靶向谷氨酸消旋酶对变异链球菌HSP100/ClpATPase的调控作用及机制研究

Streptococcus mutans (S. mutans) is the primary causative agent of dental caries. Its virulence is tightly integrated with stress tolerance. Coping with environmental insults and fluctuations, HSP100/ClpATPase plays a vital role in cell envelope biogenesis/homeostasis and affects competence development by S. mutans, traits critical to cariogenicity. Control the pathways regulating competence development or (and) destabilize cell wall integrity may reduce cariogenic potentials of this bacterium. However, the accurate targets have not yet been characterized. Our previous study showed that inactivation of glutamate racemase (MurI), an essential enzyme in cell wall biosynthesis, had a negative effect on virulence properties and stress response in S. mutans. Further studies revealed that specifically targeting MurI regulated the expression of HSP100/ClpATPase at different levels. To in-depth investigate the exact underlying mechanism, this project will conduct a high throughput screening for small-molecule inhibitors based on the protein crystal structure of MurI from S. mutans. The effect of inhibitors on the biological phenotypes associated with cariogenicity will be examined. Based on the above, the regulatory mechanisms of HSP100/ClpATPase in S. mutans during exposure to the inhibitors, as well as the expression of critical genes and proteins of ComRS competence development pathways, will be further elucidated. This study will provide evidence for establishing MurI and HSP100/ClpATPase as useful targets for therapeutics to diminish the virulence of S. mutans.

变异链球菌的致龋毒力与对环境刺激的耐受力密切相关，应激反应过程中， HSP100/ClpATPase通过双向调控作用调节细胞壁完整性和感受态形成，促进毒力因子进化。影响细胞壁稳定性和抑制感受态形成可降低变异链球菌致龋毒力，但目前尚未发现明确和有效的作用靶点。本课题组前期研究发现缺失细胞壁合成关键酶谷氨酸消旋酶（MurI）负向调控变异链球菌致龋毒力和胁迫反应；对HSP100/ClpATPase家族成员ClpX和ClpC可能存在表达、功能与分子水平多层次的调节作用，但具体机制尚不明确。本项目拟构建以MurI蛋白为靶标的小分子抑制剂高通量筛选模型，探讨MurI小分子抑制剂对ClpX和ClpC的调控分子机制，明确其影响细菌壁膜结构和生物学行为的作用模式，分析ComRS感受态形成信号通路关键基因及功能蛋白的表达改变，为确立以MurI和HSP100/ClpATPase为龋病防治新靶点提供科学依据。

细胞壁是细菌所特有的结构，对维持细菌固有形态、抵抗外界压力和表达细菌毒力因子等生命活动至关重要，因此在寻找有效的抗菌药物时细胞壁是理想的药物作用靶点。肽聚糖是变异链球菌细胞壁的核心结构，谷氨酸消旋酶（Glutamate racemase, MurI）（EC5.1.1.3）为肽聚糖骨架成分右旋谷氨酸（D-glutamate）合成的关键酶。由于在人体中不存在此种代谢途径，谷氨酸消旋酶可能成为研制预防龋病药物的作用靶点之一。本课题建立变异链球菌谷氨酸消旋酶原核蛋白表达系统，通过圆二色谱法检测酶活性，建立筛选谷氨酸消旋酶抑制剂的分子模型。成功构建表达变异链球菌谷氨酸消旋酶蛋白的工程菌，获得可溶性S. mutans MurI蛋白；建立了快速、准确测定MurI酶活性、酶促反应动力学参数和筛选MurI酶抑制剂的分子模型。在此基础上，通过体外抗菌实验， 研究L-丝氨酸-O-硫酸酯特异性靶向抗变异链球菌的作用及机制。抑制剂L-丝氨酸-O-硫酸酯对谷氨酸消旋酶的抑制作用不呈剂量依赖关系。L-丝氨酸-O-硫酸酯对变异链球菌致龋毒力因子表达的影响主要体现在其（1）作用于细菌形态学改变上；（2）影响生物膜的方式上，后者不仅仅限于生物膜生物量的变化，还涉及生物膜表面和内部结构的改建；（3）同时在基因转录水平对HSP100/ClpATPase和ComRS信号通路关键基因clpC, clpP, comD, comX, comYA的表达调控存在差异。此外，本课题采用细胞模型和动物实验，初步探讨了L-丝氨酸-O-硫酸酯的细胞毒性及生物安全性。通过抗变异链球菌生物膜形成实验以及细胞毒性证实，L-丝氨酸-O-硫酸酯具有有效抑制变异链球菌毒力因子的特性和良好的生物相容性。研究结果为进一步阐明以谷氨酸消旋酶为靶标药物防龋的物质基础及药理机制提供了理论依据和实验基础；并为下一步寻找谷氨酸消旋酶抑制剂预防龋病的先导化合物奠定基础。.