Elucidating pathways that regulate fungal keratitis pathogenesis

项目总结/摘要 真菌性角膜炎是世界范围内眼部发病率和单眼失明的重要来源。电流 高达60%的患者的抗真菌方案失败，导致需要至少一种，有时 多次角膜移植。因此需要新的抗真菌药物，但它们的开发需要 更好地了解可以作为药物靶点的真菌蛋白质/酶。由于角膜 基质是有效的细胞外基质，由胶原蛋白和其他蛋白质组成，我们假设 支持真菌蛋白催化剂的途径对眼睛中的真菌生长至关重要， 延伸，毒力因子，可能是治疗的目标。 为了使真菌利用蛋白质作为营养源，它们必须首先分泌大量的蛋白质。 蛋白酶进入环境/宿主组织。这种分泌负担导致了 内质网（ER）内未折叠的蛋白质，如果不解决，会导致内质网的“堵塞”。 分泌途径，将严重抑制真菌生长。未折叠蛋白质反应（UPR）是一种免疫反应。 在这方面的关键作用，首先传感未折叠的蛋白质，随后调节基因， 促进ER腔内蛋白质折叠能力（例如，伴侣）。在目标1中，我们将测试 假设真菌UPR促进角膜炎的常见病原体的角膜发病， 茄病镰刀菌我们将首先产生F. solani然后测试是否 正如我们预测的那样，突变体在蛋白质底物上的生长是有缺陷的。然后我们会评估 在真菌性角膜炎小鼠模型中的突变体。观察到UPR缺陷型菌株 低毒性表明UPR的抑制剂可用作新的抗真菌剂。 真菌的转录谱随着营养源的变化而变化很大。过渡 例如，从富含葡萄糖的培养基到限制葡萄糖的培养基，导致糖酵解基因的下调， 分泌的水解酶的上调，以及参与氨基酸合成的代谢酶的上调。 新陈代谢.因此，在目标2中，我们将检验F. solani利用蛋白质在 通过比较体内真菌的转录组（来自受感染的眼睛）与 在体外确定的营养条件（胶原蛋白对葡萄糖）下生长的真菌的转录组。我们 预测体内最高表达的基因将反映体内最高表达的基因。 胶原然而，我们并不期望两个数据集之间存在一一对应关系， 眼睛所特有的环境条件，例如由炎性炎症引起的应力， 反应通过这种方式，我们将获得对角膜炎中真菌适应性反应的新见解 感染，这将导致新的毒力基因和推定的药物靶点的鉴定。

Project Summary/Abstract Fungal keratitis is an important source of ocular morbidity and unilateral blindness worldwide. Current antifungal regimes fails in up to 60% of patients, resulting in the need for at least one and sometimes repeated corneal transplants. Novel antifungals are therefore required, but their development requires a better understanding of fungal proteins/enzymes that could serve as drug targets. As the corneal stroma is effectively an extracellular matrix comprised of collagen and other proteins, we hypothesize that pathways that support fungal protein catabolism are essential for fungal growth in the eye and, by extension, virulence factors that might be targeted in treatment. In order for fungi to utilize proteins as a nutritional source, they must first secrete copious amounts of proteases into the environment/host tissue. This secretory burden leads to an accumulation of unfolded proteins within the endoplasmic reticulum (ER) that, if not resolved, leads to a “clogging” of the secretion pathway that will severely inhibit fungal growth. The unfolded protein response (UPR) plays a critical role in this regard by first sensing unfolded proteins and subsequently regulating genes that that promote the protein folding capacity within the ER lumen (e.g., chaperones). In Aim 1, we will test the hypothesis that fungal UPR promotes the corneal pathogenesis of a common agent of keratitis, Fusarium solani. We will first generate UPR-deficient mutants of F. solani and then test whether the mutants are defective for growth on protein substrates as we predict. We will then assess the virulence of the mutants in a mouse model of fungal keratitis. The observation that the UPR-deficient strains are hypovirulent would suggest that inhibitors of the UPR could be used as novel antifungals. The transcriptional profile of a fungus varies largely as a function of the nutrient source. The transition from glucose-rich to glucose-limiting media, for example, leads to a down-regulation of glycolytic genes, upregulation of secreted hydrolases, and an upregulation of metabolic enzymes involved in amino acid metabolism. Therefore, in Aim 2, we will test the hypothesis that the F. solani utilizes proteins in the cornea by comparing the transcriptome of the fungus in vivo (from infected eyes) against the transcriptome of the fungus grown under defined nutrient conditions (collagen v. glucose) in vitro. We predict that the most highly expressed genes in vivo will mirror the most highly expressed genes on collagen. However, we do not expect a one-to-one correspondence between the two datasets due to environmental conditions that are unique to the eye, such as stresses imparted by the inflammatory response. In this way, we stand to gain novel insight into the fungal adaptive response during keratitis infection, which will lead to the identification of novel virulence genes and putative drug targets.