去泛素化酶Ubp5通过网格蛋白调控新生隐球菌生长与毒力的分子机制

Production and regulation of virulence factor complex is essential for Cryptococcal pathogenicity, and thus become a hot spot at present. Our previous studies demonstrated that deubiquitinase Ubp5 played pleiotropic roles in virulence factors production (such as capsule and melanin), stress response, sexual reproduction and also infectivity in Cryptococcus neoformans, which might develop into an alternative or synergistic antifungal target. However, its molecular regulatory mechanism remains to be further explored. On the basis of preliminary work (Yeast-two-hybridization test), we further hypothesized that deubiquitinase Ubp5 may depend on clathrin mediated vesicle transport pathway to control the growth and pathogenesis of C. neoformans. To verify the above hypothesis, we try to explore the interaction between Ubp5 and clathrin via Co-Immunoprecipitation and fluorescent colocalization, and to do functional genomic analyses about clathrin pathway combined with the utilization of in-vitro phenotypes assays, phagocytosis assay, vertebrate and non-vertebrate animal models in this study. This study will provide new thoughts and theoretical basis for exploring novel antifungal strategies and finally overcoming clinical mycoses.

毒力因子复合体的表达与调控是新生隐球菌致病感染的中心环节，已成为当前学界的研究热点。我们前期研究发现，去泛素化酶Ubp5对隐球菌的毒性因子表达（荚膜、黑色素）、应激应答、有性繁殖及致病力等具有多效调控作用，具备成为新型或协同抗隐球菌治疗靶点的潜质，其内在的分子调控机制亟待进一步明确。在此基础上结合前期酵母双杂交结果，我们进一步提出：去泛素化酶Ubp5可能依赖网格蛋白介导的囊泡转运途径对隐球菌的生长繁殖与致病感染发挥调控作用。为证实以上假说，本研究拟应用免疫共沉淀和荧光共定位进一步探讨Ubp5对网格蛋白的调节作用关系，通过基因敲除与回复突变，结合体外表型检测、细胞吞噬与动物感染模型研究，阐明网格蛋白相关途径在Ubp5调控隐球菌生长与代谢中的功能机制，初步论证去泛素化酶Ubp5作为抗隐球菌药物干预靶点的理论可行性，为最终攻克临床真菌感染提供新的研究思路。

毒力因子复合体的表达与调控是新生隐球菌致病感染的中心环节，是当前学界的研究热点。去泛素化酶Ubp5对隐球菌的生长繁殖、应激应答及致病感染等具有多效调控作用，其潜在的调控机制尚不甚明确。本研究首次证实去泛素化酶Ubp5参与调节隐球菌在宿主体内的肺部增殖与血源性播散过程，其调控机制可能与其介导宿主非保护性的Th2极化方向与巨噬细胞M2激活模式密切相关。格特隐球菌Ubp5缺失后，毒力同样显著降低，但黑素和荚膜表达显著增强，提示Ubp5在不同隐球菌菌种间存在进化上的保守性与变异性。酵母双杂交与免疫共沉淀实验提示Ubp5存在庞大的蛋白作用网络，与应激应答、能量代谢、蛋白质控、核糖体发生等多种生物学过程密切相关。Ubp5重要靶向蛋白泛素前体分子Ubi1缺失后，新生隐球菌生长显著受抑、应激耐受力显著降低，细胞微观形态显著改变，致病力显著降低，其作用机制可能与核糖体发生缺陷密切相关，Ubi1可能参与调控隐球菌在宿主体内的慢性感染。我们的研究系统论证了去泛素化酶Ubp5蛋白途径对隐球菌生长增殖、致病感染及免疫逃逸过程中的重要作用，为后续探索新的隐球菌临床干预靶点奠定了基础。