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.
Demuth
D R
DR
Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104.
Davis
C A
CA
Corner
A M
AM
Lamont
R J
RJ
Leboy
P S
PS
Malamud
D
D
D000373:Agglutinins / Q000276:immunology / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D012463:Saliva / Q000276:immunology / Q000378:metabolism*; D013298:Streptococcus sanguis / Q000235:genetics* / Q000276:immunology
D000373:Agglutinins; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D006023:Glycoproteins; C045564:saliva-interacting cell wall protein, Streptococcus