具核梭杆菌与口腔链球菌之间相互作用的分子机制研究

The ability of Fusobacterium nucleatum, a “bridge-organism”, to adhere to early commensal colonizers oral streptococci determines F. nucleatum perpetuating in the oral microbial flora, contributes greatly to the development of polymicrobial communities and impacts the occurrence and progress of periodontitis. The specific adhesin-receptor pair responsible for the interspecies coaggregation between oral streptococci and F. nucleatum remains to be investigated. Therefore, the elucidation of the specific molecular interactions will provide a new role of target for interferring adherence of F. nucleatum to oral streptococci. Interspecies physical attachment initiates signal trasduction cascades that trigger important physiological changes in partner species, which could not be observed by monospecies experiments. In this study, coaggregation-induced changes in the transcriptional profiles of oral streptococci and F. nucleatum are investigated using RNA-Seq. The physilogical and pathobilogical function of differentially regulated genes involved in adhesion, coopreation in substrate utilization, environmental adaptaion and virulence features are investigated to reveal the transcriptional responses triggered by physical association of “bridge-organism” with early colonizer. From contact-inducible genes, the virulent genes are screened through analysis of gingival epithelial cell attachment and invasion as well as colonization and pathogenesis in experimental animal. Different from previous virulent gene screening method from monospecies, the virulent genes identified in this study suggest their coordinating pathogenic properties within multispecies biofilm. The results in this study will shed light on understanding of the complexity of oral polymicrobial communities and may provide novel approaches for controlling microbial community-based pathogenesis.

具核梭杆菌作为“桥梁”菌，与先驱菌-口腔链球菌之间的共聚集，是其定居于口腔菌群的先决条件，在牙菌斑生物膜形成与牙周病发生、发展中扮演重要角色。探讨口腔链球菌与具核梭杆菌共聚集的分子基础，将为干扰两者之间的相互作用提供靶位。细菌之间共聚集将起始一系列的转录调控，赋予菌群新的生理功能，这是单菌种研究观察不到的。本课题通过对口腔链球菌与具核梭杆菌共聚集前、后的转录组进行分析，筛选共聚集调控转录的基因，同时从细菌相互作用的角度研究这些基因在粘附、营养物质利用、环境适应及协同致病性的功能，将深化人们对先驱菌-“桥梁”菌共聚集调控规律的认识，为阐明相互作用的分子机制提供理论基础。较以往从单一细菌基因组中筛选致病基因不同，本研究从共聚集调控转录的基因中，通过检测其对口腔上皮细胞的侵入及实验动物的致病性，筛选致病基因，能够更真实地诠释多菌种生物膜中致病基因的作用，同时为牙周病的预防和治疗提供新的靶位。

具核梭杆菌作为“桥梁”菌，与先驱菌-口腔链球菌之间的共聚集，是其定居于口腔菌群的先决条件，在牙菌斑生物膜形成与牙周病发生、发展中扮演重要角色。细菌之间共聚集将起始一系列的转录调控，导致菌群生理功能和代谢状态的改变。本课题首先通过体外实验探究具核梭杆菌与口腔链球菌共聚集对其菌群定植能力和协同致病性的影响，证实具核梭杆菌与口腔链球菌共聚集通过抑制牙菌斑菌群分泌H2O2增强具核梭杆菌活性，且有利于逃避人巨噬细胞的杀灭机制，促进人牙龈上皮细胞分泌促炎细胞因子TNF-α和IL-6，抑制抑炎细胞因子TGF-β1分泌，激活NF-κB及MAPK信号通路。其次通过对口腔链球菌与具核梭杆菌共聚集前、后的转录组及代谢产物进行分析，发现共聚集作用导致变异链球菌精氨酸合成相关基因表达上调，氧化应激相关基因表达下调；格氏链球菌和具核梭杆菌多种代谢途径相关基因差异表达，代谢组学揭示两者短链脂肪酸（SCFAs）丙酸、丁酸的分泌浓度降低。同时临床样本显示牙周炎背景下牙周炎患者和牙周炎合并糖尿病患者龈沟液中SCFAs乙酸、丙酸、丁酸含量显著高于健康者，通过对SCFAs的生理功能分析，证实了SCFAs抑制牙周膜细胞和巨噬细胞的增殖和活性，并且诱导细胞分泌促炎细胞因子。本研究从细菌相互作用的角度研究共聚集调控转录的差异基因在环境适应及协同致病性的功能，同时研究差异代谢产物在牙周炎背景下的生理功能，诠释了多菌种生物膜中致病基因的作用，为牙周病的预防和治疗提供新的靶位。