Enzymatic approach for targeting mannans/EPS to disrupt cross-kingdom cariog

摘要 微生物学研究揭示了儿童早期龋病(ECC)与 白色念珠菌，以及菌斑生物膜中高水平的变形链球菌。先前的体外和体内实验 活体研究表明，白色念珠菌和变形链球菌发展了一种共生关系，增强了 龋齿严重程度。这种细菌-真菌的相互作用是由变形链球菌外酶介导的，称为 葡萄糖转移酶(GTFS)。GTFS与真菌表面紧密结合，并产生胞外多糖。 (EPS)，促进致龋性跨王国生物膜的发展。我们之前的R03支持 (DE025728)研究表明，白念珠菌细胞壁上的N-和O-连接甘露聚糖在 这一过程。甘露聚糖缺陷突变株的GTFB结合(与野生型相比)严重降低， 进而损害EPS的产生和体内混合生物膜的形成，显示出潜力 抗生素靶标。因此，我们建议进一步阐明GTFB结合/EPS产生的机制，以及 评估以配体结合功能为靶点的酶策略是否可以预防致龋性生物被膜 发展。我们将使用现成的α-(和β-)甘露糖苷酶在假丝酵母细胞上降解甘露聚糖 WALL和葡聚糖水解酶用于EPS的原位消化。我们假设酵素联合疗法 会破坏白念珠菌表面的GTFB结合位点，同时消化S。 变形杆菌GTFS，从而阻止跨王国生物膜的形成，防止严重龋病的发生 活着。为了支持我们的假设，目标1将使用遗传学来表征GTF结合功能机制 (突变菌株)和生化(酶)方法结合光谱-荧光和 生物物理学方法。具体来说，我们将评估甘露聚糖裂解对GTF结合/活性的影响，以及 EPS生产。同时，我们将评估破坏C. 白念珠菌-S。变种相互作用与生物膜形成。优化剂量对生物膜的效果将是 然后，我们将评估生物膜发育的中断和对牙齿的致龋性- 使用我们最新开发的超分辨率共焦面形貌系统进行牙釉质处理。实时动态 跨王国相互作用、生物膜的形成、原位pH、代谢活性、釉质病变的发展、 并观察生物膜脱落情况。此外，我们还将检测临床分离的变形链球菌和白色念珠菌。 来自ECC-患者。最有效的酶剂量/组合将在体内进行评估。在目标3中，我们 将使用一只成熟的啮齿动物来确定酶疗法的抗菌膜和防龋效果 ECC模型。我们将研究这种治疗方法在预防发病和严重程度方面的影响。 龋损的比例。还将评估对细菌-真菌水平和菌斑微生物群的影响。 成功完成这些目标将导致一种非微生物杀灭和抗菌剂独立的方法 减少一种普遍且代价高昂的生物膜引起的口腔疾病，这种疾病影响脆弱的儿童人口。

ABSTRACT Microbiological studies reveal a direct association between early-childhood caries (ECC) and the presence of Candida albicans, along with high levels of Streptococcus mutans in plaque-biofilms. Previous in vitro and in vivo studies demonstrated that C. albicans and S. mutans develop a symbiotic relationship, enhancing the severity of dental caries. This bacterial-fungal interaction is mediated by S. mutans exoenzymes termed glucosyltransferases (Gtfs). The Gtfs binds avidly to the fungal surface and produces exopolysaccharides (EPS) that promotes the development of cariogenic cross-kingdom biofilms. Our previous R03 supported (DE025728) studies demonstrated that N- and O-linked mannans on the C. albicans cell wall play key roles in this process. Mutant strains defective in mannans showed severely reduced GtfB binding (vs wild type), which in turn impaired EPS production and abrogated mixed-species biofilm formation in vivo, revealing potential antibioflm targets. Thus, we propose to further elucidate the mechanisms of GtfB binding/EPS production, and assess whether an enzymatic strategy targeting the ligand-binding function could prevent cariogenic biofilm development. We will use readily available α- (and β-) mannosidases for mannan degradation on Candida cell wall and glucanohydrolases for EPS digestion in situ. We hypothesize that the enzyme combination therapy will disrupt the GtfB binding sites on C. albicans surface and concomitantly digest the EPS produced by S. mutans Gtfs, thereby blocking cross-kingdom biofilm formation and preventing the onset of severe caries in vivo. To support our hypothesis, Aim 1 will characterize the Gtf binding-function mechanism using genetics (mutant strains) and biochemical (enzymatic) approaches in conjunction with spectroscopy-fluorescence and biophysical methods. Specifically, we will assess the impact of mannan-cleavage on Gtf binding/activity and EPS production. In parallel, we will assess the optimal amounts and combinations of enzymes to disrupt C. albicans-S. mutans interactions and biofilm formation. The efficacy of optimized dosages to biofilms will be evaluated in Aim 2. Then, we will assess the disruption of biofilm development and cariogenicity on tooth- enamel using our newly developed super-resolution confocal-surface topography system. Real-time dynamics of cross-kingdom interaction, biofilm formation, in situ pH, metabolic activity, development of enamel lesions, and biofilm detachment will be observed. In addition, we will test clinical isolates of S. mutans and C. albicans from ECC-patients. The most effective dosage/combination of enzymes will be evaluated in vivo. In Aim 3, we will determine antibiofilm and anticaries efficacy of the enzymatic therapy using a well-established rodent model of ECC. We will investigate the impact of this therapeutic approach in preventing the onset and severity of caries lesions. The influences on bacterial-fungal levels and plaque microbiome will be also assessed. Successful completion of these aims will lead to a non-microbiocidal and antimicrobial independent approach to reduce a prevalent and costly biofilm-induced oral disease that affect a vulnerable children population.