粪肠球菌大量增殖并形成生物膜是根管治疗失败的主要原因之一。申请者前期研究从难治性根尖周炎患牙根管中获得4个生物膜形成能力显著增高的粪肠球菌临床分离菌株。进一步的基因表达谱测序研究发现，氮代谢通路的表达与对照组相比有显著性差异，其中编码谷氨酸合成酶的gltA基因表达显著上调，它是谷氨酸代谢途径中的重要分子。提示gltA基因及谷氨酸代谢途径在粪肠球菌生物膜形成中可能发挥重要的调控作用。本项目拟在此基础上，深入研究gltA基因在粪肠球菌生物膜形成过程中的表达状态，明确其表达变化对粪肠球菌生物膜形成的影响，进而筛选其相互作用的上下游分子，揭示gltA基因及谷氨酸代谢途径在调控粪肠球菌生物膜中的作用和机制，阐明其调控细菌生物膜形成的功能意义。这些研究可以提高我们对细菌生物膜调控的认识，为研发新型抗细菌生物膜药物和抑制剂提供可能的靶位和靶通路；还可以发现粪肠球菌生物膜调控的新机制，具有重要的理论意义。

Enterococcus faecalis (E. faecalis) is a prominent pathogen causing root canal treatment failure. The inherent ability of E. faecalis to survive in endodontically treated teeth and forming biofilm has made it eradication challenging. In the previous studies, we got four clinical isolates that showing the ability to forming much more biofilm than the Enterococcus faecalis ATCC V583. Digital Gene Expression Profiling (DGE) indicating that the nitrogen metabolism pathway has significant difference between the clinical isolates and V583 strain. The expression of gltA gene encoding glutamate synthase was up regulated significantly. Glutamate synthase is the key molecule in glutamate metabolism. The gltA gene and glutamate metabolism pathway may play a role in the biofilm formation of Enterococcus faecalis. But the molecular mechanisms of this process are still unknown. This study will investigate the expression profile of gltA gene during biofilm formation of Enterococcus faecalis by Realtime PCR and Western blot, in mRNA level and protein level respectively. Then the effects of up or down regulation of gltA gene expression on the biofilm formation of Enterococcus faecalis by Knock in or Knock out methods will be assessed. The genome-scale molecular changes will be screened out using DGE sequencing. HPLC/MS will be used to examine the glutamate metabolites changes between the gltA Knock in or Knock out strains. Finally, the transcript factor regulating gltA expression will be analyzed by bioinformatics analysis and be confirmed with EMSA and ChIP technology. By these experiments, the relationship between gltA gene expression and Enterococcus faecalis biofilm formation will be confirmed, and the possible molecular mechanism of gltA gene and glutamate metabolism pathway on biofilm formation will be elucidated. The findings in this research will provide us new targets or methods to develop novel anti-biofilm bactericidal and inhibitory agents.