Chromosomal DNA from Streptococcus mutans 6715 (serotype g) was cloned into Escherichia coli K-12 by using the cosmid pJC74 cloning vector and a bacteriophage X in vitro packaging system. Rabbit antiserum against S. mutans extracellular proteins was used for immunological screening of the clone bank. Twenty-one clones produced weak to strong precipitin bands around the colonies, but only after the X c1857 prophage was induced by being heated to lyse the E. coli cells. None of the clones expressed enzyme activity for several known S. mutans extracellular enzymes. One of these clones contained a 45-kilobase recombinant plasmid designated pYA721. An 8.5-kilobase fragment of S. mutans DNA from pYA721 was isolated and recloned into the BamHI restriction site of the plasmid vector pACYC184 to construct pYA726. pYA726 contained all, or nearly all, of the gene for a surface protein antigen (the spaA protein) of S. mutans 6715. This was deduced from immunological studies in which extracts of cells harboring pYA726 reacted with antisera against both purified 6715 spaA protein (about 210,000 daltons) and the immunologically similar antigen I/II of serotype c strains of S. mutans. In addition, the S. mutans spaA protein was found to possess at least one antigenic determinant not present on the protein specified by pYA726. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of E. coli clone extracts revealed that pYA726 produced a polypeptide with a molecular mass of about 180,000 daltons which was predominantly found in the periplasmic space of E. coli cells. Antisera to the spaA protein of S. mutans reacted with extracellular protein from representative strains of S. mutans serotypes a, c, d, e, f, and g, but not b.
Streptococcus mutans is the etiologic agent of dental caries and is a causative agent of infective endocarditis. While the mechanisms by which S. mutans cells colonize heart tissue is not clear, it is thought that bacterial binding to extracellular matrix and blood components is crucial in the development of endocarditis. Previously, we have demonstrated that S. mutans cells have the capacity to bind and activate plasminogen to plasmin. Here we report the first cloning and characterization of an alpha-enolase of S. mutans that binds plasminogen. The functional identity of the purified recombinant alpha-enolase protein was confirmed by its ability to catalyze the conversion of 2-phosphoglycerate to phosphoenolpyruvate. The protein exhibited a Km of 9.5 mM and a Vmax of 31.0 mM/min/mg. The alpha-enolase protein was localized in the cytoplasmic, cell wall and extracellular fractions of S. mutans. Binding studies using an immunoblot analysis revealed that human plasminogen binds to the enolase enzyme of S. mutans. These findings identify S. mutans alpha-enolase as a binding molecule used by this oral pathogen to interact with the blood component, plasminogen. Further studies of this interaction may be critical to understand the pathogenesis of endocarditis caused by S. mutans.
A sequence of 1,647 base pairs in length of S. mutans DNA that encodes for a 63 kDa protein with significant amino acid similarity with fibronectin-binding proteins of S. pyogenes and S. gordonii was cloned. The putative recombinant fibronectin-binding protein of S. mutans was purified using affinity chromatography and the cloned protein was used to prepare polyclonal antibodies against the recombinant protein. In immunoblot assays, antibodies against the S. pyogenes fibronectinbinding protein, FBP54, were cross-reactive with the S. mutans protein that was designated SmFnB. Additionally, antibodies to the S. mutans SmFnB protein reacted with the S. pyogenes FBP54 protein.The S. mutans SmFnB protein was found to bind to immobilized fibronectin in a concentration dependant manner. A mutant strain of S. mutans M51 that was constructed by alleleic exchange did not express the SmFnB protein. This mutant strain, S. mutans ΔSmFnB, was determined in an ELISA to bind to immobilized fibronectin 30% less when compared to the parental strain S. mutans M51. The results are consistent with the conclusion that the 63 kDa SmFnB protein of S. mutans is a fibronectin-binding protein that may contribute to the interaction of S. mutans with damaged heart tissue during pathogenesis of infective endocarditis. Also, the study suggests that multiple molecules may mediate the interaction of S. mutans with fibronectin.
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