera were classified as HP+ve and HP-ve by culture of biopsy and immunoblotting. Sera were considered positive for H. pylori if they detected the specific 120kD antigen or 4-5 other bands. 49 sera were HP+ve; the 51 HP-ve sera did not react in immunoblotting. 35/44 sera (80%) that reacted with the 120kD antigen demonstrated high titers in ELISA with all antigen preparations, and the remaining 9(20%) sera gave discordant results. 4/5 HP+ve sera that did not react with the 120kD antigen, demonstrated high ELISA titers with all 5 antigen preparations. Glycine extracts of 3 isolates did not exhibit the 120kD protein, but were equally sensitive in ELISA. The role of 120kD antigen in our ELISA was not clear. Immunoblotting demonstrated that the 5 antigen preparations share similar antigenic components. All preparations were similarly high in sensitivity and specificity, indicating that surface antigens could be satisfactorily used in our ELISA. Our ELISA using the glycine extract was compared with commercial H. pylori ELISAs developed by Bio-Rad Laboratories, USA (GAP ELISA), Roche, Switzerland (EIA 2G), and Whittaker Bioproducts, USA (Pyloristat).(ABSTRACT TRUNCATED AT 250 WORDS)

Aeromonas and Plesiomonas as food- and waterborne pathogens

Aeromonas and Plesiomonas have become increasingly recognized as human enteropathogens. Plesiomonas shigelloides has mainly been recovered from various sea foods, whereas Aeromonas sp. have also been cultured from pigs, broilers, eggs, milk and vegetables. Aeromonas sp. also multiply rapidly at +4 degrees C which is a significant risk in food storage. Aeromonas sp. have furthermore been recovered from fresh water sources, and some isolates are resistant to chlorination which makes it a further risk factor. No large food- or waterborne outbreaks have been reported so far with Aeromonas sp. Various virulence factors involved in intestinal infections are described such as enterotoxins, cytotoxins, and adhesins.