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。这种疾病已经蔓延到整个北美，有可能导致几种本土蝙蝠物种灭绝。除了对生态的破坏外，这种疾病预计还会严重影响农业，因为食虫蝙蝠对控制害虫至关重要。WNS的病因是破坏假淋巴曲霉的真菌，这种真菌具有高度侵袭性，导致组织严重破坏，特别是对蝙蝠的膜状翅膀造成破坏。关于这种真菌物种的生物学或它如何能在蝙蝠身上引起疾病，其他方面知之甚少。该项目将集中在鉴定和鉴定蛋白酶，这是蛋白质降解的催化剂，由破坏性巴氏杆菌分泌，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，因为这些酶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了一种预防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期间破坏性巴氏杆菌分泌的蛋白质，重点是鉴定分泌的蛋白酶。将使用最先进的方法来鉴定和随后鉴定蛋白酶，包括结合ICOLOGO分析的多重底物图谱技术，以揭示分泌的蛋白酶的切割特异性。利用这种方法进行的初步实验已经从破坏假单胞菌的条件培养基中鉴定出一种新的类枯草杆菌蛋白水解酶，并对其进行了部分性质的研究。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。此外，还将在破坏性巴氏杆菌中鉴定更多的蛋白水解酶，并将其与非传染性物种中的酶进行比较，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的疾病，将定义蛋白酶的抑制剂，并在感染模型中测试这些抑制剂，看看它们是否阻止WNS的进展。还将为这种真菌物种开发新的遗传工具，以实现通过删除目标基因进行分析。总而言之，这些方法将为了解破坏性巴氏杆菌真菌的生物学以及它在哺乳动物宿主中致病的方式提供新的见解。