Immunologic characteristics of a Streptococcus mutans glucosyltransferase B sucrose-binding site peptide-cholera toxin B-subunit chimeric protein.
Glucosyltranferases (Gtfs) produced by the mutans streptococci are recognized as virulence factors in dental caries, and the inhibition of Gtfs by secretory immunoglobulin A is predicted to provide protection against this disease. The basis of such mucosal immunity is linked to the ability to reliably stimulate production of secretory immunoglobulin A against Gtfs. In this regard, we are exploring the immunogenicities of various Gtf peptides genetically fused to the B subunit of cholera toxin (CTB), a known mucosal adjuvant. In this work, we have created a gene fusion linking the GtfB active-site (AS) peptide DANFDSIRVDAVDNVDADLLQIA to the amino terminus of CTB. This sequence, deduced from the nucleotide sequence of gtfB from Streptococcus mutans GS5, has been found to be strongly conserved in Gtfs from several mutans streptococci. We have purified this recombinant protein (AS:CTB) from Escherichia coli carrying the fusion gene under the control of the lactose operon promoter. This protein was immunogenic in rabbits and produced specific serum antibodies against both the Gtf peptide and the CTB moiety. The antiserum was tested for its ability to inhibit GtfB activity obtained from a mutant of S. mutans able to make only this enzyme and none of the other usual Gtfs or fructosyltransferase. Approximately 50% of the GtfB activity was inhibited in such assays. These results suggest that the AS of this enzyme is accessible to antibody binding and that this region of the protein may be considered a vulnerable target for vaccine design and development. The AS:CTB was able to bind GM1, ganglioside in enzyme-linked immunosorbent assays, indicating that the recombinant protein retained this property, which is though to be critical to the mucosal immunoadjuvant properties of CTB. Thus, this protein may be promising as a candidate anticaries vaccinogen alone or in combination with other Gtf peptides or conjugates.
Laloi
P
P
Centre de Génetique Moléculaire et Cellulaire, UMR CNRS 106, Université Claude Bernard, Villeurbanne, France.
Munro
C L
CL
Jones
K R
KR
Macrina
F L
FL
D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001483:Base Sequence; D001665:Binding Sites / Q000276:immunology; D002772:Cholera Toxin / Q000096:biosynthesis / Q000276:immunology*; D003731:Dental Caries / Q000517:prevention & control; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D008297:Male; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology*; D013295:Streptococcus mutans / Q000201:enzymology / Q000235:genetics / Q000276:immunology*; D013395:Sucrose / Q000378:metabolism
D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D002772:Cholera Toxin; C036655:1,3-alpha-D-glucan synthase; D005964:Glucosyltransferases