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

靶向甘露聚糖/EPS 的酶法可破坏跨界 cariog

基本信息

批准号：
10436198
负责人：
Geelsu Hwang
金额：
$ 37.86万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10436198
关键词：
Adhesions Affect Anabolism Antibiotics Binding Binding Sites Biochemical Biological Candida Candida albicans Caries prevention Cell Wall Cells Child Clinical Combined Modality Therapy Communicable Diseases Complement Consumption Data Dental Enamel Dental caries Development Devices Digestion Enzymes Evaluation Exhibits Fluconazole Fluorescence Spectroscopy Fluorides Genes Genetic Genetic Databases Glucosyltransferases Goals Gold Impairment In Situ In Vitro Infection Lesion Ligand Binding Link Mannans Mannosidase Mechanics Mediating Metabolic Methods Microbial Biofilms Microbiology Mouth Diseases National Institute of Dental and Craniofacial Research Oral cavity Pain Pathogenicity Patients Play Population Povidone-Iodine Process Production Resolution Rodent Model Role Severities Site Streptococcus mutans Sucrose Surface System Testing Therapeutic Therapeutic Agents Time Toddler Tooth structure Topical application Treatment Protocols United States National Institutes of Health Virulence Virulent Visualization anticaries antimicrobial base biophysical techniques clinical application clinically relevant cost demineralization dental biofilm dosage early childhood efficacy evaluation exoenzyme fungus in vivo microbiome microbiota mutant novel therapeutic intervention oral microbial community polymicrobial biofilm prevent real-time images single molecule spatiotemporal sugar tooth surface

项目摘要

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.

抽象的微生物学研究揭示了儿童早期龋齿 (ECC) 与存在的直接关系先前在体外和体内的斑块生物膜中存在白色念珠菌和高水平的变形链球菌。体内研究表明，白色念珠菌和变形链球菌形成了共生关系，增强了这种细菌-真菌相互作用是由称为变形链球菌外酶介导的。葡萄糖基转移酶 (Gtfs) Gtfs 与真菌表面紧密结合并产生胞外多糖。（EPS），促进致龋跨界生物膜的发展，我们之前的 R03 支持。 (DE025728) 研究表明，白色念珠菌细胞壁上的 N- 和 O- 连接甘露聚糖在甘露聚糖缺陷的突变株表现出 GtfB 结合严重减少（与野生型相比）。反过来，EPS 的产生受损并消除了体内混合物种生物膜的形成，揭示了潜在的潜力因此，我们建议进一步阐明 GtfB 结合/EPS 产生的机制，以及评估针对配体结合功能的酶策略是否可以预防致龋生物膜我们将使用现成的 α-（和 β-）甘露糖苷酶在念珠菌细胞上降解甘露聚糖。我们捕获了用于原位 EPS 消化的酶组合疗法。将破坏白色念珠菌表面的 GTfB 结合位点，并同时消化白色念珠菌产生的 EPS。 mutans GTfs，阻断跨界生物膜形成并防止严重龋齿的发生为了支持我们的假设，目标 1 将利用遗传学来表征 Gtf 结合功能机制。（突变菌株）和生化（酶）方法与光谱荧光和具体来说，我们将评估甘露聚糖切割对 Gtf 结合/活性的影响和与此同时，我们将评估破坏 C. 的酶的最佳数量和组合。白色念珠菌-变形链球菌相互作用和生物膜形成的优化剂量将是。在目标 2 中进行评估。然后，我们将评估生物膜发育的破坏和牙齿的致龋性使用我们新开发的超分辨率共焦表面形貌实时动力学系统进行牙釉质。跨界相互作用、生物膜形成、原位 pH 值、代谢活动、牙釉质病变的发展、此外，我们还将测试变形链球菌和白色念珠菌的临床分离株。在目标 3 中，我们将在体内评估最有效的酶剂量/组合。将使用成熟的啮齿动物来确定酶疗法的抗生物膜和防龋功效我们将研究这种治疗方法对预防 ECC 发病和严重程度的影响。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杀菌和抗菌独立的方法减少影响弱势儿童群体的普遍且昂贵的生物膜引起的口腔疾病。