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齿（ECC）与 白色念珠菌，沿着在菌斑生物膜中出现高水平的变形链球菌。以前的体外和体内 体内研究表明，C. albicans和S.变异体发展出一种共生关系， 龋齿的严重程度。这种细菌-真菌相互作用是由S.变形杆菌胞外酶 葡糖基转移酶（Gtfs）。Gtfs与真菌表面结合并产生胞外多糖 (EPS)促进了致龋跨界生物膜的形成。我们之前的R 03支持 （DE 025728）的研究表明，C.白念珠菌细胞壁在 这个过程甘露聚糖缺陷的突变株显示出严重降低的GtfB结合（相对于野生型）， 反过来又削弱了EPS的生产和废除体内混合物种生物膜的形成，揭示了潜在的 瞄准目标。因此，我们建议进一步阐明GtfB结合/EPS产生的机制， 评估靶向配体结合功能的酶促策略是否可以预防致龋生物膜 发展我们将使用容易获得的α-（和β-）甘露聚糖酶在念珠菌细胞上降解甘露聚糖 壁和葡聚糖水解酶用于EPS原位消化。我们假设酶联合疗法 将破坏C上的GtfB结合位点。白色念珠菌表面并伴随消化由S. 变形杆菌Gtfs，从而阻断跨界生物膜的形成，并防止严重龋齿的发生， vivo.为了支持我们的假设，目的1将使用遗传学来表征Gtf结合功能机制 （突变菌株）和生物化学（酶）方法结合光谱-荧光和 生物物理方法。具体而言，我们将评估甘露聚糖切割对Gtf结合/活性的影响， EPS生产。同时，我们将评估酶的最佳数量和组合来破坏C。 白色链霉菌S.变形菌相互作用和生物膜形成。优化剂量对生物膜的功效将是 在目标2中评估。然后，我们将评估牙齿上生物膜发育和致龋性的破坏- 使用我们新开发的超分辨率共焦表面形貌系统对牙釉质进行分析。实时动态 跨界相互作用、生物膜形成、原位pH、代谢活性、釉质损伤的发展， 并且将观察到生物膜脱离。此外，我们将测试临床分离的S。mutans和C.白色 ECC患者。将在体内评价酶的最有效剂量/组合。在目标3中， 将使用成熟的啮齿类动物确定酶疗法的防龋膜和防龋功效 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。 龋齿病变。还将评估对细菌-真菌水平和菌斑微生物组的影响。 这些目标的成功完成将导致非杀微生物剂和抗微生物剂独立的方法 减少一种影响弱势儿童群体的流行且昂贵的生物膜引起的口腔疾病。

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.