Als3调节饥饿状态下白色念珠菌和粪肠球菌联合致病机制的研究

Persistent periapical infection is caused by microbial colonization in root canals with environment lack of oxygen and nutrition. As major pathogens, Candida albicans and Enterococcus faecalis are always isolated concurrently. Our pervious study declared that C. albicans and E. faecalis could form mature dual-species biofilms in anaerobic and starvation conditions, with significant differences in biofilm volume, architecture features and drug-resistance compared with single-specie biofilms, which indicted that C. albicans and E. faecalis may have interaction during the pathogenic process of persistent periapical infection. Our microarray assay found that Als3 (one of the cell wall proteins), a related gene of C. albicans, was significantly up-regulated in dual-species biofilms as compared to single-specie biofilms under starvation conditions. Thus we hypothesized that Als3 might play an important role in the interaction of C. albicans and E. faecalis during the pathogenic process. We plan to construct C. albicansΔAls3 mutant, and form C. albicans (ΔAls3 mutant)-E. faecalis dual-species biofims under starvation condition, using CFU-counting, laser scanning confocal microscope and atomic force microscope to detect the effect of Als3 on interaction of C. albicans and E. faecalis, RT-PCR and Western blotting are adopted to analyze the gene and protein expression of virulence factors. The animal model of persistent periapical infection using SD rats will also be established. Then radiographic examination, HE staining and immunohistochemistry are used to study the effect of Als3 on pathogenicity of C. albicans and E. faecalis. Our results may provide a new view to understand the pathogenesis of persistent periapical infection.

微生物定殖于营养匮乏的根管系统可致根尖周持续感染，白色念珠菌（C.a）和粪肠球菌（E.f）检出率最高且常被同时发现。本课题组发现饥饿态的C.a-E.f双菌种与单菌种生物膜生物量、结构特征和耐药性有明显差异，提示两者存在交互作用。基因芯片比较饥饿态双菌种和单菌种生物膜中C.a基因表达，发现胞壁蛋白Als3在双菌种生物膜中表达上调。据此提出假说：Als3参与调节饥饿态C.a和E.f联合致病机制。本课题组拟构建C.aΔAls3突变株，诱导形成饥饿态C.aΔAls3-E.f双菌种生物膜，通过菌落计数、共聚焦和原子力显微镜等方法探讨Als3对两者交互作用的影响，用RT-PCR和Western blotting分析毒力因子表达差异，构建大鼠根尖周持续感染模型并使用X线测量、HE染色和免疫组化分析，以阐明Als3对饥饿态C.a和E.f致病性的影响，为根尖周持续感染发病机制研究提供新思路。

微生物定殖于营养匮乏的根管系统可致根尖周持续感染，白色念珠菌(C.a)和粪肠球菌 (E.f)检出率最高且常被同时发现。本课题组发现饥饿态的C.a-E.f双菌种与单菌种生物膜生物量和结构特征有明显差异，提示两者存在交互作用。基因芯片比较饥饿态双菌种和单菌种生物膜中C.a基因表达，发现胞壁蛋白Als3在双菌种生物膜中表达上调。据此提出假说：Als3参与调节饥饿态C.a和E.f联合致病机制。为此我们首先尝试构建白色念珠菌Als3突变株，在反复多次实验无法突破技术难题后转而构建Als3多克隆抗体，并与后期获得国外专家赠与的白色念珠菌DAY185野生株、 Als3基因缺失突变株、补偿株一起构建了双菌种生物膜，进行相关检测发现：Als3参与饥饿态白色念珠菌—粪肠球菌双菌种生物膜中两者的粘附过程，敲除或多克隆抗体拮抗Als3的作用后，饥饿期双菌种生物膜中两者的生物量均明显降低(n=14，p< 0.05)。构建突变株方式较多克隆抗体对白色念珠菌Als3的拮抗作用更为显著。同期我们进行了相关其他实验，发现白色念珠菌诱导进入饥饿状态过程中，14.45%~29.13%的基因表达发生改变(p< 0.05)，涉及氨基酸代谢、糖代谢、脂类代谢、核酸代谢和细胞周期等多个通路。提示白色念珠菌在诱导进入饥饿状态过程中涉及代谢和增殖水平的基因表达下降，对外界刺激和药物耐受相关基因表达上调，此改变有助于保持白色念珠菌在饥饿状态的存活力和致病性。饥饿态白色念珠菌-粪肠球菌双菌种生物膜的药物敏感性实验证实双菌种生物膜对不同浓度次氯酸钠的抵抗力均强于单菌种生物膜(n=10，p< 0.05)，采用5.25%的次氯酸钠作用30 min可完全杀灭饥饿态双菌种生物膜。同期进行关于变形链球菌的实验发现，茶多酚提取物EGCG可增强NaF对浮游态变形链球菌的抑制作用，降低致龋相关毒力因子ldh,、aguD和atpD的表达，同时ECGC还可协同作用于NaF对变形链球菌生物膜形成的抑制，降低多糖合成相关基因gtfB、 gtfC和 gtfD的表达水平。此外，确定了LmrB外排转运体的存在，并提示其可参与调节变形链球菌的应激反应和胞外多糖合成。