白色念珠菌与格氏链球菌共生平衡对机会性致儿童龋病风险的调控作用及机制研究

The consistently high incidence of childhood caries harms the overall health and development of children in China. The opportunistic cariogenic pathogen Candida albicans (Ca) can be detected from the oral cavity of most children, especially those with caries. Ca can specifically adhere to salivary proteins, co-aggregate with Streptococcus gordonii (Sg) and co-colonize in oral cavity. However, the research on association of this interaction with caries is few. We have first demonstrated the advantageous growth of Sg in saliva benefits from the serial hydrolysis of the glycosidic side chains of the proline-rich proteins. In addition, according to our preliminary experiments, Sg inhibited the planktonic growth and biofilm formation of cocultured Ca under saliva-mimic condition. Therefore, we hypothesize that, Sg forms a symbiotic relationship with Ca under conditions with low cariogenic risks, the homeostasis of which can modulate the cariogenic capability of Ca to a certain degree. To the contrary, under high cariogenic risks, Ca may gain overgrowth and enhance its virulent expressions while Sg loses the symbiotic modulation. When the symbiotic homeostasis is failed to maintain, the caries incidence will rise. We plan to study the phenotypes and underlying molecular mechanisms in the symbiotic homeostasis between Ca and Sg. The growth, biofilm formation, metabolism, virulence and involved molecular signaling pathways will be studied through clinical cross-sectional study, in vitro coculture model and childhood caries animal model, aiming to get symbiotic indices and construct the caries risks prediction model. Based on multi-factors including oral microbes, host and microenvironment, this study may offer new ideas and targets for future childhood caries early screening and prevention, also pave the way for improving current prevention and treatment strategies for childhood caries.

我国儿童龋病发病率高，严重危害儿童生长发育及身心健康。部分儿童口腔可检出具有机会致龋性的白色念珠菌（Ca）,而患龋儿童检出率更高。Ca与格氏链球菌（Sg）特异粘附共同定植，是否与致龋风险相关还未有研究。本项目负责人首次阐明Sg可在唾液中水解富脯蛋白糖侧链优势生长，发现模拟唾液共培养时Sg抑制Ca生长及生物膜形成。因此提出假设，低龋风险时Sg与Ca共生平衡，调控Ca致龋性；当龋风险升高Ca过度生长毒力增强，Sg失去调控作用，共生关系失衡, 龋病易发生。拟通过临床横断面研究、体外共培养模型及低龄龋动物模型结合Ca突变株库，从生长、生物膜形成、代谢、毒力、信号通路等角度系统研究及验证Ca-Sg共生平衡表型及分子机制，总结共生平衡龋风险相关系数，建立预测模型。本课题基于口腔微生物-宿主-微环境综合因素，为评估预警儿童龋风险提供新思路和新靶点，为完善儿童龋病防治策略提供研究基础和理论依据。

龋病是我国儿童最常患的慢性疾病，严重影响其生长发育，危害身心健康。患龋儿童口腔可检出较高水平的白色念珠菌，其可与牙菌斑生物膜早期定植菌戈登链球菌特异黏附，在口腔微生境中共同定植。前期研究发现，缺乏游离糖低龋风险的唾液环境中糖苷水解酶对S.gordonii生长有重要作用，糖苷水解酶是否参与类唾液环境C.albicans-S.gordonii交互作用、相关唾液微环境标志物是否可反映儿童龋风险尚未见研究报道。本课题通过临床横断面研究，采用16sDNA及ITS2测序进行儿童唾液微生物多样性分析，采用非标记LC-MS进行唾液蛋白谱分析，并检测常见唾液生物标记物差异。通过体外模型对C.albicans-S.gordonii在类唾液及高糖环境下不同交互模式及潜在分子机制进行探究，测定单独及共培养生长、生物膜、黏附共聚表型及GHs活性，探究交互作用模式，筛选互作机制相关基因，最后采用机器学习算法建立儿童龋风险预测模型并验证。研究发现，1）HIB共培S.gordonii生长及生物膜较单培显著增加，而C.albicans生长受抑；高糖HIBS共培两菌各自生物膜较单培无差异。HIB中S.gordonii具多种GHs活性，而C.albicans未检出；共培养GHs较S.gordonii单培显著上升。HIB共培中，C.albicans可上调S.gordonii GH2、GH20及GH85表达水平，其无细胞上清液具类似效应。C.albicans突变株ash1Δ/Δ、fcr3Δ/Δ、tec1Δ/Δ、upc2Δ/Δ促进S.gordonii生长及上调GHs活性能力较WT显著减弱，可能参与二者相互作用。2）结合多组学生物信息学筛选及算法建模分析，本课题建立儿童龋风险预测模型，AUC达0.9031。综上本课题发现，低糖环境下C.albicans上调S.gordonii GHs活性并促生长；C.albicans黏附/菌丝形成相关基因ASH1，FCR3，TEC1，UPC2可能参与调控其与S.gordonii共生关系，该共生平衡关系可能是C.albicans在低龋环境中的生存机制之一，可为儿童龋病风险机制研究提供依据。本研究建立的唾液标记物儿童龋风险预测模型具有一定临床应用前景。