Elucidating pathways that regulate fungal keratitis pathogenesis

项目摘要/摘要 真菌性角膜炎是世界范围内眼部疾病和单眼失明的重要来源。当前 高达60%的患者抗真菌治疗失败，导致至少需要一种，有时甚至是 反复的角膜移植。因此需要新的抗真菌药物，但它们的开发需要 更好地了解可作为药物靶标的真菌蛋白质/酶。作为角膜 我们推测，间质实际上是一种由胶原和其他蛋白质组成的细胞外基质 支持真菌蛋白质分解代谢的途径对眼睛中真菌的生长是必不可少的，通过 延伸，毒力因子，可能是治疗的靶点。 为了让真菌利用蛋白质作为营养来源，它们必须首先分泌大量的 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 会严重抑制真菌生长的分泌途径。未折叠蛋白反应(UPR)发挥着重要作用 首先感知未折叠的蛋白质，然后调节基因，从而在这方面发挥关键作用 促进内质网管腔内的蛋白质折叠能力(例如，伴侣)。在目标1中，我们将测试 假设真菌UPR促进一种常见角膜炎病原体的角膜发病， 茄病镰刀菌。我们将首先产生缺乏UPR的Solani突变体，然后测试 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。然后我们将评估病毒的毒性 在真菌性角膜炎的小鼠模型中的突变。观察到UPR缺陷菌株是 低毒力表明UPR的抑制剂可能被用作新型抗真菌药物。 真菌的转录图谱随着营养来源的不同而有很大的不同。过渡时期 例如，从富含葡萄糖的介质到限制葡萄糖的介质，会导致糖酵解基因的下调， 分泌型水解酶和涉及氨基酸的代谢酶上调 新陈代谢。因此，在目标2中，我们将检验这样的假设，即立枯丝核菌利用 通过比较体内真菌的转录组(来自受感染的眼睛)与 在特定营养条件(胶原与葡萄糖)下生长的真菌的体外转录组。我们 预测体内最高表达的基因将反映出最高表达的基因 胶原蛋白。但是，我们并不期望两个数据集之间存在一对一的对应关系，因为 眼睛所特有的环境条件，如炎症所带来的压力 回应。通过这种方式，我们将获得对角膜炎期间真菌适应性反应的新见解。 感染，这将导致识别新的毒力基因和假定的药物靶标。

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.