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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.

摘要