Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment
Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment
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DOI：
10.1128/jb.185.4.1236-1244.2003
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发表时间：
2003-02-01
影响因子：
3.2
通讯作者：
Berche, P
中科院分区：
文献类型：
--
作者：
Herz, K;Vimont, S;Berche, P
霍乱弧菌是霍乱的病原体，是水生环境的正常居民，它在广泛的pH和盐度条件下生存。在这项工作中，我们研究了三个Na+/H+反向转运蛋白在盐环境中对霍乱弧菌存活的作用。我们以前已经克隆了编码大肠杆菌F：霍乱同源物的Vc-nhaA基因。在这里，我们确定了两个额外的反向转运蛋白基因，命名为Vc-nhaB和Vc-nhaD，编码两个推定的蛋白质的530和477个残基，分别高度同源的弧菌物种和E.杆菌我们证明了Vc-NhaA和Vc-NhaB具有Na+抗性，Vc-NhaA在E. roll的大小、动力学参数和pH调节作用与E.可乐为了确定Na+/H+反向转运蛋白在霍乱弧菌中的作用，我们构建了nhaA、nhaB和nhaD突变体（单突变体、双突变体和三突变体）。与E. colt的研究表明，在高Na+浓度下，霍乱弧菌中三个推定的反向转运蛋白基因（Vc-nhaABD）的失活并不改变细菌的指数生长，并且在稳定期仅具有轻微的影响。与此相反，一个显着的和类似的Li+敏感的表型被发现与所有的突变体缺乏VC-nhaA在指数期的增长，也与三重突变体在静止期的增长。通过使用2-n-壬基-4-羟基喹啉N-氧化物，一种电子传递连接的Na+泵NADH-醌氧化还原酶（NQR）的特异性抑制剂，我们确定在NQR活性不存在的情况下，Vc-NhaA Na+/H+反向转运蛋白活性对于霍乱弧菌在碱性pH下对Na+的抗性是必不可少的。我们认为它的活性掩盖了Na+/H+而不是Li+/H+逆向转运蛋白的活性。我们的研究结果表明，人类病原体霍乱弧菌的Na+抗性需要一个复杂的分子系统，涉及多个反向转运蛋白和NQR泵。
Vibrio cholerae, the causative agent of cholera is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na+/H+ antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc-nhaA gene encoding the F: cholerae homolog of Escherichia colt. Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that troth Vc-NhaA and Vc-NhaB confer Na+ resistance anti that Vc-NhaA displays an antiport activity in E. roll, which is similar in magnitude, kinetic parameters, and pH regulation to that of E. cola NhaA. To determine the roles of the Na+/H+ antiporters in V. cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants). In contrast to E. colt, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li+ -sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ antiporter activity becomes essential for the resistance of V. cholerae to Na+ at alkaline pH. Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ antiporter activities. Our results indicate that the Na+ resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.