马尔尼菲青霉DOPA黑素生成的基因调控及靶向干预研究

The yeast cells of Penicillium marneffei that can survive within the host macrophages are essential to enable the fungus to produce invasive infection. Currently, P.marneffei infection is becoming an indicator disease of AIDS in the endemic regions.The commonly used antifungal agents are difficult to completely kill the pathogen survive in macrophages. Our previous research confirmed that yeast cells of P. marneffei can synthesize DOPA melanin, the melanized yeast cells increased resistance to macrophage phagocytosis and decreased susceptibility to amphotericin B. The research is going to clarify the mechanisms of gene regulation on P.marneffei DOPA-melanin synthesis and the effects of melanin targeted intervention on macrophages in clearing the yeast cells. Construction of double-stranded RNA interference vector on P. marneffei tyrosinase gene will carry out mediated by Agrobacterium tumefaciens, and the phenotype and virulence of P. marneffei with RNA interference vector in comparison with wild-type P. marneffei will be evaluated through vitro experiments and vivo animal experiments. Our previous study showed tha kojic acid had the ability to suppress melanin synthesis of P. marneffei yeast cells.It is worth to assess whether kojic acid be able to increase the ability for macrophages in clearing the yeast cells，and whether kojic acid combined with amphotericin B have synergic interaction of antifungal activity against P. marneffei isolates. After preparation of the monoclonal antibody against P.marneffei melanin / Amphotericin B immuno-liposomes, the targeted killing effect of immuno-liposome to P. marneffei yeast cells will also be evaluated in vitro experiments and in vivo animal experiments. The research expect to clarify the role of tyrosinase gene in P.marneffei DOPA-melanin synthesis and to uncover the potential drug targets in the pathway of the fungus melanin synthesis for treatment of P.marneffei infection.

马尔尼菲青霉(PM)酵母相孢子能在宿主的巨噬细胞内存活并导致侵袭性感染，常用的抗真菌药物难以彻底杀灭在巨噬细胞内寄存的PM孢子。我们前期的研究证实PM酵母相孢子可以合成DOPA黑素，黑素化的PM孢子抵抗巨噬细胞吞噬能力增强和对两性霉素B敏感性下降。为进一步阐明PM的DOPA黑素生成的基因调控机制和靶向干预黑素生成对巨噬细胞清除PM孢子的作用，本研究拟通过根癌农杆菌介导的双链RNA 干扰技术构建PM酪氨酸酶基因RNA干扰载体，观测分析RNA干扰载体对PM表型和毒力的影响；体外实验检测曲酸是否能促进巨噬细胞清除孢子以及曲酸和两性霉素B对PM是否有协同抗真菌作用；制备PM黑素单克隆抗体/两性霉素B免疫脂质体，从体外实验和体内动物实验评价免疫脂质体靶向杀灭PM的效应。明确酪氨酸酶基因在PM的DOPA黑素生成中的作用，发掘PM黑素生成途径中可以作为潜在的治疗PM感染的药物靶点。

马尔尼菲蓝状菌(TM)能在宿主的巨噬细胞内存活并导致侵袭性感染，常用的抗真菌药物难以彻底杀灭在巨噬细胞内寄存的TM。我们前期的研究证实酵母相TM可以合成DOPA黑素，黑素化的TM抵抗巨噬细胞吞噬能力增强和对两性霉素B(AMB)敏感性下降。为进一步阐明TM的DOPA黑素生成的调控机制,本研究从TM的黑素生成与酪氨酸酶（TYR）基因的关系、相关蛋白组调控通路、靶向抑制剂对黑素生成途径的作用等方面进行探索。TM合成黑素过程中TYR基因表达显著升高，TM的黑素生成与TYR基因关联；蛋白组学分析显示TM进行DOPA黑素化后有显著的38个差异点，功能富集分析生物通路富集分析发现热休克蛋白(HSP)家族显著富集在TM重要生物代谢途径中。针对酪氨酸酶调控黑素生成的途径，采用曲酸靶向抑制TM的黑素生成，曲酸增加TM对AMB的敏感性；针对HSP调控TM黑素生成途径，采用HSP90抑制剂格尔德霉素（GDA）靶向抑制HSP90活性，透射电镜观察见TM细胞壁明显变薄、细胞内吞噬体减少和电子致密颗粒减少，培养基肉眼观察见TM黑素生成明显减少，药敏检测显示GDA增加TM对AMB的敏感性。TM相关蛋白组分析显示DOPA黑素化后细胞内吞噬体生物学通路显著增强，提示黑素化TM抗氧化能力增强。我们同步的研究发现在TM感染巨噬细胞过程中酵母相TM能诱导巨噬细胞极化，这些结果和DOPA黑素化后TM细胞内吞噬体生物学通路显著增强互相吻合，进一步阐释TM抵抗巨噬细胞氧化杀伤的机制。本研究明确TM黑素生成与TYR基因相关，首次发现HSP调控通路与TM黑素生成相关，HSP抑制剂能有效抑制TM黑素生成和影响酵母相TM细胞结构，发掘出TM黑素生成途径中可以作为潜在的治疗TM感染的药物靶点。