Role of extracellular matrix proteins in Candida biofilm formation

描述（由申请人提供）：白色念珠菌（CA）是最常见的分离真菌，与生物膜[细胞外基质（ECM）包裹的微生物群落相关，这些微生物群落形成于留用医疗器械上，对常用的抗真菌药物具有抗性。富含碳水化合物的ECM被认为在不同细菌的生物膜形成能力中起关键作用，但其在真菌生物膜形成和抗真菌抗性中的作用尚未得到研究。因此，真菌生物膜ECM的研究存在明显的知识空白。在初步研究中，我们：(1)标准化了一种基于edta处理的方法，从CA生物膜中分离ECM相关蛋白；(2)证明了在生物膜发育过程中，总碳水化合物（TC）、总蛋白（TP）和细胞外DNA （eDNA）的水平增加；(3)优化了一种凝胶过滤方法，分离ECM蛋白；(4)使用传统的二维凝胶电泳（2DGE）鉴定了15种在生物膜ECM中差异表达的蛋白。(5)利用最先进的二维凝胶电泳（DIGE）蛋白质组学技术，证实了231种ECM蛋白在生物膜和浮游CA的ECM中存在差异表达。这些分析还显示了10种独特的蛋白在生物膜的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)采用初步质谱（MS）分析鉴定出5种差异表达蛋白。基于这些初步数据，我们假设CA生物膜特异性ECM蛋白在生物膜形成和抗真菌抗性中起关键作用。为了验证这一假设，在本次拨款申请中，我们提出了基于以下具体目标的研究：目的1 .使用2D-DIGE蛋白质组学确定成熟生物膜基质中唯一表达的蛋白质的身份。目的二世。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。第三目标。确定：(A) EDTA处理生物膜对其抗真菌敏感性的影响，以及(B)靶向基因缺失对CA形成生物膜的能力以及这些生物膜的形态、结构和抗真菌敏感性的影响。本提案中描述的研究将使我们能够识别生物膜特异性ECM蛋白，这些蛋白在体外和体内对念珠菌生物膜的形成至关重要，可能代表新的抗生物膜治疗靶点和/或诊断标记。白色念珠菌（CA）是最常见的分离真菌，与生物膜[细胞外基质（ECM）包裹的微生物群落有关，这些微生物群落形成于留置医疗器械上，对常用的抗真菌药物具有抗性。本提案中描述的研究将使我们能够鉴定生物膜特异性ECM蛋白，这些蛋白在体外对念珠菌生物膜的形成至关重要，可能代表新的抗生物膜治疗靶点和/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.