申克孢子丝菌形态转换的多基因调控机制及致病性形成的相关研究

The morbidity with sporotrichosis increases constantly, as well as disseminated systemic infection. Sporothrix schenckii is a dimorphic pathogenic fungus with the pathogenic yeast-phase. Dimorphic transformation is one of the most important factors of infection, which results from complicated multi-gene interactions. The dimorphism reflects a developmental switch in morphology and lifestyle that is necessary for pathogenesis. The T-DNA insertional mutant library of Sporothrix schenckii has been established. Several morphological mutants were received. Various methods were applied to construct knock-out and complementary strains. And associated biological characteristics and pathogenesis were primarily studied. In this study, Sporothrix schenckii T-DNA insertional mutants, knock-out strains, complementary strains, overexpression strains, RNAi mutants, and heterologous expression strains should be used to investigate key genes and multi-gene regulatory networks for dimorphic transformation, with the several methods of transcriptomics, reversed genetics, bioinformatics and molecular biology. Co-incubation of Sporothrix schenckii with HaCaT cells, tissue-engineered human skin substitutes and murine disseminated sporotrichosis infection model should be established to study dimorphic switching and its role in pathogenicity. Our results could have potential advantages for systemic analysis the correlation of dimorphism and pathogenicity. These would be useful for prevention and control sporotrichosis and other dimorphic fungal infection.

孢子丝菌病发病率不断增高，全身感染亦呈上升趋势。病原菌申克孢子丝菌是一种双相型真菌，其酵母相有致病性。形态转换是感染发生的重要因素，是复杂的多基因共同作用的结果，但其调控机制尚不明确，与致病性形成的关系亦需深入探讨。本项目组已构建申克孢子丝菌T-DNA插入突变体库，获得若干形态转换相关突变体；利用多种技术构建了基因敲除株和回复突变株，并对其生物学性状和致病性进行了初步研究。本项目拟利用转录组学、反向遗传学、生物信息学及分子生物学等手段，以申克孢子丝菌T-DNA插入突变株、基因敲除株、回复突变株、过表达株、RNAi突变株、异源表达株为材料，探讨形态转换关键基因、作用方式及信号转导通路的调控机制；利用菌株与HaCaT细胞共孵育体系、组织工程皮肤真菌感染模型及小鼠感染模型，探讨形态转换与致病性形成的关系。为申克孢子丝菌及其它双相型真菌的形态转换、致病机制、感染防治等研究提供理论依据和技术支撑。

申克孢子丝菌是一种双相型真菌，其酵母相具有致病性。该菌可引起免疫受损人群严重的皮肤外播散性及全身感染。温度依赖性形态转换是二相型真菌能够适应宿主新环境生存的关键，是复杂的、多基因相互作用的结果，然而目前对其分子遗传基础及与宿主的关系尚不清晰。.本课题以分离自临床播散性肺孢子丝菌病患者标本的申克孢子丝菌高毒力菌株为研究对象，对其菌丝相和酵母相进行了二相型转录组测序分析。结果表明两者存在显著差异，这些差异表达基因影响着压力应激、生长发育、信号调控及粘附定植等重要的生物学过程，进而调控菌株的二相型转换及致病性。.进一步扩大申克孢子丝菌T-DNA插入突变体库，筛选出形态及二相型转换异常突变体，并对T-DNA打断基因进行了生物信息学分析，发现与糖代谢、有性生殖、离子转运及信号转导等功能相关。结合二相型转录组数据及T-DNA插入突变基因信息，解析了MAPK信号转导通路的Stpk和Ste20基因、铜离子转运蛋白编码基因Ctp、泛素结合酶编码基因E2h、Ras/cAMP信号通路的Ras-Gap基因功能，发现这些基因可菌株的影响生长繁殖、合成代谢、二相型转换及毒力产生，与致病性形成密切相关。.本课题研究结果加深了对申克孢子丝菌二相型转换及毒力产生和调控的认知，为揭示该菌的致病机制奠定有力的基础，为制定防控孢子丝菌病的有效策略提供依据，亦可为其他二相型真菌的相关研究提供参考。