聚酮体合成酶介导的黑素合成在疣状瓶霉毒力和致病中的作用

黑素是致病真菌的代谢产物，与真菌的形态、毒力和抗药性有关。疣状瓶霉在免疫正常及免疫缺陷者可引起着色芽生菌病、暗色丝孢霉病、足菌肿等皮下或播散性感染。申请者所在课题组基于分类学明确了疣状瓶霉复合体内7个种的特异性及致病力差异。申请者前期对复合体内8个临床株和环境株行全基因组测序，对序列作了注释，对它们的分子差异与致病性作了相关性分析，发现真菌黑素合成的关键酶聚酮体合成酶基因pks与致病性有关。本项目拟用CRISPR/Cas9基因编辑方法敲除疣状瓶霉的pks基因，用只转入Cas9的菌株作为对照，体外观察△pks株形态、药物敏感性的改变和对巨噬细胞功能的影响；用野生型和card9-/-小鼠的感染模型检测其生存率、病变程度、吞噬和杀伤力、以及诱导产生细胞因子能力的改变。所得结果将有助于明确pks介导的黑素合成在疣状瓶霉致病中的作用，进一步阐明真菌感染的发病机制；也为开发新型的抗真菌药提供新思路。

Melanin is a metabolic product of pathogenic fungi, which is closely related to the morphology, virulence and drug resistance of fungi. Phialophora verrucosa can cause subcutaneous or disseminated infections such as chromoblastomycosis, phaeohyphomycosis and mycetoma in both immunocompetent and deficient hosts.Multilocus analysis revealed the existence of seven species apparently having different infectious potentials in the Phialophora verrucosa complex. The applicant completed genome sequencing of 8 clinical and environmental strains in the complex, annotated the sequences, and analyzed the molecular differences and pathogenicity. It was found that the key enzyme of melanin biosynthesis, polyketide synthase(pks), was related to pathogenicity. This project intends to use CRISPR/Cas9 gene editing method to knock out the pks gene of Phialophora verrucosa. The morphology, drug resistance and macrophage function of △pks strain were observed in vitro, and the survival rate, pathological changes, phagocytosis and killing ability, as well as the ability to induce cytokines were detected by wild-type and card9-/-mice infection models. The results will help to clarify the role of melanin synthesis in the pathogenesis of Phialophora verrucosa, further elucidate the pathogenesis of fungal infection, and provide new ideas for developing new antifungal drugs.