龋病抗毒力生态防治的应用基础研究

Anti-virulence therapy is a novel and alternative approach to the control of microbial infectious diseases, especially for those chronic multi-species bacterial infections where a specific pathogen is not clearly identified but virulence factors involved are well elucidated. The "ecological plaque hypothesis" on the etiology of dental caries indicates that anti-virulence therapy may represent a promising approach to the management of this worldwide costly infectious disease. The current study is to construct a genetic recombinant Streptococcus mutans strain and to investigate its anti-virulence potential in the management of dental caries. In detail, the lactate dehydrogenase gene (ldh) of a potent BLIS (bacteriocin-like inhibitory substances)-producing S. mutans strain is to be inactivated by inserting into it an alkali-producing gene (ure) from Streptococcus salivarius as well as an alcohol dehydrogenase gene (adhB) from Zymomonas mobilis. The resulting strain is thought to possess enhanced colonizing ability and lessened cariogenicity. Importantly, the alkali-producing ability of this recombinant strain will impose significant positive effect on microbial ecology by countering the acidity within the dental biofilm. Therefore, the in vitro physiological properties of the recombinant strain will be investigated regarding its acidogenicity, aciduricity and hard tissue dissociation ability. A multi-species biofilm model will also be established and fluorescent in situ hybridization (FISH) as well as other bio-molecular techniques will be employed to investigate the effect of the recombinant strain on the oral microbial ecology. An in vivo rat model to further investigate the effect of the recombinant strain on the in situ oral microbial ecology as well as the incidence and severity of dental caries will also be established. The anticipated result will be that the recombinant strain not only possesses lessened cariogenicity, but also imposes significant effect on the commensal bacteria in the oral cavity, and thus reverses the imbalanced oral microbial ecology and provides a new paradigm in the anti-virulence treatment of dental caries.

"抗毒力"生态防治是针对多菌种生物膜微生态失衡所致感染性疾病的新兴控制理论与方法。龋病作为一种经典的多菌种微生态失衡所致慢性感染性疾病，传统针对特异性病原菌的抗菌和替代治疗效果并不理想。本研究拟以"抗毒力"生态防龋理论为依据，在强效产"细菌素样抑菌物质"的变异链球菌株基因组中插入外源性乙醇脱氢酶基因（adhB）和尿素酶基因（ure），失活其乳酸脱氢酶基因（ldh），构建新型产碱"抗毒力"效应菌株用于龋病生态防治。通过体外研究模型评价效应菌株生物表型及其对口腔常驻菌群的影响；借助大鼠龋病模型和锁槽式原位菌斑采集装置，采用菌种特异性荧光原位杂交（FISH）等微生态研究技术，深入研究效应菌株对口腔原位生物膜微生态的影响及其"抗毒力"防龋效果。研究结果将有助于阐明利用细菌产碱代谢系统进行"抗毒力"防龋的潜在优势及作用模式，更将为探索龋病生态防治有效途径提供依据。

龋病是口腔微生态失衡，产酸耐酸菌过度生长所导致的牙体硬组织多细菌感染性疾病。本研究拟以“抗毒力”生态防龋理论为依据，探索龋病生态防治新途径。一方面我们从口腔主要产酸致龋菌—变异链球菌入手，通过构建低毒力菌株对龋病进行替代治疗；另一方面，我们从口腔共生菌入手，拟通过外源性干预手段，促进口腔产碱共生菌生长，利用细菌间相互竞争关系，抑制主要致龋菌生长，逆转口腔微生态失衡，防治龋病。代表性研究结果包括：1） 构建变异链球菌低毒力菌株并进行体外验证。2）通过人群临床流行病学调查，发现细菌精氨酸代谢对维持口腔微生态平衡具有重要作用；通过外源性精氨酸干预，可促进口腔产碱共生菌生长，抑制产酸致龋菌的过度生长，逆转高龋人群口腔微生态失衡，对龋病防治具有重要意义。3）通过体外生物膜模型研究，发现氟化物与精氨酸联用可协同调节致龋牙菌斑生物膜内细菌组成及酸碱代谢酶活性。项目研究成果署名发表第一/通讯作者SCI论文8篇，中文核心期刊2篇，在项目资助下参编中文学术专著5部，英文学术专著1部，获授权国家发明专利一项。通过本项目对牙菌斑生物膜微生态干预调节作用模式与机制的系统研究，为合理利用各种外源性干预手段（如氟化物与精氨酸）调节口腔微生物群落的酸碱代谢水平，进而逆转口腔微生态失衡，实现龋病抗毒力生态防治奠定了理论基础和潜在途径。