Biomarkers of Fusarium biofilm formation in vitro and in vivo

描述（由申请方提供）：眼（或眼部）组织的真菌感染是发病的重要原因，可导致失明，眼部感染的最常见表现影响角膜，导致角膜炎。真菌性角膜炎主要由丝状真菌镰刀菌引起，但酵母菌（如念珠菌）也可引起这种疾病。肌性角膜炎的危险因素包括外伤、眼部和全身缺陷以及先前应用皮质类固醇。此外，接触透镜配戴也与镰刀菌角膜炎有关。镰刀菌性角膜炎对大多数抗真菌药物无反应，需要穿透性角膜移植术，失败和眼内炎的风险很高。镰刀菌在接触镜/透镜上形成生物膜的能力被认为是导致最近镰刀菌角膜炎爆发的因素之一。这些发现强调了对镰刀菌角膜炎的生物学和发病机制进行详细调查的必要性。在初步研究中，我们发现：（1）镰刀菌在软接触镜上形成生物膜，其对透镜清洁溶液具有抗性，（2）在接触透镜相关镰刀菌角膜炎的鼠模型中，在硅酮水凝胶接触镜上形成的镰刀菌生物膜诱导损伤角膜上的角膜炎，（3）镰刀菌的爆发分离株形成对抗真菌剂具有抗性的坚固生物膜，和（4）在鼠角膜炎模型中观察到爆发分离株形成生物膜的能力与疾病严重程度之间的直接相关性，（5）蛋白质组学分析鉴定了与培养生长的细胞相比在镰孢菌生物膜中差异表达的29种蛋白质，（6）甘油醛-3-磷酸脱氢酶（GAPDH）在这些蛋白中表达最高（7）GAPDH的抑制导致减弱的生物膜，（8）生物膜含有更高数量的凋亡细胞，和（9）GAPDH在真菌生物膜中从胞质溶胶易位到细胞核。在本申请中，我们决定研究GAPDH在生物膜形成中的作用，因为：（a）它是镰刀菌属和念珠菌属生物膜中过表达最多的蛋白质，表明它可能是真菌生物膜的常见调节剂，（B）GAPDH在得自真菌性角膜炎患者的眼组织中过表达，和（c）除了其酶活性外，GAPDH还表现出调节关键细胞过程（例如细胞凋亡、细胞内信号传导）的非酶活性。基于这些观察，我们假设GAPDH作为镰刀菌生物膜的调节剂。我们提出以下具体目标：使用生物化学方法来确定GAPDH的酶活性和非酶活性在镰刀菌生物膜形成中的作用。Aim II.使用分子方法来（a）构建缺乏GAPDH的镰刀菌属的同基因突变体菌株，和（B）确定靶向基因破坏和过表达对镰刀菌属生物膜的影响。

DESCRIPTION (provided by applicant): Fungal infections of ophthalmic (or ocular) tissue are an important cause of morbidity and can result in blindness, and most common manifestations of ocular infections affect the cornea, resulting in keratitis. Fungal keratitis is predominantly caused by filamentous fungi Fusarium, but yeasts (e.g. Candida) can also cause this disease. Risk factors for mycotic keratitis include trauma, ocular and systemic defects and prior application of corticosteroids. Additionally, contact lens wear is also associated with Fusarium keratitis. Fusarium keratitis is not responsive to most antifungal agents, necessitating penetrating keratoplasty, with a high risk of failure and endophthalmitis. The ability of Fusarium to form biofilms on contact lenses/lens cases was suggested to be one of the factors that contributed to the recent Fusarium keratitis outbreak. These findings underscore the need for detailed investigations into the biology and pathogenesis of Fusarium keratitis. In preliminary studies, we showed that: (1) Fusarium forms biofilms on soft contact lenses, which were resistant to lens cleaning solutions, (2) Fusarium biofilms formed on silicone hydrogel contact lenses induce keratitis on injured corneas in our murine model of contact lens associated Fusarium keratitis, (3) outbreak isolates of Fusarium form robust biofilms that are resistant to antifungal agents, and (4) a direct correlation was observed between the ability of outbreak isolates to form biofilms and disease severity in the murine keratitis model, (5) proteomics analysis identified 29 proteins that were differentially expressed in Fusarium biofilms compared to planktonically grown cells, (6) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most overexpressed among these proteins (by 9-fold) in Fusarium biofilms, (7) inhibition of GAPDH resulted in attenuated biofilms, (8) biofilms contained higher number of apoptotic cells, and (9) GAPDH is translocated from the cytosol to the nucleus in fungal biofilms. In this application, we decided to investigate the role of GAPDH in biofilm formation because: (a) it is the most over-expressed protein in both Fusarium and Candida biofilms, suggesting that it may be a common regulator of fungal biofilms, (b) GAPDH was overexpressed in ocular tissues obtained from patients with fungal keratitis, and (c) in addition to its enzymatic activity, GAPDH also exhibits non-enzymatic activities that modulate key cellular processes (e.g. apoptosis, intracellular signaling). Based on these observations, we hypothesize that GAPDH acts as a modulator of Fusarium biofilms. We propose the following specific aims: Aim I. Use a biochemical approach to determine the role of enzymatic and non-enzymatic activities of GAPDH in Fusarium biofilm formation. Aim II. Use a molecular approach to (a) construct isogenic mutant strains of Fusarium lacking GAPDH, and (b) determine the effect of targeted gene disruption and overexpression on Fusarium biofilms.