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)：对幼儿菌斑生物膜的微生物学研究表明，幼儿龋病(ECC)与白色念珠菌的存在以及高水平的变形链球菌之间存在直接联系。以往的体内外研究表明，变形链球菌与白色念珠菌的相互作用可能影响ECC的发病机制。使用这种疾病的啮齿动物模型，与单独感染任何一个物种的动物相比，在混合感染动物的菌斑生物膜中观察到变形链球菌和白色念珠菌携带量增加的感染水平。重要的是，混合感染动物的菌斑生物膜的毒力显著增强，导致牙齿光滑表面上猖獗的龋病病变(与ECC中发现的相似)。进一步的体外研究已经确定了一株合作的、依赖蔗糖的变形链球菌C。白念珠菌相互作用是由变形链球菌胞外酶介导的，称为葡萄糖转移酶(GTFS)。在三种GTF中，GTFB最能与白念珠菌表面结合，在体外蔗糖存在的情况下保持酶活性，并在白念珠菌表面产生胞外多糖(EPS)。表面形成的EPS增强了变形链球菌和白色念珠菌之间的黏附作用，促进了富含EPS的细胞外基质的组装，并增加了变形链球菌的定植和唾液涂层磷灰石表面的共生生物膜的形成。我们推测，通过GTFB介导的变形链球菌-白色念珠菌联合作用调节牙齿上高龋性生物膜的形成。富含EPS的基质的组装和微生物积累的增加可能增强了生物膜的机械稳定性/粘聚性，并促进了生物膜内酸性微环境的创建，从而影响体内龋病的发生和发展。为了验证我们的假设，我们将(目标1)在体外使用遗传方法(例如筛选白色念珠菌的突变株)结合光谱/荧光和AFM方法(用于GTFB结合和活性测量)来表征变形链球菌衍生的GTFB与白色念珠菌的相互作用。真菌表面的GTFB结合和葡聚糖合成酶调节变形链球菌-C。白念珠菌共粘和共种生物被膜的形成。因此，我们将鉴定在GTFB结合和酶活性方面都有缺陷的白色念珠菌突变株。在目标2中，这种真菌-细菌相互作用的影响通过 GTFB对生物膜发育和机械稳定性的影响将使用一种新的工程工具来确定，而生物膜内酸性生态位的时空发展将使用体外时移pH图来评估。最后，在目标3中，我们将研究GTFB介导的变形链球菌-C的作用。在我们的啮齿动物模型中，利用GTFB结合/活性缺陷的白色念珠菌或GTFB缺陷的变形链球菌(及其亲本菌株)，白念珠菌在疾病发病机制中的相互作用。提供了一个从实验室研究到活体研究的全面计划，以提供对变形链球菌-C白色念珠菌相互作用的机制及其在增强龋病毒力方面的影响的关键见解。