83 - Verification of Collagenase Production by Streptococcus Mutans

Abstract

The oral environment contains multiple collagenases which are believed to play a role in dentin caries formation, as collagen is one of the major components of dentin. In the laboratory, biofilm and collagenase-containing chemical models have been used to study dental degradation. However, the numerous collagenase sources in the oral cavity pose a challenge when it comes to comparing in vitro and in vivo data. Streptococcus mutans, one of the important cariogenic oral streptococcu, contains genes for at least two collagenases (SMU_759 and SMU_761), which have been shown to be expressed in samples collected from active caries. Here, we investigate if the collagenases produced by S. mutans contribute to dentin degradation. S. mutans UA159 (ATCC700610) was cultured in BHI with/without KHCO3 using different concentrations of sucrose (0%, 0.05%, 0.2%, and 1%) at 37 C and 5% CO2 for 48 hrs. The pH of the media at 48 hrs was measured by using pH test paper strips. RNA from S. mutans biofilms cultured without KHCO3 was collected to measure collagenase gene expression by RT qPCR. To measure collagenase activity, KHCO3-buffered BHI was used for culturing to avoid collagenase denaturing caused by low pH. Supernatants of the same culture were centrifuged, filter sterilized and dialyzed, and exposed to a commercially available colorimetric assay kit. Absorbance readings were collected every 20 min for 16 hrs. With KHCO3 buffering, the pH of the media containing 48-hrs S. mutans cultured with 0%, 0.05%, 0.2%, and 1% sucrose were around 7.6, 6.5, 6.0, and 5.5, respectively. The corresponding pH values of media without KHCO3 were around 7.0, 5.5, 5.0, and 4.5. Addition of 1% W/V sucrose in BHI led to an approximately 20-fold increase in the expression of both SMU_759 and 761 collagenases compared to BHI only. Collagenase expression increased by less than 3-fold when 0.05% and 0.2% W/V sucrose was added. Although quite slow, a continuous reduction in absorbance was observed in the 16-hr collagenase activity test, indicating collagenase activity of the media containing S. mutans. The collagenase activities of the samples with 0%, 0.05%, 0.2%, and 1% sucrose were: 0.00106, 0.000679, 0.000679, and 0.000931 U/ml, respectively. Gene expression and collagenase activity test showed that S. mutans produced active collagenase(s) in response to sucrose during the 48 hrs of culturing, which supports the idea that S. mutans contributes to dentin degradation.