Cloning and expression of a Streptococcus sanguis surface antigen that interacts with a human salivary agglutinin

Human saliva contains a high-molecular-weight glycoprotein (agglutinin) which binds to specific streptococci in a calcium-dependent reaction leading to the formation of bacterial aggregates. We report the cloning of a gene encoding a surface antigen from Streptococcus sanguis M5 and show that the expressed protein inhibits agglutinin-mediated aggregation and specifically binds the salivary agglutinin in a calcium-dependent fashion. Clones isolated from the immunological screening of S. sanguis M5 genomic libraries with polyclonal antibodies against whole cells were assayed for the ability to compete with S. sanguis for agglutinin. One clone, pSSP-5, expressed antigens of 165 and 130 kilodaltons (kDa) possessing this activity. A 3-kilobase-pair (kbp) insert fragment from this clone was used to screen a genomic library in lambda EMBL3 which resulted in the isolation of clone SSP-5A. This clone contained an insert of 17 kb and expressed proteins of 170 to 205 kDa that reacted with the anti-S. sanguis antibodies. Subcloning of a 5.3-kbp EcoRI-BamHI fragment from SSP-5A produced pEB-5, which expressed streptococcal components that were indistinguishable from SSP-5A. The streptococcal antigen was purified by gel permeation and ion exchange chromatography and shown to potently compete with S. sanguis M5 cells for agglutinin. The antigen also bound purified salivary agglutinin in the presence of 1 mM CaCl2. This binding was inhibited by EDTA. Both the SSP-5 antigen and a 205-kDa protein in surface protein extracts from S. sanguis M5 cross-reacted with antibodies directed against antigen B from S. mutans and SpaA from S. sobrinus 6715. These results indicate that a 205-kDa surface protein that is antigenically related to SpaA and antigen B is involved in the binding of salivary agglutinin to S. sanguis M5.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  R. Lamont,et al.  Streptococcus sanguis surface antigens and their interactions with saliva , 1988, Infection and immunity.

[3]  J. Ogier,et al.  Cloning of the saliva-interacting protein gene from Streptococcus mutans , 1987, Journal of bacteriology.

[4]  J. Cisar,et al.  Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V , 1987, Journal of bacteriology.

[5]  R. Arnold,et al.  Differentiation and interaction of secretory immunoglobulin A and a calcium-dependent parotid agglutinin for several bacterial strains , 1987, Infection and immunity.

[6]  P. Fives-Taylor,et al.  Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae , 1987, Infection and immunity.

[7]  E. J. Morris,et al.  Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein , 1987, Journal of bacteriology.

[8]  L. Tabak,et al.  Preparation of a sialic acid-binding protein from Streptococcus mitis KS32AR , 1986, Infection and immunity.

[9]  R. Curtiss.  Genetic Analysis of Streptococcus mutans Virulence and Prospects for an Anticaries Vaccine , 1986, Journal of dental research.

[10]  J. Slot,et al.  Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril , 1986, Journal of Bacteriology.

[11]  H. C. van der Mei,et al.  Structural properties of fibrillar proteins isolated from the cell surface and cytoplasm of Streptococcus salivarius (K+) cells and nonadhesive mutants , 1986, Journal of bacteriology.

[12]  D. Malamud,et al.  Identification of a Streptococcus sanguis Receptor for Salivary Agglutinins , 1986, Journal of dental research.

[13]  J. Haselgrove,et al.  A rapid, inexpensive, quantitative, general-purpose densitometer and its application to one-dimensional gel electrophoretograms. , 1985, Analytical biochemistry.

[14]  E. J. Morris,et al.  Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity , 1985, Journal of bacteriology.

[15]  D. Malamud,et al.  A comparison of bacterial aggregation induced by saliva, lysozyme, and zinc , 1985, Infection and immunity.

[16]  D. W. Thompson,et al.  Surface properties of Streptococcus sanguis FW213 mutants nonadherent to saliva-coated hydroxyapatite , 1985, Infection and immunity.

[17]  C. Douglas,et al.  Effect of specific antisera upon Streptococcus mutans adherence to saliva-coated hydroxylapatite , 1984 .

[18]  J. Messing,et al.  Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. , 1983, Gene.

[19]  K. Knox,et al.  Characteristics of a high molecular weight extracellular protein of Streptococcus mutans. , 1983, Journal of general microbiology.

[20]  J. Rundegren,et al.  Characterization of a salivary agglutinin reacting with a serotype c strain of Streptococcus mutans. , 1983, European journal of biochemistry.

[21]  D. Malamud,et al.  Enhanced saliva-mediated bacterial aggregation and decreased bacterial adhesion in caries-resistant versus caries-susceptible individuals , 1982, Infection and immunity.

