Role of extracellular matrix proteins in Candida biofilm formation

细胞外基质蛋白在念珠菌生物膜形成中的作用

• 批准号: 7386259
• 负责人: Pranab Kumar Mukherjee
• 金额: $ 23.55万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386259
• 关键词: Antifungal Agents; Applications Grants; Architecture; Arts; Bacteria; Biological Assay; Candida albicans; Carbohydrates; Catheters; Cell surface; Consultations; DNA; Data; Databases; Development; Diagnostic; Early identification; Edetic Acid; Electron Microscopy; Extracellular Matrix; Extracellular Matrix Proteins; Extracellular Protein; Fluorescence Microscopy; Fungal Drug Resistance; Gel Chromatography; Gene Deletion; Gene Targeting; Genes; In Vitro; Infection; Knowledge; Laser Scanning Microscopy; MALDI-TOF Mass Spectrometry; Mass Spectrum Analysis; Measurement; Medical Device; Medical center; Mentors; Metabolic; Methods; Microbial Biofilms; Modeling; Molecular; Morphology; Oryctolagus cuniculus; Phase; Play; Predisposition; Prevention; Production; Proteins; Proteomics; Publications; Research Personnel; Resistance; Role; Scanning; Southern Blotting; Spottings; Techniques; Testing; The science of Mycology; Thick; Two-Dimensional Gel Electrophoresis; Weight; base; candida biofilm; extracellular; follow-up; fungus; gel electrophoresis; in vitro Model; in vivo; microbial community; mutant; novel; therapeutic target

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.

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置医疗器械上，对常用的抗真菌药物具有抗药性。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发挥着关键作用，但其在真菌生物膜形成和抗真菌耐药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) 证明了生物膜形成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) 优化了凝胶过滤方法来分离 ECM 蛋白，(4) 使用传统的二维 凝胶电泳（2DGE）鉴定了生物膜ECM中差异表达的15个蛋白质，（5）使用凝胶电泳（DIGE）蛋白质组学中最先进的二维差异技术证明了231个ECM蛋白质在生物膜和浮游CA的ECM中差异表达。这些分析还表明，10 种独特的蛋白质在生物膜的 ECM 中表达，其中 8 种在成熟生物膜中表达，两种在早期生物膜中表达。 (6)利用初步的质谱(MS)分析鉴定出5个差异表达的蛋白质。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耐药性中发挥关键作用。为了检验这一假设，在本次拨款申请中，我们提出基于以下具体目标的研究： 目标 I. 使用 2D-DIGE 蛋白质组学确定成熟生物膜基质中独特表达的蛋白质的身份。目标二。构建缺乏生物膜基质特异性蛋白质的白色念珠菌同基因突变株。目标三。确定：(A) 生物膜的 EDTA 处理对其抗真菌敏感性的影响，以及 (B) 靶向基因删除对 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。该提案中描述的研究将使我们能够识别对体外和体内念珠菌生物膜形成至关重要的生物膜特异性 ECM 蛋白，这可能代表新的抗生物膜治疗靶点和/或诊断标记物。白色念珠菌 (CA) 是最常见的分离真菌，与留置医疗器械上形成的生物膜 [细胞外基质 (ECM) 包裹的微生物群落相关，这些微生物群落对常用的抗真菌药物具有抗药性。该提案中描述的研究将使我们能够识别对体外念珠菌生物膜形成至关重要的生物膜特异性 ECM 蛋白，这可能代表新的抗生物膜治疗靶点和/或诊断标记物。