Streptococcus mutans is a recognized principal etiologic agent in coronal caries. Although S. mutans has the ability to bind collagen and degrade FALGPA, a synthetic peptide mimicking collagen substrate, its role in dental root caries has not yet been fully elucidated. Degradation of collagen fibrils in dentin was attributed to S. mutans, but a collagenase enzyme has not yet been isolated from this organism. Considering the increased incidence of dental root decay among the elderly, an understanding of the role of the pathogenic factors is necessary to the development of preventive measures. The present study has focused on the cloning and analysis of S. mutans collagenase enzyme. Toward this goal, a putative collagenase gene was identified in S. mutans UA159 by genomic analysis and a primer set was designed and used to amplify the corresponding gene in S. mutans GS-5 used as a model organism. The PCR product was cloned into the vector pCR 2.1 TOPO-TA, and the gene sequenced and analyzed. Alignment of the S. mutans GS-5 and UA159 putative collagenase genes showed 99% homology. The gene was next cloned in frame into the inducible expression vector pET100/D TOPO. Induction and expression of recombinant protein in E. coli were confirmed by SDS-PAGE and Western immunoblotting, while biochemical analysis indicated that it was a calcium- dependent metalloproteinase. Enzyme analysis of the recombinant enzyme showed both gelatinolytic and collagenolytic activity. Further analysis of the GS5 gene using databases such as ExPASy, Pfam, and SMART indicated that it was highly homologous to the U32 peptidase family, which includes the PrtC collagenase of Porphyromonas gingivalis, a bacterium causing periodontitis. The present study was the first to unequivocally demonstrate the existence of a collagenase gene in S. mutans, and to identify it as a member of the U32 peptidase family. The obtaining of the S. mutans collagenase gene should help in further investigation of the role of this enzyme in dental root decay and its potential use as a dental root caries vaccine.

Analysis of the genomic sequences of Streptococcus mutans UA159 and Group B Streptococcus (GBS) strains Streptococcus agalactiae NEM316 and S. agalactiae 2603V/R indicated the presence of two putative collagenase genes in each organism. smcol1 from S. mutans was previously cloned and analyzed and the results indicated that the enzyme belonged to the U32 family of collagenases/peptidases. This enzyme shares homology with the prtC of Porphyromonas gingivalis, one of the principal examples of the U32 family of peptidases. Considering the potential role of these enzymes in the pathogenicity of P. gingivalis (periodontitis or gum disease), GBS (premature rupture of the amniochorionic membrane) and S. mutans (dental root decay), it is necessary to study these enzymes and establish their role in the virulence of these organisms. Toward this goal the present study has focused on cloning collagenase 2 (smcol2) from S. mutans and cloning collagenase 1 (gbscol1), and collagenase 2 (gbscol2), from GBS. The information obtained will contribute to a further understanding of the U32 peptidase family.