液泡-ATP酶作为氟康唑与小檗碱协同抗耐药白念珠菌新靶点的分子机制研究

Multidrug resistance aggravates treatment failure of invasive infections with Candida albicans. It became more important to discover new antifungal targets which is capable against multidrug-resistant isolates. We found that"fluconazole (FLC)+berberine(BBR)"has a powerful synergism against Candida albicans resistant to FLC for the first time in the world. However, the key links of the synergism has not been elucidated. Our previous study including observation of cell ultrastructure, synergistic antimicrobial spectrum and analysis of membrane sterol composition showed that FLC can further inhibit the function of vacuolar–ATPase(V-ATPase)by inhibiting the synthesis of ergosterol so as to improve the sensitivity of Candida albicans on BBR, which give expression to synergistic effect of the two drugs. Based on this theory, we supposed that the V-ATPase can be further studied as a new target of the synergism of FLC and BBR against multidrug-resistant Candida albicans. In this study, we plan to investigate the mechanism of V-ATPase as a target against multidrug-resistant Candida albicans by performing following experiments:①investigation of the alteration of pH value or Ca2+ concentration in cytoplasm after FLC and/or BBR treatment by using pH fluorescent probe or Ca2+ fluorescent probe;②investigation of the change of sensitivity of Candida albicans treated with FLC and/or BBR after changing pH value or Ca2+ concentration in cytoplasm;③determination of the specific V-ATPase genes participated synergistic mechanism of FLC and BBR against multidrug-resistant Candida albicans. This research provides a new perspective to find new antifungal targets against multidrug resistance in Candida albicans.

耐药性已成为临床白念珠菌感染治疗失败最主要原因，寻找新型抗耐药白念珠菌靶点尤为重要。我们在国际上首次发现氟康唑(FLC)+小檗碱(BBR)具有强大协同抗耐药白念珠菌作用。前期细胞超微结构观察、药物敏感性实验及甾醇成分分析等提示FLC可通过抑制麦角甾醇合成进一步抑制V-ATPase功能从而提高白念珠菌对BBR的敏感性，体现两药协同作用，认为V-ATPase可作为FLC与BBR协同抗耐药白念珠菌新靶点进行深入研究。本项目拟研究V-ATPase作为抗耐药白念珠菌新靶点的分子机制。①考察FLC与BBR单用及合用于白念珠菌后细胞内环境(pH、Ca离子浓度)的变化②考察改变细胞内环境(pH、Ca离子浓度)后白念珠菌对FLC与BBR单用及合用时敏感性的变化③确定参与"FLC与BBR协同抗耐药白念珠菌机制"的具体V-ATPase相关基因，最终在基因水平阐明其机制。本项目为发现新型抗真菌药物靶点提供了思路。

本课题组前期研究发现氟康唑（FLC）与小檗碱（BBR）合用具有强大的协同抗FLC耐药白念珠菌的作用，且FLC可通过抑制麦角甾醇合成进一步抑制V-ATPase功能从而提高白念珠菌对BBR的敏感性，体现两药协同作用。本项目旨在研究V-ATPase作为抗耐药白念珠菌新靶点的分子机制。首先，我们考察了FLC与BBR单用及合用于临床耐药白念珠菌后细胞内环境的变化，发现BBR单用后白念珠菌细胞内pH值升高、V-ATPase活性升高以及甾醇含量增加，说明V-ATPase参与了白念珠菌对BBR的耐受。随后，我们考察了加入麦角甾醇后白念珠菌对BBR敏感性的变化，发现加入麦角甾醇后白念珠菌对BBR的耐受性增强、细胞内BBR蓄积量减少。由于V-ATPase功能的发挥依赖于麦角甾醇的参与，这进一步证明了V-ATPase是FLC与BBR协同抗耐药白念珠菌的重要靶点。上述工作发表于Frontiers in Cellular and Infection Microbiology。最后，我们考察了V-ATPase相关基因VMA11敲除后白念珠菌对FLC与BBR敏感性的变化及相关表型变化。我们发现VMA11基因敲除菌液泡形态呈碎片状，细胞膜结构被破坏，生长速率减慢，细胞内pH值升高，对碱性环境耐受力下降，对BBR的敏感性有所增加，对FLC的敏感性增加显著，ERG11、MDR1、CDR1及CDR2基因表达均发生显著变化，麦角甾醇含量明显减少。VMA11基因敲除相关工作已投稿到Antimicrob Agents Chemother。在上述工作开展期间，我们开辟了新的研究方向，发现白念珠菌可通过形成染色体1三倍体及染色体5单倍体2种非整倍体对5-氟胞嘧啶产生耐药性，而2种非整倍体对其他类抗真菌药物的敏感性亦发生了不同变化。我们认为对该研究方向进行深入、系统的研究具有较大的临床意义。目前该研究的部分结果已分别投稿在Antimicrobial Agents and Chemotherapy及mBio杂志。该项目的科学意义在于：①为唑类药物与BBR联合抗耐药白念珠菌的临床治疗提供了理论依据；②为发现抗真菌药物新靶点、寻找新型抗耐药白念珠菌药物及治疗方法提供了思路。