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

白鼻综合征（WNS）是一种毁灭性的疾病，自2007年被发现以来，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，并有可能导致几种本地蝙蝠物种灭绝。除了生态破坏外，预计这种疾病还会严重影响农业，因为食虫蝙蝠对控制害虫至关重要。WNS的病因是真菌Pseudogymnoascus destructans，这种真菌具有很强的侵入性，会对组织造成严重的破坏，尤其是对蝙蝠的膜状翅膀。人们对这种真菌的生物学特性知之甚少，也不知道它是如何引起蝙蝠疾病的。本项目将重点鉴定和表征蛋白酶，这是蛋白质降解的催化剂，由P. destructans分泌，可能促进蝙蝠组织的入侵和破坏。特别是，蛋白酶的存在将被评估，因为它们代表了几种哺乳动物真菌病原体的重要毒力因素。因此，阻断蛋白酶活性可能是预防WNS的一种治疗策略。本提案中使用的方法将首先定义在实验室培养过程中由P. destructans分泌的蛋白质，特别强调鉴定分泌的蛋白酶。最先进的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，结合冰岛分析来揭示分泌蛋白酶的裂解特异性。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和部分表征了一种新的枯草杆菌样蛋白酶。这种活性被命名为Destructin-1，因为它能够降解胶原蛋白，胶原蛋白是动物结缔组织的主要成分。将在P. destructans中鉴定更多的蛋白酶，并将其与非感染性物种进行比较，以确定潜在的毒力因素。为了测试这些蛋白酶是否与蝙蝠的疾病有关，将定义蛋白酶抑制剂，并在感染模型中测试这些抑制剂，以观察它们是否阻断WNS的进展。新的遗传工具也将为这种真菌物种开发，以便通过删除目标基因进行分析。总之，这些方法将为P. destructans真菌的生物学及其在哺乳动物宿主中引起疾病的手段提供新的见解。

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.