Biomarkers of Fusarium biofilm formation in vitro and in vivo

描述(由申请人提供)：眼(或眼)组织的真菌感染是发病的重要原因，可导致失明，最常见的眼部感染表现会影响角膜，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。真菌性角膜炎的危险因素包括创伤、眼部和全身缺陷以及事先使用皮质类固醇。此外，戴隐形眼镜也与镰刀菌角膜炎有关。镰刀菌角膜炎对大多数抗真菌药物没有反应，需要进行穿透性角膜移植，失败和眼内炎的风险很高。镰刀菌在隐形眼镜/镜盒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生物膜的镰刀菌对晶状体清洁液具有抵抗力；(2)在我们的隐形眼镜相关性角膜炎小鼠模型中，硅胶隐形眼镜上形成的镰刀菌生物膜在受损的角膜上引起角膜炎；(3)镰刀菌暴发分离株形成对抗真菌药物具有抵抗力的生物膜；(4)在小鼠角膜炎模型中，暴发分离株形成生物膜的能力与疾病严重程度直接相关，(5)蛋白质组学分析确定了在镰刀菌生物膜中与浮游植物细胞相比差异表达的29种蛋白质。(6)在这些蛋白中，3-磷酸甘油醛脱氢酶(GAPDH)在镰刀菌生物膜中的表达最高(9倍)，(7)抑制GAPDH导致生物膜变薄，(8)生物膜中含有更多的凋亡细胞，(9)GAPDH在真菌生物膜中从胞浆转移到细胞核。在这一应用中，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表达最多的蛋白质，这表明它可能是真菌生物膜的共同调节因子，(B)GAPDH在真菌性角膜炎患者的眼组织中过表达，以及(C)除了它的酶活性外，GAPDH还具有调节关键细胞过程(如细胞凋亡、细胞内信号)的非酶活性。基于这些观察结果，我们推测GAPDH作为镰刀菌生物膜的调节器。我们提出了以下具体目标：目的I.使用生化方法来确定GAPDH的酶活性和非酶活性在镰刀菌生物膜形成中的作用。目的II.利用分子生物学方法(A)构建缺乏GAPDH的镰刀菌等基因突变株，以及(B)确定靶基因干扰和过表达对镰刀菌生物膜的影响。 公共卫生相关性：镰刀菌角膜炎是发病率的重要原因，可导致失明，并与最近的一次全球暴发有关。镰刀菌形成生物膜的能力被认为是导致这次疫情爆发的因素之一，我们发现这些生物膜过度生产糖酵解蛋白GAPDH。在目前的应用中提出的研究将揭示GAPDH在镰刀菌生物膜中的作用，并可能促进该蛋白作为生物膜感染的潜在生物标志物的使用。

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. PUBLIC HEALTH RELEVANCE: Fusarium keratitis is an important cause of morbidity and can result in blindness, and was associated with a recent global outbreak. The ability of Fusarium to form biofilms is believed to be one of the factors that contributed to this outbreak, and we found that these biofilms overproduce the glycolytic proteins GAPDH. Studies proposed in the current application will reveal the role of GAPDH in Fusarium biofilms, and may facilitate the use of this protein as a potential biomarker for biofilm infections.