A functional genomic analysis of type 3 Streptococcus pneumoniae virulence.

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature-tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature-tagged mutants was created by insertion-duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty-six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty-two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.

The SpeB virulence factor of Streptococcus pyogenes, a multifunctional secreted and cell surface molecule with strepadhesin, laminin-binding and cysteine protease activity.

The interactions between pathogenic bacteria and the host need to be resolved at the molecular level in order to develop novel vaccines and drugs. We have previously identified strepadhesin, a novel glycoprotein-binding activity in Streptococcus pyogenes, which is regulated by Mga, a regulator of streptococcal virulence factors. We have now identified the protein responsible for the strepadhesin activity and find that (i) strepadhesin activity is carried by SpeB, streptococcal pyrogenic exotoxin with cysteine protease activity; (ii) SpeB carries laminin-binding activity of the bacteria; and (iii) SpeB is not only a secreted molecule but also occurs unexpectedly tightly bound to the bacterial cell surface. Thus, in contrast to the previous view of SpeB as mainly an extracellular protease, it is also present as a streptococcal surface molecule with binding activity to laminin and other glycoproteins.

The effects of sialic acid on the gene expression of fibrillar collagens: different changes in normal and fibrotic scar derived fibroblasts.

OBJECTIVE: To investigate the effect of free sialic acid on collagen gene expression in fibroblasts. DESIGN: Cell culture study. SETTING: University hospital, Finland. CELL LINES: Human granulation tissue fibroblasts, human hypertrophic scar fibroblasts and human keloid fibroblasts. INTERVENTIONS: Treatment of cell cultures with 3 microM, 30 microM and 300 microM N-acetyl-neuraminic acid. MAIN OUTCOME MEASURES: The measurement of steady state level of mRNA for type I and type III collagen. RESULTS: Fibroblast lines react dissimilarly under the influence of sialic acid. Granulation tissue fibroblasts showed decrease in the gene expression of type I and III collagen, while keloid fibroblasts contrastingly showed an increase. Hypertrophic scar derived fibroblasts showed no change. CONCLUSIONS: Sialic acids may decrease collagen gene expression in granulation tissue and that disturbed wound healing in diabetics and smokers may in part be due to direct effect of sialic acids on fibroblasts. Sialic acids may in part induce keloid formation.

The effects of sialic acid on the gene expression of fibrillar collagens: different changes in normal and fibrotic scar derived fibroblasts.

OBJECTIVE: To investigate the effect of free sialic acid on collagen gene expression in fibroblasts. DESIGN: Cell culture study. SETTING: University hospital, Finland. CELL LINES: Human granulation tissue fibroblasts, human hypertrophic scar fibroblasts and human keloid fibroblasts. INTERVENTIONS: Treatment of cell cultures with 3 microM, 30 microM and 300 microM N-acetyl-neuraminic acid. MAIN OUTCOME MEASURES: The measurement of steady state level of mRNA for type I and type III collagen. RESULTS: Fibroblast lines react dissimilarly under the influence of sialic acid. Granulation tissue fibroblasts showed decrease in the gene expression of type I and III collagen, while keloid fibroblasts contrastingly showed an increase. Hypertrophic scar derived fibroblasts showed no change. CONCLUSIONS: Sialic acids may decrease collagen gene expression in granulation tissue and that disturbed wound healing in diabetics and smokers may in part be due to direct effect of sialic acids on fibroblasts. Sialic acids may in part induce keloid formation.

Determination of the cell adhesion specificity of Streptococcus suis with the complete set of monodeoxy analogues of globotriose.

Streptococcus suis causes meningitis and other serious infections in pigs and humans, and binds to host cell globotriosylceramide. In order to determine the essential hydroxyls involved in binding, the complete set of monodeoxy derivatives of the receptor trisaccharide Gal alpha1-Gal beta1-4Glc were tested as inhibitors of bacterial hemagglutination. Removal of the 4''-, 6'', 2' or 3'-hydroxyls abolished inhibitory activity, which indicated that they were critically involved in binding. The same results were obtained using synthetic lipid-linked monodeoxy derivatives of the trisaccharides in a thin-layer overlay assay. The P(N) and P(O) subtypes of the S. suis adhesin showed similar binding patterns. The hydroxyls of the glucose moiety were not critical for binding, although the adhesin binds better to the trisaccharide Gal alpha1-4Gal beta1-4Glc than the disaccharide Gal alpha1-4Gal.

The galactosyl-(alpha 1-4)-galactose-binding adhesin of Streptococcus suis: occurrence in strains of different hemagglutination activities and induction of opsonic antibodies.