[22]  Y. Abiko,et al.  Streptococcus mutans Genes That Code for Extracellular Proteins in Escherichia coli K-12 , 1982, Infection and immunity.

[23]  L. Tabak,et al.  Specificity of salivary-bacterial interactions: II. Evidence for a lectin on Streptococcussanguis with specificity for a NeuAcα2,3Ga1β1,3Ga1NAc sequence , 1982 .

[24]  R. Arnold,et al.  Bactericidal Activity of Human Lactoferrin: Differentiation from the Stasis of Iron Deprivation , 1982, Infection and immunity.

[25]  D. Malamud,et al.  Characteristic differences between saliva-dependent aggregation and adhesion of streptococci , 1982, Infection and immunity.

[26]  R. Arnold,et al.  Inhibition of dental plaque acid production by the salivary lactoperoxidase antimicrobial system , 1981, Infection and immunity.

[27]  D. Malamud,et al.  Bacterial aggregating activity in human saliva: comparisons of bacterial species and strains , 1981, Infection and immunity.

[28]  T. Lehner,et al.  Separation and characterization of a protein antigen from cells of Streptococcus mutans. , 1981, Journal of general microbiology.

[29]  T. Lehner,et al.  Affinity purification and characterization of protease-susceptible antigen I of Streptococcus mutans , 1980, Infection and immunity.

[30]  R. Russell.  Distribution of cross-reactive antigens A and B in Streptococcus mutans and other oral streptococci. , 1980, Journal of general microbiology.

[31]  T. Lehner,et al.  Protein antigens of Streptococcus mutans: purification and properties of a double antigen and its protease-resistant component , 1980, Infection and immunity.

[32]  D. Malamud,et al.  Bacterial aggregating activity in human saliva: simultaneous determination of free and bound cells , 1979, Infection and immunity.

[33]  R. Russell.  Wall-associated protein antigens of Streptococcus mutans. , 1979, Journal of general microbiology.

[34]  D. Bratthall,et al.  Salivary agglutinin and secretory IgA reactions with oral streptococci. , 1978, Scandinavian journal of dental research.

[35]  R. Gibbons,et al.  Comparative Estimates of Bacterial Affinities and Adsorption Sites on Hydroxyapatite Surfaces , 1978, Infection and immunity.

[36]  M. Levine,et al.  Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans , 1978, Infection and immunity.

[37]  W. D. Benton,et al.  Screening lambdagt recombinant clones by hybridization to single plaques in situ. , 1977, Science.

[38]  W. Liljemark,et al.  Competitive Binding Among Oral Streptococci to Hydroxyapatite , 1977, Journal of dental research.

[39]  I. Mandel.  Nonimmunologic Aspects of Caries Resistance , 1976, Journal of dental research.

[40]  K. Pruitt,et al.  The reaction of salivary substances with bacteria. , 1975, Journal of oral pathology.

[41]  R. Eisenberg,et al.  A method for gentle lysis of Streptococcus sanguis and Streptococcus mutans. , 1975, Biochemical and biophysical research communications.

[42]  J. Summers.  Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius, endonuclease R-HaeIII , 1975, Journal of virology.

[43]  E. Stowell,et al.  Effects of Salivary Proteins on the Adsorption of Cariogenic Streptococci by Hydroxyapatite , 1971, Journal of dental research.

[44]  J. van Houte,et al.  Sorption of bacteria to human enamel powder. , 1970, Archives of oral biology.

[45]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[46]  C. Douglas,et al.  The adsorption of human salivary components to strains of the bacterium Streptococcus mutans. , 1984, Archives of oral biology.

[47]  D. Helfman,et al.  Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[48]  S. Brenner,et al.  New bacteriophage lambda vectors with positive selection for cloned inserts. , 1983, Methods in enzymology.

[49]  S. Kashket,et al.  Saliva-induced aggregation of Streptococci. Solubilization of a bacterial surface receptor. , 1982, Caries research.

[50]  D. Malamud,et al.  A MODEL FOR SALIVA-MEDIATED BACTERIAL AGGREGATION , 1981 .

[51]  S. D. Hogg,et al.  Surface fibrils may be responsible for the salivary glycoprotein-mediated aggregation of the oral bacterium Streptococcus sanguis. , 1981, Archives of oral biology.

[52]  V. Iacono,et al.  Bacteriolysis of Streptococcus mutans BHT by lysozyme and inorganic anions normally present in human saliva. , 1981, Archives of oral biology.

[53]  T. Ericson,et al.  Effect of salivary agglutinins of reactions between hydroxyapatite and a serotype c strain of Streptococcus mutans. , 1976, Caries research.

[54]  L. Heppel.  THE CONCEPT OF PERIPLASMIC ENZYMES , 1971 .