Role of extracellular matrix proteins in Candida biofilm formation

描述(由申请人提供)：白色念珠菌(CA)是与生物膜[细胞外基质(ECM)包裹的微生物群落]相关的最常见的真菌，形成于留置医疗器械上，对常用抗真菌药物具有耐药性。富含碳水化合物的ECM被认为在不同细菌的生物被膜形成能力中起着关键作用，但它在真菌生物被膜形成和抗真菌耐药性中的作用尚未被研究。因此，在真菌生物膜ECM的研究中存在着明显的知识空白。在初步研究中，我们：(1)标准化了一种基于EDTA处理的分离CA生物膜中ECM相关蛋白的方法，(2)证明了在生物膜发育过程中总碳水化合物(TC)、总蛋白(TP)和胞外DNA(EDNA)水平的增加，(3)优化了凝胶过滤分离ECM蛋白的方法，(4)使用传统的双向凝胶电泳法(2DGE)鉴定了15种在生物膜ECM中差异表达的蛋白质，(5)应用双向差异凝胶电泳法(DGE)蛋白质组学技术，研究了231个ECM蛋白在生物膜和浮游CA细胞外基质中的差异表达。这些分析还显示，10种独特的蛋白质在生物膜的ECM中表达，8种在成熟生物膜中表达，2种在早期生物膜中表达。(6)利用初步的质谱学分析鉴定了5个差异表达蛋白质。基于这些初步数据，我们推测CA生物膜特异性ECM蛋白在生物膜形成和抗真菌耐药性中起关键作用。为了验证这一假设，在这次拨款申请中，我们提出了基于以下特定目标的研究：目的I.利用2D-DGE蛋白质组学确定成熟生物膜基质中唯一表达的蛋白质的同一性。目的II.构建缺乏生物膜基质特异性蛋白的白色念珠菌等基因突变株。目的：(A)EDTA处理生物膜对其抗真菌敏感性的影响；(B)靶向基因缺失对CA生物膜形成能力以及这些生物膜的形态、结构和抗真菌敏感性的影响。这项研究将使我们能够确定在体外和体内对念珠菌生物被膜形成至关重要的生物被膜特异性ECM蛋白，这些蛋白可能是新的抗生物被膜治疗靶点和/或诊断标记。白色念珠菌(CA)是与生物被膜(ECM)相关的最常见的真菌，这些微生物群落形成在留置医疗器械上，对常用抗真菌药物具有耐药性。这项研究将使我们能够识别在体外对念珠菌生物被膜形成至关重要的生物被膜特异性ECM蛋白，这些蛋白可能是新的抗生物被膜治疗靶点和/或诊断标记。

DESCRIPTION (provided by applicant): Candida albicans (CA) is the most commonly isolated fungus associated with biofilms [extracellular matrix (ECM)-encased microbial communities formed on indwelling medical devices which are resistant against commonly used antifungals. The carbohydrate-rich ECM is believe to play critical role in the biofilm forming ability of different bacteria, but its role in fungal biofilm formation and antifungal resistance has not been investigated. Therefore, obvious knowledge gaps exist in the study of fungal biofilm ECM. In Preliminary Studies, we: (1) standardized an EDTA-treatment based method to isolate ECM associated proteins from CA biofilm, (2) demonstrated that the levels of total carbohydrates (TC), total protein (TP), and extracellular DNA (eDNA) increase during biofilm development, (3) optimized a gel filtration method to isolate ECM proteins, (4) used traditional two-dimensional gel electrophoresis (2DGE) to identify 15 proteins differentially expressed in biofilm ECM, (5) used state-of-the-art technique of 2D difference in gel electrophoresis (DIGE) proteomics to demonstrate that 231 ECM proteins were differentially expressed in ECM of biofilm and planktonic CA. These analyses also showed 10 unique proteins to be expressed in ECM of biofilms eight in mature biofilms, and two in early phase biofilms. (6) Used preliminary mass spectrometry (MS) analyses to identify five differentially expressed proteins. Based on these Preliminary Data, we hypothesize that CA biofilm-specific ECM proteins play critical roles in biofilm formation and antifungal resistance. To test this hypothesis, in this grant application, we propose studies based on the following specific aims: Aim I. Determine the identity of proteins uniquely expressed in matrix of mature biofilms, using 2D-DIGE proteomics. Aim II. Construct isogenic mutant strains of C. albicans lacking the biofilm matrix-specific proteins. Aim III. Determine the effect of: (A) EDTA treatment of biofilms on their antifungal susceptibility, and (B) Targeted gene deletions on the ability of CA to form biofilms and the morphology, architecture, and antifungal susceptibility of these biofilms. The studies described in this proposal will allow us to identify biofilm-specific ECM proteins that are critical to Candida biofilm formation in vitro and in vivo, which may represent novel anti-biofilm therapeutic targets and/or diagnostic markers. Candida albicans (CA) is the most commonly isolated fungus associated with biofilms [extracellular matrix (ECM)-encased microbial communities formed on indwelling medical devices, which are resistant against commonly used antifungals. The studies described in this proposal will allow us to identify biofilm-specific ECM proteins that are critical to Candida biofilm formation in vitro, which may represent novel anti-biofilm therapeutic targets and/or diagnostic markers.