Li Hui, Wang Yingyu, Chen Qiyan, Xia Xi, Shen Jianzhong, Wang Yang, Shao Bing
Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control, Beijing, China.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Front Microbiol. 2021 Jan 25;11:583185. doi: 10.3389/fmicb.2020.583185. eCollection 2020.
The emergence and worldwide dissemination of plasmid-mediated colistin resistance gene has attracted global attention. The MCR-1 enzyme mediated colistin resistance by catalyzing phosphoethanolamine (PEA) transfer onto bacterial lipid A. However, the interaction partners of MCR-1 located in membrane protein in are unknown. Co-immunoprecipitation (Co-IP) and Mass Spectrometry were performed to define the interacting proteins of MCR-1. A total of three different anti-MCR-1 monoclonal antibody (mAbs) were prepared and 3G4 mAb was selected as the bait protein by compared their suitability for Co-IP. We identified 53, 13, and 14 interacting proteins in BL21 (DE3) (pET28a-), BL21 (DE3) (pET28a-), and DH5α (pUC19-), respectively. Six proteins, including the stress response proteins DnaK (chaperone protein) and SspB (stringent starvation protein B), the transcriptional regulation protein H-NS, and ribosomal proteins (RpsE, RpsJ, and RpsP) were identified in all these three strains. These MCR-1-interacting proteins were mainly involved in ribosome and RNA degradation, suggesting that MCR-1 influences the protein biosynthesis through the interaction with ribosomal protein. Multidrug efflux pump AcrA and TolC were important interacting membrane proteins of MCR-1 referred to drug efflux during the PEA modification of the bacterial cell membrane. Overall, we firstly identified the functional interactome profile of MCR-1 in and discovered that two-component AcrA-TolC multidrug efflux pump was involved in -mediated colistin resistance.
质粒介导的黏菌素耐药基因的出现及在全球范围的传播引起了全球关注。MCR-1酶通过催化磷酸乙醇胺（PEA）转移至细菌脂多糖A上介导黏菌素耐药。然而，位于膜蛋白中的MCR-1的相互作用伙伴尚不清楚。通过免疫共沉淀（Co-IP）和质谱分析来确定MCR-1的相互作用蛋白。共制备了三种不同的抗MCR-1单克隆抗体（mAb），通过比较它们对Co-IP的适用性，选择3G4 mAb作为诱饵蛋白。我们分别在BL21（DE3）（pET28a-）、BL21（DE3）（pET28a-）和DH5α（pUC19-）中鉴定出53种、13种和14种相互作用蛋白。在所有这三种菌株中均鉴定出六种蛋白，包括应激反应蛋白DnaK（伴侣蛋白）和SspB（严格饥饿蛋白B）、转录调节蛋白H-NS以及核糖体蛋白（RpsE、RpsJ和RpsP）。这些与MCR-1相互作用的蛋白主要参与核糖体和RNA降解，表明MCR-1通过与核糖体蛋白相互作用影响蛋白质生物合成。多药外排泵AcrA和TolC是MCR-1重要的相互作用膜蛋白，在细菌细胞膜PEA修饰过程中参与药物外排。总体而言，我们首次确定了MCR-1在中的功能相互作用组图谱，并发现双组分AcrA-TolC多药外排泵参与介导黏菌素耐药。
