ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.
ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.
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DOI：
10.3390/ijms19041000
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发表时间：
2018-03-27
影响因子：
5.6
通讯作者：
Lin HV
中科院分区：
文献类型：
--
作者：
Lu WJ;Lin HJ;Janganan TK;Li CY;Chin WC;Bavro VN;Lin HV
霍乱弧菌 ATP 结合盒转运蛋白 VcaM（霍乱弧菌 ABC 多药耐药泵）此前已被证明可对多种医学上重要的药物产生耐药性。在这项研究中，我们着手分析其在去污剂溶解状态下的体外特性和体内特性，以区分其功能对辅助蛋白的依赖性。我们报告了纯化的 VcaM 的第一个详细动力学参数和磷酸盐 (Pi) 的产生速率。为了确定 VcaM 对三方外排泵可能的功能依赖性，我们利用了缺乏主要二级转运蛋白 AcrB（丙烯黄素抗性蛋白）的不同大肠杆菌菌株，以及缺乏外膜因子 (OMF) TolC（大肠菌素耐受性）的细胞。与 VcaM 的 ATP 酶功能一致，我们发现它对原钒酸钠 (NaOV) 敏感，然而，我们还发现 VcaM 功能对 TolC 具有明显的依赖性。针对次级活性转运蛋白的抑制剂对 VcaM 赋予的抗性或 Hoechst 33342 积累没有影响，这表明 VcaM 可能能够与 TolC 通道结合，而无需额外转运蛋白的周质介导。我们的研究结果表明，VcaM 能够利用 TolC 通道将底物从细胞质一步转移到细胞外空间，使其成为 MacB 家族之外唯一具有明显 TolC 依赖性的多药 ABC 转运蛋白。
Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.
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