Proteomic identification of putative enzymes for dental plaque deconstruction

Conference poster, 2024

Oral diseases are estimated to affect 3.5 billion people in the world and almost all people have dental caries at some point in their life. The cause of many oral diseases, including caries, gingivitis and periodontitis, is untreated dental plaque. Dental plaque is a biofilm infection and is, like most biofilms, difficult and costly to treat because of the resilient nature of the biofilm. Arguably, the most important characteristic of dental plaque is the extracellular polysaccharide matrix, which makes up around 10-20 % of its dry weight and builds up the protective structure where bacteria eventually create an acidic environment causing demineralization of the teeth. The majority of the polysaccharides constituting the biofilm are insoluble glucans, mutan (α-1,3-glucan) and alternan (α-1,3/α-1,6-glucan) as well as soluble dextran (α-1,6-glucan), which are all synthesized from sucrose by glucosyltransferases secreted by oral bacteria such as Streptococcus mutans. While dextran is believed to mainly act as an energy storage molecule, both mutan and alternan have been shown to be crucial for the biofilm’s attachment to the teeth and to the bacteria in the plaque. Although enzymatic degradation of mutan has been shown to disrupt the biofilm, mutanases remain understudied and unused and alternanases have not been studied at all for this purpose. In this study, we have used a bioprospecting approach to find new enzymes for biofilm control and have successfully isolated six Streptomyces strains from soil with the ability to utilize either mutan or alternan as the sole carbon source. Genome sequencing and phylogenetic analysis identified one strain as S. laculatispora, three as of S. poriferorum and two as new species for which we propose the names Streptomyces castrisilvae and Streptomyces glycanovorans. Comprehensive genomic and biochemical characterizations were conducted, highlighting typical features of Streptomyces, such as large genomes (8-9.6 Mb) with high GC-content (70.5-72%). All six strains also encode a wide repertoire of putative carbohydrate-active enzymes, indicating a capability to utilize various complex polysaccharides as carbon sources such as starch, mutan, and cellulose, which was confirmed experimentally. Interestingly, the strains isolated on mutan could not utilize alternan while the strains isolated on alternan could degrade both alternan and, although to a lesser degree, mutan. To explore the differences in degrative ability, we are currently pursuing analyses of the secretomes of three of the strains during growth on these polysaccharides.