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年发现以来，已经在美国杀死了大约600万只蝙蝠。 这种疾病已经蔓延到整个北美，并有可能使几种本土蝙蝠物种灭绝。 除了生态破坏外，预计这种疾病还将严重影响农业，因为食虫蝙蝠对控制害虫至关重要。 WNS的原因是真菌Pseudogymnocellulodestructans，这是高度侵入性的造成重大组织破坏，特别是对蝙蝠的膜翼。 人们对这种真菌物种的生物学特性以及它如何导致蝙蝠疾病知之甚少。 该项目将侧重于蛋白酶的鉴定和表征，蛋白酶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，将评估蛋白酶的存在，因为这些蛋白酶代表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预防WNS的治疗策略。本提案中使用的方法将首先确定实验室培养期间由破坏性变形杆菌分泌的蛋白质，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随后的表征，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导致了一种新的枯草杆菌蛋白酶样蛋白酶的鉴定和部分表征的条件培养基的破坏变形杆菌。 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。 将在破坏变形杆菌中鉴定其他蛋白酶，并与非感染性种属中的蛋白酶进行比较，以鉴定潜在的毒力因子。为了测试这些蛋白酶是否有助于蝙蝠的疾病，将定义蛋白酶抑制剂，并在感染模型中测试这些抑制剂，以查看它们是否阻止WNS的进展。 还将为这种真菌物种开发新的遗传工具，以便通过删除靶基因进行分析。 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方式提供新的见解。

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.