Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA.
mBio. 2012 Dec 4;3(6):e00446-12. doi: 10.1128/mBio.00446-12.
MtrA is a member of the AraC family of transcriptional regulators and has been shown to play an important role in enhancing transcription of the mtrCDE operon, which encodes a tripartite multidrug efflux pump, when gonococci are exposed to a sublethal level of antimicrobials. Heretofore, the DNA-binding properties of MtrA were unknown. In order to understand how MtrA activates mtrCDE expression, we successfully purified MtrA and found that it could bind specifically to the mtrCDE promoter region. The affinity of MtrA for the mtrCDE promoter increased 2-fold in the presence of a known effector and substrate of the MtrCDE pump, the nonionic detergent Triton X-100 (TX-100). When placed in competition with MtrR, the transcriptional repressor of mtrCDE, MtrA was found to bind with apparent lower affinity than MtrR to the same region. However, preincubation of MtrA with TX-100 prior to addition of the promoter-containing DNA probe increased MtrA binding and greatly reduced its dissociation from the promoter upon addition of MtrR. Two independent approaches (DNase I footprinting and a screen for bases important in MtrA binding) defined the MtrA-binding site 20-30 bp upstream of the known MtrR-binding site. Collectively, these results suggest that the MtrA and MtrR-binding sites are sterically close and that addition of an effector increases the affinity of MtrA for the mtrCDE promoter such that MtrR binding is negatively impacted. Our results provide a mechanism for transcriptional activation of mtrCDE by MtrA and highlight the complexity of transcriptional control of drug efflux systems possessed by gonococci.
Antibiotic resistance in Neisseria gonorrhoeae has been increasing in recent years, such that in 2007 the Centers for Disease Control and Prevention listed N. gonorrhoeae as a "superbug." One of the major contributors to antibiotic resistance in N. gonorrhoeae is the MtrCDE efflux pump. Until now, most work on the regulation of the genes encoding this efflux pump has been done on the transcriptional repressor, MtrR. This study is the first one to purify and define the DNA-binding ability of the transcriptional activator, MtrA. Understanding how levels of the MtrCDE efflux pump are regulated increases our knowledge of gonococcal biology and how the gonococcus can respond to various stresses, including antimicrobials.
MtrA 是 AraC 家族转录调控因子的成员，当淋病奈瑟菌暴露于亚致死水平的抗生素时，已证实其在增强 mtrCDE 操纵子的转录中发挥重要作用，该操纵子编码一个三联多药外排泵。迄今为止，MtrA 的 DNA 结合特性尚不清楚。为了了解 MtrA 如何激活 mtrCDE 的表达，我们成功地纯化了 MtrA，并发现它可以特异性地结合 mtrCDE 启动子区域。当存在 MtrCDE 泵的已知效应物和底物非离子去污剂 Triton X-100（TX-100）时，MtrA 与 mtrCDE 启动子的亲和力增加了 2 倍。当与 mtrCDE 的转录抑制剂 MtrR 竞争时，与相同区域相比，发现 MtrA 与 MtrR 的结合亲和力明显较低。然而，在加入包含启动子的 DNA 探针之前，用 TX-100 预孵育 MtrA 可增加 MtrA 的结合，并在加入 MtrR 时大大减少其从启动子上的解离。两种独立的方法（DNase I 足迹法和鉴定 MtrA 结合中重要碱基的筛选）定义了在已知 MtrR 结合位点上游 20-30bp 的 MtrA 结合位点。总的来说，这些结果表明 MtrA 和 MtrR 结合位点在空间上是接近的，并且效应物的添加增加了 MtrA 对 mtrCDE 启动子的亲和力，从而对 MtrR 结合产生负面影响。我们的结果为 MtrA 对 mtrCDE 的转录激活提供了一种机制，并强调了淋病奈瑟菌药物外排系统转录控制的复杂性。
近年来，淋病奈瑟菌对抗生素的耐药性一直在增加，以至于 2007 年疾病控制与预防中心将淋病奈瑟菌列为“超级细菌”。淋病奈瑟菌对抗生素耐药性的一个主要原因是 MtrCDE 外排泵。到目前为止，对编码该外排泵的基因的调控大部分是在转录抑制剂 MtrR 上进行的。这项研究是第一个纯化并定义转录激活剂 MtrA 的 DNA 结合能力的研究。了解 MtrCDE 外排泵水平的调节方式增加了我们对淋病奈瑟菌生物学的认识，以及淋病奈瑟菌如何应对各种压力，包括抗生素。
