变异链球菌耐氟菌的耐氟调控机制及其对不同龋易感人群的致龋作用研究

Fluoride is a commonly used caries-preventive agent. However, the widespread and prolonged application of fluoride may lead to the emergence of fluoride-resistant species, which will decrease the effectiveness of fluoride on anti-caries. Currently, the mechanism of fluoride resistance in the fluoride-resistant strains is not clear. Our previous studies found that the fluoride-resistant Streptococcus mutans strain UAFR showed more resistant to fluoride and had enhanced biofilm formation as well as lactic acid production, compared to its parental stain UA159 and the single-nucleotide mutated fluoride-resistant strain UF35. In addition, strain UAFR was identified to contain multiple genetic mutations by comparing its genome sequence with that of the parental strain UA159, including the promoter mutp of the fluoride antiporter-coding genes, enolase gene eno and pyruvate kinase gene pyk. These data indicated that the three gene sites may be involved in the modulation of fluoride resistance. In this project, we attempt to construct S. mutans strains containing one single mutation or combined genes mutation and then examine their resistance to fluoride, to reveal the regulatory mechanism of the three genes on fluoride resistance. We also investigate the effects of these single or combined genes mutant strains on the biological characteristics and microbial communities of saliva microcosms in different caries-susceptible populations. In vitro artificial carious models will be established to confirm the shift of cariogenic potential in the microcosms. This project will reveal one or more genes involved in the higher resistance to fluoride and the enhanced virulence of S. mutans strains. It also provides the strategy for the application of fluoride and a new approach to keeping the balance of oral microbial ecology.

氟化物是预防龋病最常用的方法，但长期使用氟制剂容易导致细菌耐氟菌株的产生，降低其防龋效果。目前关于耐氟菌的耐氟调控机制尚不明确。我们前期研究发现，变异链球菌耐氟菌UAFR的耐氟水平、生物膜形成及产酸能力均强于亲代菌UA159和单基因突变耐氟菌UF35；UAFR的基因序列与亲代菌相比存在多个突变位点，分布于氟逆向转运蛋白基因启动子mutp，烯醇酶基因eno和丙酮酸激酶基因pyk，提示UAFR的耐氟机制可能由多个基因共同调控。本项目拟构建变异链球菌上述基因位点单独及联合突变的菌株并检测其耐氟特性，揭示多基因位点协同的耐氟调控机制；从生物学特性和群落水平上探讨单独及联合突变株对不同龋易感人群唾液生物膜的影响，分析唾液生物膜致龋状态的变化，并通过体外人工龋模型进行验证。本课题可揭示增强变异链球菌耐氟性及致龋力的多基因位点协同作用，为临床合理使用氟化物提供实验依据，也为维护口腔微生态平衡提供新途径。

龋病是由于牙菌斑微生态系统的稳态受到破坏，以变异链球菌为主的致龋菌成为菌斑中的优势菌，产生大量有机酸造成牙体硬组织脱矿而引起。氟化物是目前预防龋病最常用的方法，但长期、广泛使用氟制剂可能导致变异链球菌耐氟菌的出现，且耐氟菌的致龋毒力明显增强，在口腔微环境中更具竞争优势，进一步影响菌群的稳态及多样性，加速龋病的发生发展。本项目聚焦于变异链球菌的耐氟机制及唾液微生态在不同pH环境下的生物学特性和菌群结构的变化，通过构建变异链球菌UA159 mutp单基因突变耐氟菌UF36M，并与多基因突变耐氟菌UAFR比较，发现在正常的中性环境和致龋的pH循环环境下单基因突变耐氟菌的生物膜形成能力及代谢活性均显著低于多基因突变耐氟菌UAFR，表明多个耐氟基因对变异链球菌的致龋毒力具有协同调控作用；收集健康人群的非刺激性唾液分别在正常和致龋环境下培养生物膜，发现致龋环境下唾液生物膜的形成及产酸能力与正常环境相比均显著增强，菌群多样性则明显降低，菌群组成以唾液链球菌为主，典范对应分析显示致龋环境下唾液生物膜的生物量及产酸量与唾液链球菌和殊异韦荣球菌密切相关，提示致龋环境下具有较强生物膜形成及产酸能力的细菌如唾液链球菌等可能成为优势菌种，进而影响菌群稳态，促进龋病的发生发展；此外，本研究发现唾液生物膜中过氧化氢的产生可能受pH环境、受试者等多种因素影响，还与唾液菌群中的殊异韦荣球菌、产黑色素普氏菌、丛毛单胞菌科、柄杆菌属等密切相关。本项目不仅深化了对变异链球菌耐氟机制及龋病发生发展的认识，也为调控唾液微生态的pH环境及其相关菌群标记物以防治龋病提供了科学依据，并为维护口腔微生态的稳态开拓新思路。发表SCI学术论文1篇，在国内核心期刊《口腔疾病预防》发表专论1篇，培养硕士研究生1名。