Characterization of the Secretome of P. Destructans, the Causative Agent of White-Nose Syndrome in Bats

蝙蝠白鼻综合症的病原体破坏性 P. Destructans 的分泌组特征

• 批准号: 1456787
• 负责人: Richard Bennett
• 金额: $ 50万
• 依托单位: Brown University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1456787&HistoricalAwards=false
• 关键词: Characterization; Secretome; P.; Destructans; Causative

White-Nose Syndrome (WNS) is a devastating disease that has killed around 6 million bats in the US since its discovery in 2007. The disease has spread across North America and has the potential to drive several native bat species to extinction. In addition to ecological damage, the disease is predicted to seriously impact the agricultural industry, as insectivorous bats are critical for the control of insect pests. The cause of WNS is the fungus Pseudogymnoascus destructans, which is highly invasive causing significant tissue destruction particularly to the membranous wings of bats. Little else is known about the biology of this fungal species or how it is able to cause disease in bats. This project will focus on identification and characterization of protease enzymes, which are catalysts in protein degradation, secreted by P. destructans that may promote the invasion and destruction of bat tissue. In particular, the presence of proteases will be evaluated as these represent important virulence factors in several mammalian fungal pathogens. The blocking of protease activities may therefore represent a therapeutic strategy for the prevention of WNS.The approach used in this proposal will first define the proteins secreted by P. destructans during laboratory culture, with a particular emphasis on identifying secreted proteases. Cutting-edge approaches will be used for the identification and subsequent characterization of proteases, including a multiplex substrate profiling technique coupled with iceLogo analysis to reveal the cleavage specificity of secreted proteases. Preliminary experiments using this approach have led to the identification and partial characterization of a novel subtilisin-like protease from the conditioned medium of P. destructans. This activity has been named Destructin-1 as it is able to degrade collagen, the major component of animal connective tissue. Additional proteases will be identified in P. destructans and compared to those in non-infectious species to identify potential virulence factors. To test if these proteases contribute to disease in the bat, inhibitors of proteases will be defined and these inhibitors tested in infection models to see if they block the progression of WNS. New genetic tools will also be developed for this fungal species to enable analysis by deletion of target genes. Together, these approaches will provide new insights into the biology of the P. destructans fungus and the means by which it causes disease in the mammalian host.

白鼻综合症 (WNS) 是一种毁灭性的疾病，自 2007 年发现以来，已导致美国约 600 万只蝙蝠死亡。这种疾病已蔓延到整个北美，并有可能导致多种本土蝙蝠物种灭绝。 除了生态破坏之外，预计该疾病还将严重影响农业，因为食虫蝙蝠对于控制害虫至关重要。 WNS 的病因是真菌 Pseudogymnoascus destructans，这种真菌具有高度侵入性，会导致严重的组织破坏，尤其是对蝙蝠的膜翅。 人们对这种真菌的生物学特性以及它如何引起蝙蝠疾病知之甚少。 该项目将重点鉴定和表征蛋白酶，这些酶是蛋白质降解的催化剂，由破坏性 P. destructans 分泌，可能促进蝙蝠组织的入侵和破坏。 特别是，将评估蛋白酶的存在，因为它们代表了几种哺乳动物真菌病原体中的重要毒力因子。 因此，阻断蛋白酶活性可能代表一种预防 WNS 的治疗策略。本提案中使用的方法将首先定义实验室培养过程中破坏性假单胞菌分泌的蛋白质，特别强调鉴定分泌的蛋白酶。 尖端方法将用于蛋白酶的鉴定和后续表征，包括多重底物分析技术与iceLogo分析相结合，以揭示分泌蛋白酶的切割特异性。 使用这种方法的初步实验已经从 P. destructans 的条件培养基中鉴定并部分表征了一种新型枯草杆菌蛋白酶。 这种活性被命名为 Destructin-1，因为它能够降解动物结缔组织的主要成分胶原蛋白。 将在破坏性假单胞菌中鉴定出其他蛋白酶，并将其与非感染性物种中的蛋白酶进行比较，以确定潜在的毒力因子。为了测试这些蛋白酶是否会导致蝙蝠疾病，我们将定义蛋白酶抑制剂，并在感染模型中测试这些抑制剂，看看它们是否能阻止 WNS 的进展。 还将针对这种真菌物种开发新的遗传工具，以便通过删除目标基因进行分析。 总之，这些方法将为了解 P. destructans 真菌的生物学及其在哺乳动物宿主中引起疾病​​的方式提供新的见解。