The occurrence of the galactose-(alpha 1-4)-galactose-specific adhesin in Streptococcus suis, a pig and human pathogen causing sepsis, meningitis, and other serious infections, was studied. Poly- and monoclonal anti-bodies to the purified adhesin, as well as pigeon ovomucoid, a specific probe for the adhesin activity, detected one single protein band in extracts of S. suis. The adhesin was detected in all 23 strains studied, representing pathogenic serotypes (1, 2, 4, 5, 7, 8, and nontypeable) and including several weakly hemagglutinating or nonhemagglutinating strains and phase variants. The amount of adhesin detected was not correlated with the hemagglutination activity of the intact bacteria. Extraction of cells showing no binding of pigeon ovomucoid by ultrasonic treatment resulted in extracts with pigeon ovomucoid binding activity, suggesting that the adhesin was not accessible to the probe on the intact cells. Analysis of the amount of capsular polysaccharide revealed an inverse relationship between the hemagglutination activity and expression of capsular polysaccharide, thus suggesting a factor influencing adhesin accessibility. The purified adhesin was highly immunogenic and induced in preliminary experiments bactericidal activity in mice. Thus, the adhesin, with its specific binding mechanism to host cells and a proposed pathogenic role, is widely expressed among strains of different serotypes and therefore appears to represent a novel promising candidate for the development of a vaccine against S. suis.

The Gal alpha 1-4 Gal-binding adhesin of Streptococcus suis, a gram-positive meningitis-associated bacterium.

Streptococcus suis causes septicaemia and meningitis in pigs and occasionally in humans. A major galactose-inhibitable adhesin recognizing the blood group P-related disaccharide Gal alpha 1-4 Gal beta 1--present in the GbO3 glycolipid was identified in S. suis. Two variant adhesins, inhibitable by galactose and N-acetylgalactosamine (type PN) or galactose only (type Po) both preferred the disaccharide in terminal position. The hydrogen bonding patterns were determined using deoxy and other derivatives of the receptor disaccharide, and were compared to that of E. coli PapG396 adhesin. The essential hydroxyls were the HO-4', HO-6', HO-2 and HO-3 hydroxyls; type Po adhesin also weakly interacted with HO-6 and HO-3'. The mechanism differed from that of E. coli which binds to a cluster of five hydroxyls, HO-6, HO-2', HO-3', HO-4' and HO-6'. The purified adhesin had a molecular weight of 18 kDa and an isoelectric point of 6.4. The agglutination of latex-bound purified adhesin was inhibited by the same inhibitors as agglutination with whole bacteria. The adhesin was detected by immunoblot analysis in all 23 S. suis strains examined representing different serotypes, was highly immunogenic and showed opsonizing activity. This represents the first example of the comparison of the saccharide receptor hydrogen bondings of two bacteria of different origin and shows that the same saccharide may be recognised by two different mechanisms. As a potential virulence factor present in different serotypes the adhesin represents a potential vaccine against S. suis infections.

Purification of a galactosyl-alpha 1-4-galactose-binding adhesin from the gram-positive meningitis-associated bacterium Streptococcus suis.

Streptococcus suis causes meningitis, sepsis, and other serious infections in newborn and young pigs and in adult humans. The Gal alpha 1-4Gal-binding adhesin of S. suis was purified to homogeneity by ultrasonic treatment, fractional ammonium sulfate precipitation, and preparative polyacrylamide gel electrophoresis. Pigeon ovomucoid, a glycoprotein with Gal alpha 1-4Gal terminals, was used to detect the adhesin by blotting. The purified adhesin appeared as single band of an apparent size of 18 kDa and of a pI of 6.4; no disulfide bridges were present. The amount of adhesin as revealed by pigeon ovomucoid binding correlated with the hemagglutination activity of different S. suis strains. The purified adhesin bound to latex particles induced hemagglutination which was specifically inhibited with the same inhibitors as hemagglutination by the intact bacteria, thus demonstrating that the purified protein was the Gal alpha 1-4Gal-recognizing adhesin of S. suis. Two adhesin variants (PN and PO) with differing Gal alpha 1-4Gal binding specificity had the similar electrophoretic mobilities and the same N-terminal peptide sequences, indicating that they were closely related. This represents the first isolation of an adhesin with well-defined cell surface carbohydrate binding activity from Gram-positive bacteria associated with meningitis.

Oligosaccharide-receptor interaction of the Gal alpha 1-4Gal binding adhesin of Streptococcus suis. Combining site architecture and characterization of two variant adhesin specificities.

