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（由申请方提供）：幼儿菌斑生物膜的微生物学研究显示，幼儿龋齿（ECC）与白色念珠菌（沿着高水平变形链球菌）的存在直接相关。先前的体外和体内研究强烈表明，S。mutans与C.白念珠菌可能影响ECC的发病机制。使用啮齿类动物的疾病模型，提高感染水平与提高携带的S。mutans和C.与单独感染任一种的动物相比，在来自共感染动物的噬菌斑生物膜中观察到白色念珠菌。重要的是，在共感染动物中菌斑-生物膜的毒力显著增强，导致牙齿光滑表面上的猖獗的龋损的发展（类似于在ECC中发现的那些）。进一步的体外研究已经确定了一个合作和蔗糖依赖性的S。突变C.白念珠菌的相互作用是由S.变形杆菌胞外酶称为葡糖基转移酶（Gtfs）。在三种Gtf中，GtfB与C结合最强烈。白色念珠菌细胞表面，保留酶活性并产生胞外多糖（EPS）。白色念珠菌表面的蔗糖在体外的存在。表面形成的EPS增强了S. mutans和C.白念珠菌，促进富含EPS的细胞外基质的组装，并增加S.在唾液涂覆的磷灰石表面上形成变形菌和同种生物膜。我们推测，通过GtfB介导的变形链球菌-白色念珠菌缔合调节牙齿上的高度致龋生物膜的发展。可能的是，富含EPS的基质和升高的微生物积累的组装增强了生物膜机械稳定性/内聚性，并促进了生物膜内酸性微环境的产生，这可以影响体内龋齿的发作和进展。为了验证我们的假设，我们将（目的1）描述S的相互作用。变形杆菌GtfB与C.使用遗传学方法（例如筛选C.白色念珠菌）结合光谱/荧光和AFM方法（用于GtfB结合和活性测量）。GtfB结合和真菌表面上的酶的葡聚糖合成调节S。突变C.白色念珠菌共粘附和同种生物膜形成。因此，我们将确定C。白色念珠菌突变株的GtfB结合和酶活性都有缺陷。在目标2中，通过以下方式来研究这种真菌-细菌相互作用的影响： GtfB对生物膜发展和机械稳定性的影响将使用新的工程工具来确定，而生物膜内酸性小生境的时空发展将使用体外时间推移pH映射来评估。最后，在目标3中，我们将研究GtfB介导的S.突变C.使用GtfB-结合/活性缺陷型C.白色念珠菌或gtfB缺陷型S.变异株（沿着它们的亲本菌株）与我们的啮齿动物模型。一个全面的计划，从实验室研究，在体内调查提供关键的见解，这S。mutans与白色念珠菌的相互作用及其在增强龋病毒力中的意义