Antifungal Resistance Mechanism in Biofilm Growing candida albicans

描述(申请人提供)：白色念珠菌是一种装备精良的机会性病原体，能产生多种毒力因子。最近认识到的一个毒力属性是在生物膜中生存和保持的能力。念珠菌生物被膜感染通常与医疗器械有关。中心静脉导管(CVC)相关的血流感染是最重要的生物被膜感染之一。由于与生物膜生长相关的耐药性，在不移除医疗设备的情况下，这些感染极难治疗。导致生物膜耐药的机制还不是很清楚。我们的长期目标是阐明念珠菌生物被膜耐药的机制，作为开发可用于减轻这种疾病过程的治疗策略的先决条件。这项拟议的研究背后的具体假设是，在白色念珠菌生物膜生长过程中产生的2-葡聚糖分子与抗真菌药物相互作用，并防止与细胞内药物靶点结合。这一假设基于以下观察结果。首先，生物膜过程与细胞壁的变化有关，这种变化与2-1，3葡聚糖含量的增加有关。此外，细胞外基质的组成主要是碳水化合物，包括2-葡聚糖。第二，分离的生物膜基质能够与抗真菌药物氟康唑结合。此外，将生物膜基质和其他外源2-葡聚糖添加到自由漂浮的(浮游)白色念珠菌中，使细胞对抗真菌药物氟康唑和两性霉素B产生抗药性。此外，生物膜生长过程中2-葡聚糖的酶促变化增强了抗真菌药物的活性。第三，葡聚糖合成和修饰途径中三个基因的突变导致对抗真菌药物氟康唑的敏感性增加。基于这些观察，这一提议的实验重点是葡聚糖途径。其具体目的是(I)确定葡聚糖合成和修饰途径中的基因对生物膜形成和耐药性的意义，(Ii)表征亲本和葡聚糖途径中与生物膜相关的胞外碳水化合物的结构和数量，以及(Iii)提供生物膜葡聚糖与抗真菌药物相互作用的结构和定量评估。公共卫生相关性：医疗器械的白色念珠菌生物被膜感染很常见，并导致大量患者死亡。与生物被膜相关的耐药性与不良结局有关。耐药表型的潜在机制尚不清楚。目前的提案旨在确定分泌型碳水化合物在生物被膜耐药中的作用。

DESCRIPTION (provided by applicant): Candida albicans is a well-armed opportunistic pathogen that produces a diverse array of virulence factors. One recently recognized virulence attribute is the ability to live and persist in a biofilm. Candida biofilm infections are commonly associated with medical devices. Central venous catheter (CVC) related bloodstream infections are among the most important biofilm infections. These infections are extremely difficult to treat without removal of the medical device due to the drug resistance associated with biofilm growth. The mechanisms responsible for biofilm resistance are not well-understood. Our long term goal is to elucidate the mechanisms responsible for drug resistance in Candida biofilms as a prerequisite to the development of therapeutic strategies that can be used to attenuate this disease process. The specific hypothesis behind the proposed research is that 2-glucan molecules produced during C. albicans biofilm growth interact with antifungal drugs and prevent binding to intracellular drug targets. This hypothesis is based on the following observations. First, the biofilm process is associated with cell wall changes that are associated with increased 2-1,3 glucan content. In addition, the composition of the extracellular matrix is predominantly carbohydrate, including 2-glucan. Second, isolated biofilm matrix is capable of binding to the antifungal fluconazole. Furthermore, addition of biofilm matrix and other exogenous sources of 2 -glucan to free floating (planktonic) C. albicans renders the cells resistant to the antifungals, fluconazole and amphotericin B. Also, enzymatic alteration of 2- glucan during biofilm growth enhances the activity of the antifungal drugs. Third, mutation of three genes in the glucan synthesis and modification pathway results in enhanced susceptibility to the antifungal fluconazole. Based on these observations, the experimental focus of this proposal is on the glucan pathway. The specific aims are designed to (i) define the significance of genes in the glucan synthesis and modification pathways for biofilm formation and drug resistance, (ii) characterize the structure and quantity of the biofilm associated extracellular carbohydrate in parent and mutants in the glucan pathways, and (iii) provide a structural and quantitative evaluation of the biofilm glucan and antifungal drug interaction. PUBLIC HEALTH RELEVANCE: Candida albicans biofilm infection of medical devices is common and causes significant patient mortality. Drug-resistance associated with biofilms is associated with poor outcomes. The mechanisms underlying the resistance phenotype are poorly understood. The current proposal is designed to determine the role of a secreted carbohydrate in biofilm resistance.