A Competence-Induced Bacteriocin Reduces Growth of Oral Pathogenic Bacteria

Objectives: Streptococcus mitis is a commensal bacterium that colonizes all surfaces of the oral cavity. In streptococci, pheromone signaling mediated by competence-stimulating peptides (CSP) is associated with competence for genetic transformation and production of bacteriocins, which may provide an important advantage in the competitive environment of biofilms. Bacteriocins play a major role in this scenario, inhibiting growth of bacterial strains in the surrounding environment. The aim was to characterize S.mitis response to CSP and investigate the inhibition spectrum of bacteriocins whose production is induced by CSP.
Methods:
Transcriptome analysis using RNA sequencing was performed for a thorough assessment of up-regulated genes by CSP. RT-PCR was conducted to establish whether the upregulated genes were early or late CSP-induced. Antimicrobial peptides with purity >80% were purchased from GenScript and diluted 2-fold prior to use. Bacteriocin activity assays were performed in 96-well plates and growth was measured spetrophotometrically at 600nm. Indicator strains were S.oralis, S.sanguinis, S.salivarius, S.mutans and P.gingivalis.
Results: RNA-seq revealed up-regulated sequences accounting for 6.1% of S.mitis genome. We identified two up-regulated genes which code for bacteriocin production (~240-fold). By protein sequence analysis, we classified the peptides as a part of class IIb bacteriocins. Synthetic purified bacteriocins inhibited growth of S.mutans and P.gingivalis significantly (62% and 50%, respectively). Such effect was achieved with concentrations lower than 70nM. There was no inhibitory activity against S.oralis, S.salivarius and S.sanguinis.
Conclusions: This is the first transcriptome analysis of S.mitis regarding the influence of the CSP in gene expression. Among the up-regulated sequences, we identified a two-peptide bacteriocin that may be involved in killing other species. Bacteriocin activity assays indicated that this bacteriocin is species-specific and may be used to target exclusively S.mutans and P.gingivalis. Understanding mechanisms used by S.mitis to successful colonize the oral cavity may lead to novel strategies to control dental biofilm associated diseases.