State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong University, Qingdao, China.
Poultry Institute, Shandong Academy of Agricultural Sciences, Jinan, China.
Antimicrob Agents Chemother. 2024 Apr 3;68(4):e0167823. doi: 10.1128/aac.01678-23. Epub 2024 Mar 13.
Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived strains, with a higher efflux capacity for various drugs than the AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623) previously identified in the AcrB (EcAcrB), a relatively unconservative loop (L665-675) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.
水平基因转移已被证明是多药耐药病原体出现的重要驱动因素。最近,在动物源性菌株的质粒中发现了一个可转移的耐药-结节-分裂(RND)超家族基因簇,其对各种药物的外排能力比 AcrAB-TolC 同源系统更高。在本研究中,我们专注于这两个系统的内膜泵的差异,并确定了一些关键残基,这些残基有助于 TMexCD1 系统的强大外排活性。通过同源建模和分子对接,选择了来自近端结合口袋(PBP)的 8 个残基和来自远端结合口袋(DBP)的 9 个残基,并进行了定点突变。其中一些残基,如 S134、I139、D181 和 A290,被证明在 DBP 区域对底物结合很重要,而 PBP 和 DBP 中的所有残基都表现出一定的底物偏好。除了先前在 AcrB(EcAcrB)中鉴定的保守开关环(L613-623)外,还提出了 PBP 底部相对非保守的环(L665-675)作为 TMexD1 强大活性的关键元素,因为与 EcAcrAB 相比,该环在 E669、G670、N673 和 S674 位点发生了变异,并且由于这些突变,其外排活性发生了显著改变。这些关键因素的保守性和灵活性有助于 RND 外排泵的进化,因此可以作为开发抑制剂的潜在靶点,以阻止 TMexCD1 系统的广泛传播。