The sugar binding specificities of two groups of Streptococcus suis, a pig pathogen that causes meningitis also in man, were determined. Both the group represented by a recently characterized strain inhibitable by galactose and N-acetylgalactosamine (type PN) and the group inhibitable by galactose (type PO) were found by hemagglutination and solid-phase binding inhibition experiments to recognize the disaccharide Gal alpha 1-4Gal of the P1 and Pk blood group antigens. Both types preferred the disaccharide in terminal position. PN showed some, whereas PO showed almost no, binding to the globoside oligosaccharide containing an additional GalNAc beta 1-3 residue. The complete hydrogen bonding patterns were determined by using deoxy and other synthetic derivatives of the receptor disaccharide, and the constructed models of the interactions were compared with that of Escherichia coli PapG396 adhesin. The essential hydroxyls for binding were the HO-4', HO-6', HO-2, and HO-3 hydroxyls on the beta' alpha-side of the Gal alpha 1-4Gal molecule. Type PO adhesin also formed weak interactions with the hydroxyls HO-6 and HO-3'. The mechanism differed from that of E. coli, which binds to a cluster of five hydroxyls (HO-6, HO-2', HO-3', HO-4', and HO-6') and thus to a different part of the receptor disaccharide. These results represent the first example of the comparison of the saccharide receptor hydrogen bonding patterns of two bacterial organisms of different origin and show that the same saccharide may be recognized by two different binding mechanisms.

Characterization of a novel bacterial adhesion specificity of Streptococcus suis recognizing blood group P receptor oligosaccharides.

Streptococcus suis causes sepsis, meningitis, and other serious infections in piglets, and meningitis in humans. Hemagglutination inhibition experiments with mono- and oligosaccharides and glycoproteins indicated that galactose-binding strains of S. suis recognized the Gal alpha 1-4Gal sequence present in the P1 and Pk blood group antigen structures. In thin-layer chromatography overlay assays the bacteria bound to trihexosylceramide (GbO3) but not to globoside (GbO4) or Forssman glycolipid (GbO5), in contrast to P-fimbriated Escherichia coli, which bound only to the latter two. The S. suis adhesin also differed from that of E. coli in that some of the hydrogen bonds formed with the receptor, as determined with chemically modified receptor analogues, were different. In agreement with the binding specificity, the S. suis bacteria agglutinated best among P blood group erythrocytes those of the P1k and P2k type, and from different animal erythrocytes those from rabbit, which express GbO3 as the predominant glycolipid. Binding to frozen sections of pig pharyngeal tissue was decreased by the free GbO3 oligosaccharide and its protein conjugate, which indicated that the corresponding glycolipid may function as receptor for galactose-binding strains of S. suis in pig pharyngeal epithelium.

Identification of N-acetylneuraminyl alpha 2-->3 poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains.

Streptococcus suis is a common cause of sepsis, meningitis, and other serious infections in young piglets and also causes meningitis in humans. The cell-binding specificity of sialic acid-recognizing strains of Streptococcus suis was investigated. Treatment of human erythrocytes with sialidase or mild periodate abolished hemagglutination. Hemagglutination inhibition experiments with sialyl oligosaccharides indicated that the adhesin preferred the sequence NeuNAc alpha 2-3Gal beta 1-4Glc(NAc). Resialylation of desialylated erythrocytes with Gal beta 1-3(4)GlcNAc alpha 2-3-sialyltransferase induced a strong hemagglutination, whereas no or only weak hemagglutination was obtained with cells resialylated with two other sialyltransferases. Binding of radiolabeled bacteria to blots of erythrocyte membrane proteins revealed binding to the poly-N-acetyllactosamine-containing components Band 3, Band 4.5, and polyglycosyl ceramides and to glycophorin A. The involvement of glycophorin A as a major ligand was excluded by the strong hemagglutination of trypsin-treated erythrocytes and En(a-) erythrocytes defective in glycophorin A. Sensitivity of the hemagglutination toward endo-beta-galactosidase treatment of erythrocytes and inhibition by purified poly-N-acetyllactosaminyl glycopeptides indicated that the adhesin bound to glycans containing the following structure: NeuNAc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-.

Hemagglutination activities of group B, C, D, and G streptococci: demonstration of novel sugar-specific cell-binding activities in Streptococcus suis.

A total of 378 streptococcal isolates of Lancefield groups B, C, D and G were tested for their ability to hemagglutinate untreated, sialidase-treated, and endo-beta-galactosidase-treated human erythrocytes. Of the 43 strains showing positive hemagglutination, 9 were inhibitable with neutral monosaccharides. Four strains were inhibited with galactose and N-acetylgalactosamine, whereas five were inhibited with galactose only. A third, sialic acid-specific adhesion activity was suggested for two additional strains on the basis of their agglutination of native and endo-beta-galactosidase-treated but not sialidase-treated erythrocytes. All the sugar-specific agglutination activities detected were confined to Streptococcus suis strains of group D streptococci, whereas streptococci of other groups did not exhibit these types of hemagglutination activities. The adhesins were sensitive to proteases and heat treatment, which indicates that they were proteins. The hemagglutinating isolates of S. suis originated from pig brain and lung, human brain, and the tonsils of healthy pigs. No clear correlation with a particular serotype was observed. These results demonstrate the occurrence of unique sugar-specific adherence activities in S. suis, an important pig pathogen with occasional human pathogenicity.