Identification and Analysis of Collagenolytic Activity in Streptococcus mutans

Objectives: To measure collagenolytic activity of S. mutans towards demineralized human dentin and identify the expressed putative collagenases and gelatinases in different bacterial fractions.
Methods: Dentin slabs (3mmx3mm) were prepared from human molars (University of Toronto Human Ethics Protocol #25793), demineralized in 10% phosphoric acid for 24hrs, then incubated (n=3/group) with 200ml of Fujawara medium alone or with one of the following: overnight (O/N) culture of S. mutans UA159; 1:100 freshly inoculated culture of S. mutans UA159; intracellular component of O/N culture; supernatant from O/N culture or bacterial membrane solution (37C, 24hrs). Media from each condition was collected for hydroxyproline assay (OH-pro) to verify collagen degradation. Eight-hour S. mutans intracellular components and supernatant were separated, concentrated, collected and analyzed for proteomics analysis by Mass spectrometry (LTQ-Orbitrap, ThermoFisher, San Jose, CA). Proteome software Scaffold (Portland, OR) was used to validate MS/MS-based peptide and protein identities. The identified collagenases were further analyzed by Protein Homology/analogy Recognition Engine V2.0 (Phyre2) (Structural Bioinformatics Group, London, UK, http://www.sbg.bio.ic.ac.uk/phyre2).
Results: The medium and bacterial membrane had no activity towards dentin collagen. The O/N culture of S. mutans had the highest degradative activity towards dentin collagen producing 17848.6±904.4pmol/100ml OH-Pro, followed by supernatant (12983.7±521.1pmol) and intracellular component (8283.9±1115.8pmol/100ml)(p<0.05). The least OH-Pro (2913.7±534.0pmol/100ml) was released from dentin slabs incubated with freshly inoculated cultures of S. mutans (p<0.05). Two putative collagenases, SMU.759 and SMU.761, were detected from S. mutans UA 159. Based on homology detection and 3D model analysis, these two collagenases belong to the U32 collagenase family.
Conclusions: S. mutans can demineralize dentin through acid production. The current study demonstrates that demineralized dentin is hydrolyzed by cariogenic bacterium. This hydrolysis of collagen potentially contributes to the progression of primary caries, to the degradation of the restoration-tooth interface, and to restoration failure.