在铜胁迫下活性氧介导的抑制新生隐球菌机制研究

Cryptococcus neoformans is one of the three deadly infectious pathogenic fungi. There are about 60,0000 people die of pneumonia and meningoencephalitis caused Cryptococcus neoformans infection each year. Recent studies have demonstrated that lung tissue used copper toxicity as natural immune strategy against the invasive fungus, but the antifungal mechanism remains unclear. Preliminary experiment suggested that copper toxicity may implement its function by improving intracellular ROS level, however, the molecular mechanism remained to be further defined. This study intended to verify the key relationship between copper toxicity and ROS metabolize, using metallothionein knockout strain cmt1/2ΔΔ as copper sensitive model. Furthermore, the metabolic pathways regulated by ROS under copper stress will be revealed, using quantitative proteomics and bioinformatics methods. And on this basis, the antifungal effect of targeted pathway will be detected by means of activating or repressing them. Finally, based on the mass spectrum data, the target proteins will be identified by constructing gene knockout strains in cmt1/2ΔΔ to verify their function and impact on virulence, both in vitro and in vivo. This study will reveal antifungal mechanisms mediated by activated ROS under copper stress from both metabolic pathways and the target proteins, and provide the theoretical foundation for Cryptococcus neoformans disease diagnosis and new antifungal drugs exploration.

新生隐球菌是人类三大致病真菌之一，每年约有60万人死于其感染引发的肺炎以及脑膜脑炎。宿主肺组织可利用铜胁迫天然免疫策略抵御病原体侵袭，然而机制尚不明确。前期工作证明，铜胁迫可能是通过提高胞内活性氧含量介导抑菌的，分子机制有待进一步明确。本研究拟以金属硫蛋白敲除菌株cmt1/2ΔΔ为铜离子敏感模型，通过调节新生隐球菌胞内ROS含量，验证铜胁迫抑菌与活性氧代谢之间的关联性。其次，利用蛋白组定量及生物信息学分析技术，挖掘铜胁迫下由活性氧介导的抑菌信号通路，并从整体水平抑制或激活目标通路，研究其在铜胁迫抑菌中的作用。最后，筛选蛋白组数据中显著差异蛋白，在cmt1/2ΔΔ菌株基础上构建目标蛋白敲除菌株，从体内体外两方面研究目标蛋白的功能及其对致病毒力的影响。本研究将从信号通路及目标蛋白两个层面，深入研究铜胁迫下活性氧介导的抑菌机制，以期为新生隐球菌感染疾病的诊断以及新型抗真菌药物的开发提供理论基础。

新生隐球菌是一种侵袭性的人类真菌病原体，每年造成超过18.1万人死亡。研究表明，肺部新生隐球菌感染可诱导铜代谢调控的先天免疫反应，而新生隐球菌的维持铜稳态能力可提高其在肺部感染中的适应性和致病性。然而，铜抑制新生隐球菌增殖的分子机制尚不清楚。我们使用对铜高度敏感的金属硫蛋白双敲除突变体cmt1/2ΔΔ，证明外源铜离子通过诱导活性氧生成抑制真菌细胞的生长。通过液相色谱-串联质谱分析，我们发现铜下调参与蛋白质翻译的蛋白，同时上调参与泛素介导的蛋白质降解的蛋白。我们认为铜抑菌的主要效应是下调蛋白合成和上调蛋白降解。铜胁迫促进了新生隐球菌总蛋白泛素修饰，并提高了蛋白酶体活性。抑制蛋白酶体途径可减轻铜的抑菌作用。我们的蛋白质组学分析为了解铜的抗真菌机制提供了新角度。