Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans

White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.白鼻综合征（White nose syndrome, WNS）是一种侵袭蝙蝠的皮肤真菌病，可导致北美洞穴蝙蝠种群出现前所未有的大规模死亡。目前针对可能参与WNS宿主-病原体互作的酶活性的研究较少，且尚无研究分离并描述过分泌型蛋白酶。为验证破坏性假裸囊菌（Pseudogymnoascus destructans）在WNS感染过程中通过分泌细胞外蛋白酶引发翅膀坏死的假说，本研究以该菌为对象：先在多种培养基上进行培养，随后分离并结构鉴定培养基中稳定积累的蛋白酶。我们在添加了蛋白质底物的基础营养肉汤中检测到单一主要的蛋白酶活性，且该活性可被苯甲基磺酰氟（phenylmethylsulfonyl fluoride）强烈抑制。通过制备型等电聚焦与伴刀豆球蛋白A凝集素亲和层析，我们分离得到了该破坏性假裸囊菌丝氨酸蛋白酶（PdSP1）。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估算，PdSP1的分子量为27900，其最适pH范围为6~8，最适温度为60℃。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）、轨道阱串联质谱（Orbitrap MS/MS）以及埃德曼N端肽测序对PdSP1进行结构表征后发现，其与已测序的破坏性假裸囊菌基因组中的一段假设蛋白登录序列完全匹配，该序列被进一步鉴定为MEROPS家族S8A类枯草杆菌蛋白酶型丝氨酸肽酶。在破坏性假裸囊菌基因组中还鉴定出另外两种同工型：PdSP2与PdSP3，二者的同源性分别为90%与53%。相较于人类致病皮肤癣菌，破坏性假裸囊菌的S8A类丝氨酸蛋白酶与蠕形假裸囊菌（P. pannorum）及植物病原真菌的序列保守性更高。基于PdSP1序列制备的肽特异性多克隆抗体可在蛋白质免疫印迹（Western blot）中检测到该蛋白。这类枯草杆菌蛋白酶型丝氨酸蛋白酶可作为后续WNS宿主-病原体互作功能研究的候选靶点。