Role of GtfB on S.mutans-C.albicans interactions and cariogenic biofilm formation

GTfB 在 S.mutans-C.albicans 相互作用和致龋生物膜形成中的作用

• 批准号: 9016967
• 负责人: Geelsu Hwang
• 金额: $ 8万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9016967
• 关键词: Adherence; Adhesions; Adhesives; Affect; Animal Feed; Animals; Binding; Candida albicans; Cell surface; Child; Clinical; Communicable Diseases; Complement; Data; Dental caries; Development; Devices; Diet; Disease; Engineering; Enzymes; Extracellular Matrix; Fluorescence; Glucans; Glucosyltransferases; Goals; In Situ; In Vitro; Infection; Ingestion; Investigation; Laboratory Study; Lesion; Maps; Measurement; Mechanics; Mediating; Methods; Microbial Biofilms; Mouth Diseases; Oral cavity; Outcome; Pain; Pathogenesis; Production; Rodent Model; Role; Saliva; Severities; Site; Streptococcus mutans; Sucrose; Surface; Systemic infection; Testing; Time; Toddler; Tooth structure; Virulence; Virulent; base; clinically relevant; early childhood; enzyme activity; exoenzyme; extracellular; genetic approach; in vivo; insight; microbial; mutant; pathogen; prevent; programs; public health relevance; screening; sugar; time use; tool; tooth surface

 DESCRIPTION (provided by applicant): Microbiological studies of plaque-biofilms from toddlers reveal a direct association between early-childhood caries (ECC) and the presence of Candida albicans, along with high levels of Streptococcus mutans. Previous in vitro and in vivo studies suggest strongly that S. mutans interactions with C. albicans may influence the pathogenesis of ECC. Using a rodent model of the disease, enhanced levels of infection with elevated carriage of S. mutans and C. albicans were observed in plaque-biofilms from co-infected animals compared to those infected with either species alone. Importantly, the virulence of plaque-biofilm in co-infected animal was dramatically enhanced, leading to the development of rampant carious lesions on smooth-surface of teeth (similar to those found in ECC). Further in vitro studies have identified a cooperative and sucrose-dependent S. mutans-C. albicans interactions that is mediated by S. mutans exoenzymes termed glucosyltransferases (Gtfs). Of the three Gtfs, GtfB binds most avidly to C. albicans cell surface, retains enzymatic activity and produces exopolysaccharides (EPS) on C. albicans surfaces in the presence of sucrose in vitro. The surface-formed EPS enhances adhesive interactions between S. mutans and C. albicans, promotes the assembly of an EPS- rich extracellular matrix, and increases colonization of S. mutans and co-species biofilm formation onto saliva- coated apatitic surfaces. We hypothesize that S.mutans-C.albicans association mediated via GtfB modulates the development of hypercariogenic biofilms on teeth. It is possible that the assembly of EPS- rich matrix and elevated microbial accumulation enhance biofilm mechanical stability/cohesiveness and facilitate the creation of acidic microenvironments within biofilms, which can influence the onset and progression of caries in vivo. To test our hypothesis, we will (Aim 1) characterize the interaction of S. mutans derived-GtfB with C. albicans using genetic approaches (e.g. screening mutants of C. albicans) combined with spectroscopic/fluorescence and AFM methods (for GtfB binding and activity measurements) in vitro. GtfB binding and glucan synthesis by the enzyme on the fungal surface modulates S. mutans-C. albicans co- adherence and co-species biofilm formation. Therefore, we will identify C. albicans mutant strains that are defective in both the GtfB binding and enzymatic activity. In Aim 2, the effects of this fungal-bacterial interaction via GtfB on biofilm development and mechanical stability will be determined using a new engineering tool, while spatio-temporal development of acidic niches within biofilms will be assessed using time-lapsed pH mapping in vitro. Lastly, in Aim 3, we will examine the role of GtfB-mediated S. mutans-C. albicans interaction in the pathogenesis of the disease in vivo using GtfB-binding/activity defective C. albicans or gtfB-defective S. mutans (along with their parental strains) with our rodent model. A comprehensive program from laboratory studies to in vivo investigations is offered to provide critical insights into the mechanisms of this S. mutans-C albicans interaction and its implications in enhancing the virulence of dental caries disease.

 描述（由申请人提供）：对幼儿牙菌斑生物膜的微生物学研究表明，早期儿童龋齿 (ECC) 与白色念珠菌以及高水平的变形链球菌之间存在直接关联。强烈认为变形链球菌与白色念珠菌的相互作用可能会影响 ECC 的发病机制。使用该疾病的啮齿动物模型，随着变形链球菌携带量的增加，感染水平也会增加。与单独感染任一物种的动物相比，在共同感染的动物的斑块生物膜中观察到变形链球菌和白色念珠菌，重要的是，共同感染的动物中斑块生物膜的毒力显着增强，导致猖獗的龋齿病变的发展。牙齿光滑表面上的相互作用（与 ECC 中发现的类似）。在三种 Gtf 中，GtfB 最热衷于与白色念珠菌细胞表面结合，保留酶活性，并在体外蔗糖存在的情况下在白色念珠菌表面产生胞外多糖 (EPS)。 EPS 增强变形链球菌和白色念珠菌之间的粘附相互作用，促进组装富含 EPS 的细胞外基质，并增加了变形链球菌和同种生物膜在唾液涂覆的磷灰石表面上的形成，我们发现通过 GtfB 介导的变形链球菌与白色念珠菌的结合可调节牙齿上高致龋生物膜的形成。富含 EPS 的基质的组装和微生物积累的增加可能会增强生物膜的机械稳定性/内聚性，并促进生物膜内酸性微环境的形成，为了检验我们的假设，我们将（目标 1）使用遗传方法（例如筛选白色念珠菌突变体）来表征变异链球菌衍生的 GtfB 与白色念珠菌的相互作用。使用光谱/荧光和 AFM 方法（用于 GtfB 结合和活性测量）在体外通过真菌表面上的酶调节 S.因此，我们将在目标 2 中鉴定出 GtfB 结合和酶活性均存在缺陷的白色念珠菌突变菌株，即这种真菌-细菌相互作用的影响。 GTfB 对生物膜发育和机械稳定性的影响将使用新的工程工具来确定，而生物膜内酸性生态位的时空发育将使用体外延时 pH 绘图进行评估。最后，在目标 3 中，我们将研究 GtfB 的作用。使用GtfB结合/活性缺陷的白色念珠菌或gtfB缺陷的体内疾病发病机制中GtfB介导的白色念珠菌相互作用。变形链球菌（及其亲本菌株）与我们的啮齿动物模型提供了从实验室研究到体内研究的综合计划，以提供对变形链球菌-白色念珠菌相互作用的机制及其对增强毒力的影响的重要见解。的龋齿疾病。