曲霉中钙信号通路参与的唑类药物多药耐药的分子调控机制

Over the past decades, the incidence of invasive fungal infections has increased, causing serious systemic disease in immunocompromised patients, especially those caused by Aspergillus species. Up to date, azoles are widely applied and largely effective; however, they are generally fungistatic and clinically resistant isolates are emerging increasingly.The main reason for drug resistance produced by fungi is the mutation or the over-expression of multidrug efflux pumps. Thus, most of previous studies have focused to search for new drug targets or obtain new drugs to solve this problem. However, the studies about how the main player-Pleiotropic drug resistance protein was controlled by its regulators during this process are limited. In this project, based on our previous finding that synergistic activity of azoles with calcium chelators EGTA against Aspergillus nidulans and Aspergillus fumigatus, we proposed that Ca2+/calmodulin/calcineurin complex was involved in the stress-response reactions. To further completely understand the regulation mechanism of efflux pumps in Pleiotropic drug resistance activity, by using calcium chelators or inhibitors combined with activated or inactivated mutations in calcium signalling pathway, we will analysis the components of complex in this calcium signalling pathway including Ca2+-transporter,Ca2+-channels and their interaction proteins,accompanied with its transcription factors or partners during responses to azole treatment in filamentous fungi Aspergillus. Especially, We will focus on the new regulator candidates which involved in regulation of drug-efflux-pump activity by inducing calcium homeostasis. Moreover, on the basis of results from this study, we proposed the finding for this project will insight in new solution for fighting for drug resistance.

很多临床上分离的多药耐药菌株中麦角固醇合成途径和药泵活力并无组成性的改变，真菌高度耐药的特征仅暴露在药物刺激下才诱导性地发生。因此, “药物逆境刺激而使真菌细胞激活药物逆境响应从而引起的耐药性”这条途径在临床上更需要引起关注。预实验结果表明采用钙信号通路阻断剂或钙信号蛋白或其转录因子的缺失菌株，曲霉表现出对唑类药物超敏感，表明钙信号系统参与曲霉对唑类药物的耐受性。本课题将从真菌感染的临床病人中收集曲霉耐药菌株，同时通过药物诱导和农杆菌T-DNA插入法筛选曲霉耐药突变株并采用非标记质谱定量比较耐药菌株和敏感菌株的蛋白质表达的差异；探索钙信号通路或其修饰、互作蛋白对于产生耐药的直接参与者-药物外排泵和药靶麦角固醇的合成的影响，阐明临床菌株多药耐药性与钙信号系统中功能基因的关系。探讨联合唑类药和钙信号通路阻断剂来改变耐药特征，为控制真菌的耐药性和改进现有治疗策略提供新思路。

目前认为条件致病菌烟曲霉是对于免疫缺陷病人具有高致死率的主要致病菌之一。临床上分离的病原真菌烟曲霉越来越多地表现出耐药特征，但是有些耐药菌株中麦角固醇合成途径和药泵活力并无组成性的改变，因此认为这些真菌高度耐药的特征可能仅暴露在药物刺激下才诱导性地发生，“药物逆境刺激而使真菌细胞激活药物逆境响应从而引起的耐药性”这条途径在临床上更需要引起关注。预实验结果表明采用钙信号通路阻断剂或钙信号蛋白或其转录因子的缺失菌株，曲霉表现出对唑类药物超敏感，表明钙信号系统参与曲霉对唑类药物的耐受性。本项目从真菌感染的临床病人中收集曲霉耐药菌株，同时通过实验室诱导曲霉耐药突变株探索钙信号通路或其修饰、互作蛋白对于产生耐药的直接参与者-药物外排泵和药靶麦角固醇的合成的影响，阐明临床菌株多药耐药性与钙信号系统中功能基因的关系。探讨联合唑类药和钙信号通路阻断剂来改变耐药特征，为控制真菌的耐药性和改进现有治疗策略提供新思路。研究发现表明：在临床曲霉耐药菌株方面，收集并鉴定了多株不同来源的感染菌株，证实了病原特征，提出了慢阻肺合并侵袭性肺曲霉病的发病机制，发现PTX3是一种保护性可溶性受体，能识别并调理吞噬曲霉孢子的作用，其多态性与肺曲霉病易感性相关。在曲霉参与应对唑类药物刺激的钙信号系统功能基因方面，发现AkrA通过调控钙信号系统棕榈酰化，进而影响胞内钙信号并影响药物抗性。证实系列功能基因McuA，MrsA，CpcB，Dap家族与烟曲霉药物易感性直接相关，并参与调控药物抗性。建立了活细胞胞内钙浓度检测方法，证明胞内钙浓度参与真菌应答唑类药物刺激，胞内钙离子平衡影响对抗真菌药的应答。在新药靶发现方面，证明病原真菌烟曲霉存在独特的抗损坏性蛋白家族以及真菌亮氨酸合成途径调控子，这些功能蛋白都影响抗真菌药物的药效，为抗真菌药的协同使用以及新药研发提供新理念和新途径。