A 23 kDa membrane glycoprotein bearing NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells.

Streptococcus sanguis colonizes several human oral surfaces, including both hard and soft tissues. Large salivary mucin-like glycoproteins bearing sialic acid residues are known to bind various S.sanguis strains. However, the molecular basis for the adhesion of S.sanguis to human buccal epithelial cells (HBEC) has not been established. The present study shows that S.sanguis OMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner. The desialylation of such cells invariably abolishes adhesion of S.sanguis OMZ 9 to the cell surface. A soluble glycopeptide bearing short sialylated O-linked carbohydrate chains behaves as a potent inhibitor of the attachment of S.sanguis OMZ 9 to exfoliated HBEC. The resialylation of desialylated HBEC with CMP-sialic acid and Gal beta 1,3GalNAc alpha 2,3-sialyltransferase specific for O-glycans restores the receptor function for S.sanguis OMZ 9, whereas a similar cell resialylation with the Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase specific for N-glycans is without effect. Finally, the same resialylation reaction carried out with CMP-9-fluoresceinyl-sialic acid as a substrate yields exfoliated HBEC bearing fluorescence on a single 23 kDa protein, when using the alpha 2,3-sialyltransferase as the catalyst. The latter finding demonstrates that this 23 kDa cell surface glycoprotein bears NeuNAc alpha 2-3Gal beta 1-3GalNAc O-linked sugar chains, a carbohydrate sequence which is recognized by S.sanguis OMZ 9 on exfoliated HBEC. In similar experiments carried out with a buccal carcinoma cell line termed SqCC/Y1, S.sanguis OMZ 9 did not attach in great numbers to such cultured cells, and these cells were shown to not express membrane glycoprotein bearing alpha 2,3-sialylated O-linked carbohydrate chains.

Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation.

In the last 30 years, 81 Streptococcus thermophilus bacteriophage isolates were collected from industrial yogurt (n = 40) and cheese (n = 41) fermentation. Forty-six distinct restriction patterns of phage DNA (11 in yogurt and 35 in cheese) were observed. The phages were investigated for host range, serological properties, and DNA homology to study whether these three independent techniques can be used to classify the phages into taxonomic groups. Yogurt factory-derived phages were classified into the same two subgroups by serology, host range analysis, and hybridization with subgroup-specific DNA sequences. Cheese factory-derived phages, however, could not be classified: the 35 cheese phage isolates with distinct restriction patterns showed 34 different host ranges. All but one cheese phage isolate showed serological cross-reactivity with yogurt phages. A phage DNA fragment that hybridized with all phage DNA samples was cloned, establishing the genetic relatedness of all S. thermophilus phages from our collection. With the sequence information from an unusually conserved S. thermophilus phage DNA element (H. Brüssow, A. Probst, M. Frémont, and J. Sidoti, Virology 200:854-857, 1994), a PCR-based phage detection method was developed for cheese whey from a factory that produced mozzarella cheese with complex undefined starter mixes. PCR allowed the detection of phages in cheese whey (detection limit, 10(3) PFU/ml) which could not be detected by dot blot hybridization techniques (detection limit, 10(7) PFU/ml).

Adhesion of colonization factor antigen II-positive enterotoxigenic Escherichia coli strains to human enterocytelike differentiated HT-29 cells: a basis for host-pathogen interactions in the gut.

Enterotoxigenic Escherichia coli are the most common cause of travelers' and infant diarrhea in less-developed countries. In the present work, among several metabolically labeled human diarrheagenic E. coli strains, enterotoxigenic strains expressing colonization factor antigen II were shown to bind to HT-29 intestinal cell monolayers when these cells were grown in conditions promoting their enterocytic differentiation. Indirect immunofluorescence with fimbrial antisera revealed that pathogen attachment was associated with the production of a specific bacterial adhesin, the E. coli surface antigen CS3. Scanning and transmission electron micrographs showed an apical pattern of colonization, characteristic of enterotoxigenic E. coli infections. The above data were consistent with all observations previously made with human enterocytes obtained from intestinal biopsies. The lectin-carbohydrate nature of this cell-cell recognition mechanism was also established. Bacterial binding to differentiated HT-29 cells was inhibited by a mixture of newborn meconium glycopeptides. By coating the cell layers with the plant agglutinin from Evonymus europaea, pathogen attachment was also prevented. Binding of 125I-labeled CS3 adhesin and E. europaea agglutinin to brush border membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose revealed three bands of about 30, 20, and 13 kilodaltons, which acted as receptors for both bacterial and plant lectins. These data suggest that the sugar units to which the bacterial colonization factor CS3 binds are synthesized as carbohydrate chains of three brush border membrane glycoproteins in HT-29 cells by a differentiation-specific pathway.

Cross-neutralizing antibodies induced by single serotype vaccination of cows with rotavirus.

Single serotype vaccination of mature cows with nine different strains of bovine, simian and human rotaviruses induced heterotypic milk and serum neutralizing antibodies against two bovine and four human rotavirus serotypes. Immunization with single-shelled simian rotavirus SA11 increased milk and serum neutralization titres fivefold over those of control cows, without inducing antibodies to outer shell polypeptides of rotavirus. Vaccination with double-shelled SA11 virions also elicited cross-reacting antibodies to the outer shell proteins VP3 and/or VP7 which neutralized rotavirus seven times more efficiently than antisera to single-shelled SA11 virus. A related rotavirus similar to simian rotavirus SA11, but from a different host, might thus be an attractive vaccine for immunization of pregnant cows to confer passive immunity to calves.

Characterization of a second bovine rotavirus serotype.

Bovine rotavirus (BRV) V 1005 was characterized by two-way cross-neutralization tests as a second serotype of BRV. Virions and inner shell particles of 65 nm and 55 nm diameter respectively, and empty capsids of 65 nm and 55 nm diameter were separated by density gradient centrifugation. Three polypeptides of molecular weight 60,000, 36,000 and 28,000 (minor protein) could be identified in the outer shell of virions and in the larger empty capsids. Inner shell particles contained three polypeptides of molecular weight 105,000, 83,000 and 43,000. Both sizes of empty capsids showed two polypeptides of molecular weight 75,000 and 55,000 not found in virions. Pulse-labelling of infected cells revealed eight major and three minor intracellular viral polypeptides. Viral polypeptides synthesis started at about 6 hours p.i. and correlated in time with double-stranded RNA synthesis. As soon as viral polypeptide synthesis was detectable, newly synthesized viral polypeptides were incorporated into intracellular viral particles. Radioactive viral polypeptides appeared without a longer lag period in extracellular viruses from 6 hours p.i. onwards.

Plasmids, Lactic Acid Production, and N-Acetyl-d-Glucosamine Fermentation in Lactobacillus helveticus subsp. jugurti.

Two lactobacillus strains, Lactobacillus helveticus subsp. jugurti S13-8 and L. helveticus subsp. jugurti S36-2, were examined for the presence of plasmids. Plasmids of 16.45, 13.03, and 11.83 kilobases (kb) were found in the first, low lactic acid-producing strain; their function is not presently known. A single plasmid species of 13.17 kb was found in the second, high lactic acid-producing strain. This plasmid was found to be associated with lactic acid production and N-acetyl-d-glucosamine fermentation.