Yeast Biofuel Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, 110067, India.
Amity Institute of Integrative Science and Health and Amity Institute of Biotechnology, Amity University, Gurgaon, 122413, Haryana, India.
FEMS Yeast Res. 2020 Jun 1;20(4). doi: 10.1093/femsyr/foaa032.
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.
鉴于 ABC 和 MFS 超家族的药物转运蛋白在致病念珠菌中的相关性，一直需要有一种过表达系统，使这些膜蛋白能够在同源背景下进行功能分析。我们可以通过构建一个对药物高度敏感的光滑念珠菌缺失株来满足这一未满足的需求，该缺失株缺失了七个主要的 ABC 转运体基因，如 CgSNQ2、CgAUS1、CgCDR1、CgPDH1、CgYCF1、CgYBT1 和 CgYOR1，并在转录因子（TF）CgPDR1 中引入一个 GOF 突变，导致 CgCDR1 基因座的过度激活。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）转运体基因得到了验证，这些基因由一个工程化的表达质粒介导整合到 CgCDR1 基因座上。适当表达和定位的转运蛋白具有完全的功能，这从它们对药物的几倍抗性增加、生长动力学、定位研究和外排活性中得到了揭示。本同源系统将有助于确定单个转运蛋白对其底物特异性、药物外排、致病性和毒力特征的作用，而不会受到其他主要转运蛋白的干扰。
