单细胞尺度的牙菌斑尿素代谢异质性机制研究

Caries, a polymicrobial infection, is mediated not by a single pathogen,but by a microbial community. Thus understanding the "functional heterogeneity" of individual members in the caries microbiota will be one key to caries etiology. However dissecting the "functional heterogeneity" in plaque microbiota has been difficult as most bacteria in plaque remain uncultured. A single cell is the atomic unit of the structure and function of a microbial community. Therefore interrogation of the activity of both microbial community and its individual members, at the resolution of "a single live cell", will be able to circumvent the need for cell culture/propagation and be capable of unraveling the "deepest" functional heterogeneity in the microbiota. I will test the hypothesis that in plaque microbiota there is functional heterogeneity in ammonia-production from urea, which is important as ammonia-production counters the activity of acid-producing bacteria and thus might be an anti-caries factors. Employing non-invasive phenotyping techniques such as Single-cell Raman Spectroscopy and Raman-activated sorting, as well as single-cell and community genomics/transcriptomics approaches, I will monitor and compare the metabolic activity (as well as the underlying genotypes) of both the plaque microbiota and individual members at single-cell resolution. These efforts will reveal how the heterogeneity of urea catabolism is manifested at the organismal and functional-gene levels，as well as how the heterogeneity changes during the simulated process of caries development. This study will provide the first single-cell model of functional heterogeneity in plaque metabolism and might provide novel approaches for investigating the etiology of caries and other polymicrobial infectious disease.

龋病是多细菌参与的慢性感染性疾病，因此认识其病因关键是理解菌群中个体间的功能差异，即"功能异质性"。而牙菌斑内多数细菌难以或尚未被培养，妨碍其异质性解析。单个活体细胞是菌群结构和功能的单位，因此在"单个活体细胞"尺度观察和比较个体功能，可望"跳过"细菌扩增培养这一环节，在最"深"的层面上揭示细胞群体内的功能异质性。针对"牙菌斑中部分成员能够代谢尿素产氨，从而拮抗菌群产酸活性而具抗龋效应"这一假设，申请人将基于原位构建的牙菌斑，运用活体单细胞拉曼光谱分析与分选等非侵害性、原位的新型细胞表征与操作技术，考察菌群中单个活体细胞之间代谢尿素的表型差异与基因型差异；并结合元基因组手段，探讨该异质性在物种、基因等角度的呈现规律，及其在模拟龋病发生过程中的动态变化机制。本研究将建立首个活体单细胞尺度的牙菌斑代谢异质性研究模型，从而为龋病病因学研究提供新思路和新方法。

本研究以儿童龋病患者和健康人儿童的唾液和牙菌斑作为口腔菌群样本来源，围绕细菌图谱在菌种多样性以及物种分布特征，重点考察不同人群之间细菌谱系构成差异，在排除时间因素诱导菌群变化的基础上考察龋病新发病例与口腔菌群结构变化之间的联系，揭示与龋活跃性相关的特异性菌种的存在，探讨应用单细胞技术对口腔菌群的代谢潜力进行筛选，针对不同代谢底物可以检测出代谢特定底物的细菌细胞，从而探索不基于牙医主观判断，基于口腔菌群检测而诊断甚至预测儿童龋病的新方法和技术路线。在儿童龋病相关的口腔微生物组病因学研究方面，我们与通过纵向研究追踪设计，监测儿童早期龋病发病情况与口腔菌群的关系，重点考察不同人群之间细菌谱系构成差异，在排除时间因素诱导菌群变化的基础上考察龋病新发病例与口腔菌群结构变化之间的联系，揭示与龋活跃性相关的特异性菌种的存在，阐明口腔菌群中各种微生物在龋病发生发展过程中的不同作用，从而探索不基于牙医主观判断，基于口腔菌群检测而诊断甚至预测儿童龋病的新方法和技术路线。本课题研究发现儿童龋病是口腔菌群的发育收到抑制，同时可以通过监测口腔菌群预测龋病的新发，为儿童龋病的研究提供新的思路(Teng, et al, Cell Host Microbiome, 2015;Xu Y, et al, Arch Oral Biol. 2014; Yang F, et al, J Int Soc Prev Community Dent. 2015)。在应用单细胞技术完成对口腔菌群的筛选过程中，课题组已经建立了活体单细胞的不同代谢水平表型测定技术，针对不同代谢底物可以检测出代谢特定底物的细菌细胞，在不需生物标识物、不需进行外加标记的情况下，定量表征与测定单个活体微生物细胞对尿素底物摄取量，进而证明能够利用单细胞拉曼光谱区分和推测口腔菌群中不同微生物细胞对尿素底物代谢的速度，从而判别哪些细菌细胞在抗龋过程中起到关键作用；同时，针对单菌在不同酸性条件下耐酸性的差异，我们设计了采用单细胞检测不同预酸化水平单菌的实验，可以不通过培养方法，借助拉曼的快速检测技术筛选特定预酸化水平的细菌细胞，为探寻具有产酸耐酸特性的致龋菌寻找提供前期实验基础(Yang F, et al, PLoS One. 2014; Yang F, et al, Springerplus, 2016)。