Biomarkers of Fusarium biofilm formation in vitro and in vivo

描述（申请人提供）：眼部（或眼部）组织真菌感染是致病的重要原因，可导致失明，眼部感染最常见的表现是影响角膜，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（如念珠菌）也可引起这种疾病。真菌性角膜炎的危险因素包括创伤、眼部和全身缺陷以及先前使用皮质类固醇。此外，佩戴隐形眼镜也与镰刀菌性角膜炎有关。镰刀菌性角膜炎对大多数抗真菌药物没有反应，需要穿透性角膜移植术，失败和眼内炎的风险很高。镰刀菌在隐形眼镜上形成生物膜的能力被认为是导致最近爆发的镰刀菌角膜炎的因素之一。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研究的必要性。在初步研究中，我们发现：(1)在软性隐形眼镜上形成镰刀菌生物膜，对镜片清洁液有抗性；(2)在硅胶水凝胶隐形眼镜上形成镰刀菌生物膜，在我们的小鼠隐形眼镜相关性镰刀菌角膜炎模型中诱发损伤角膜角膜炎；(3)爆发分离的镰刀菌形成坚固的生物膜，对抗真菌药物有抗性。(4)在小鼠角膜炎模型中观察到爆发分离物形成生物膜的能力与疾病严重程度之间存在直接相关性，(5)蛋白质组学分析发现，与浮游生长的细胞相比，29种蛋白在镰刀菌生物膜中表达差异，(6)甘油醛-3-磷酸脱氢酶（GAPDH）在镰刀菌生物膜中过度表达最多（高9倍），(7)抑制GAPDH导致生物膜减弱。(8)生物膜中凋亡细胞数量较多；(9)真菌生物膜中GAPDH从细胞质转移到细胞核。在这个应用中，我们决定研究GAPDH在生物膜形成中的作用，因为：(a)它是镰刀菌和念珠菌生物膜中过度表达最多的蛋白，这表明它可能是真菌生物膜的共同调节剂；(b) GAPDH在真菌性角炎患者的眼组织中过度表达；(c)除了酶活性外，GAPDH还表现出调节关键细胞过程（如凋亡、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：目的1 .利用生化方法确定GAPDH酶和非酶活性在镰刀菌生物膜形成中的作用。目的二世。利用分子方法(a)构建缺乏GAPDH的镰刀菌等基因突变菌株，(b)确定靶向基因破坏和过表达对镰刀菌生物膜的影响。

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.