Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters.

中文翻译：

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研究药物转运蛋白在光滑念珠菌中作用的同源过表达系统。

考虑到属于 ABC 和 MFS 超家族的药物转运蛋白在致病性念珠菌物种中的相关性，一直需要有一个过表达系统，其中这些用于功能分析的膜蛋白可以在同源背景中表达。我们可以通过构建一个高度药物敏感的光滑念珠菌菌株来解决这一未满足的需求，该菌株在七个主要的 ABC 转运蛋白基因中缺失，例如CgSNQ2、CgAUS1、CgCDR1、CgPDH1、CgYCF1、CgYBT1和CgYOR1，并在转录因子 (TF) CgPDR1中引入 GOF 突变导致CgCDR1的过度激活轨迹。该表达系统通过过表达四个 GFP 标记的 ABC（CgCDR1、CgPDH1、CaCDR1和ScPDR5）和一个 MFS（CgFLR1）转运蛋白基因进行验证，该基因由工程化的表达质粒促进整合到CgCDR1基因座。正确表达和定位的转运蛋白功能齐全，正如它们几倍增加的耐药性、生长动力学、定位研究和外排活性所揭示的那样。本同源系统将有助于确定单个转运蛋白在其底物特异性、药物流出、致病性和毒力特征方面的作用，而不受其他主要转运蛋白的干扰。