Proteome Analysis Identifies the Dpr Protein of Streptococcus mutans as an Important Factor in the Presence of Early Streptococcal Colonizers of Tooth Surfaces

Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE). After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci.口腔链球菌是牙齿表面的初代定植菌，而变形链球菌（Streptococcus mutans）是人类龋齿的主要致病菌。变形链球菌的单菌种生物被膜形成过程涉及多种蛋白质。本研究采用二维凝胶电泳（two-dimensional gel electrophoresis，2-DE）技术，分析了在存在或不存在牙齿表面初代定植菌戈登链球菌（S. gordonii）的条件下，变形链球菌生物被膜的蛋白质表达谱。本研究通过质谱分析鉴定变形链球菌蛋白质后，进一步分析了其在戈登链球菌存在条件下的表达情况。实验设置两组：分别为仅接种变形链球菌，以及同时接种变形链球菌与戈登链球菌DL1（S. gordonii DL1），两组菌种均通过0.2微升的Anopore膜进行分隔培养。收集聚苯乙烯培养板上的生物被膜，将溶解后的蛋白质通过2-DE进行分离。当变形链球菌在戈登链球菌存在的条件下形成生物被膜时，其过氧化物抗性蛋白Dpr的表达量相较于无该初代定植菌时形成的生物被膜提升了4.3倍。此外，本研究利用变形链球菌抗氧化蛋白突变株，联合戈登链球菌及其他初始定植菌开展了竞争实验。dpr基因敲除的变形链球菌突变株的生长可被戈登链球菌及血链球菌（S. sanguinis）显著抑制。此外，细胞活力实验结果显示，与野生型菌株相比，dpr缺陷型突变株在与戈登链球菌共培养时的细胞活力显著降低。综上，上述结果表明，在存在早期定植口腔链球菌的环境中，Dpr可能是变形链球菌在牙齿表面存活所需的关键蛋白质之一。