Mechanisms of Antibiotic Efflux in Campylobacter

描述(由申请人提供)：细菌抗生素外排转运体在赋予抗菌素内在和获得性耐药性方面发挥着重要作用。根据基因组序列，空肠弯曲杆菌是美国食源性腹泻的主要细菌来源，也是NIAID B类优先病原体名单中的一种病原体，它含有来自不同家族的多个抗生素外排转运体。在前面的应用阶段，我们确定了弯曲杆菌中抗性结瘤分裂(RND)家族的两个外排泵(CmeABC和CmeDEF)的功能和调节。我们的发现表明，外排系统不仅有助于产生抗菌素耐药性，而且在促进弯曲菌在动物肠道中定植方面具有重要的生理功能。我们还发现，CmeR是一种转录因子，它抑制cmeABC，胆盐(通常存在于肠道)通过抑制CmeR与cmeABC启动子的结合而诱导cmeABC的表达。我们最近的初步研究也有力地表明，CmeR是一个全球性的调节因子，调节Mf(主要促进剂)和Mate(多药和有毒化合物挤出)转运体以及C4-二羧酸转运体的表达，这些转运体可能参与弯曲杆菌对有限氧环境的适应，这些转运体存在于动物宿主中弯曲杆菌占据的生态位中。这些发现清楚地表明，抗生素外排系统的调控表达在抗菌素耐药性和促进弯曲杆菌对环境变化的适应方面发挥着重要作用。尽管最近取得了这些进展，但大多数CmeR调控的外排转运蛋白在弯曲杆菌中的功能尚未确定，调控转运蛋白表达的分子机制和CmeR调控的结构基础仍有待确定。为了弥合我们对弯曲杆菌主动外排系统的认识上的这些重要差距，我们计划在这一应用中追求三个特定的目标：1)确定空肠弯曲菌中Mf和Mate转运体的调节机制和功能；2)确定CmeR调节的C4-二羧酸运输系统在促进弯曲菌适应缺氧环境中的作用；以及3)利用X射线结晶学阐明CmeR调节的结构基础和胆盐的诱导机制。拟议的研究利用了我们实验室现有的独特资源，并利用了当代分子、遗传和生化方法以及已建立的动物模型。一旦完成，拟议的工作与前一个申请期进行的研究将揭示细菌中抗生素外排转运体的功能和调节机制的新信息。这些发现还将有助于确定控制和治疗耐抗生素弯曲杆菌的潜在分子靶点。

DESCRIPTION (provided by applicant): Bacterial antibiotic efflux transporters are important players in conferring intrinsic and acquired resistance to antimicrobials. According to the genomic sequence, Campylobacter jejuni, a leading bacterial cause of foodborne diarrhea in the United States and an agent included in the NIAID Category B Priority Pathogens list, harbors multiple antibiotic efflux transporters of different families. During the previous application period, we determined the function and regulation of two efflux pumps (CmeABC and CmeDEF) of the resistance- nodulation-division (RND) family in Campylobacter. Our findings indicate the efflux system not only contributes to antimicrobial resistance, but also has important physiological functions in facilitating Campylobacter colonization in animal intestinal tract. We have also found that CmeR, a transcriptional factor, represses cmeABC and that bile salts (normally present in the gut) induce the expression of cmeABC by inhibiting the binding of CmeR to the promoter of cmeABC. Our recent preliminary studies also strongly suggest that CmeR is a global regulator and modulates the expression of the MF (major facilitator) and MATE (multidrug and toxic compound extrusion) transporters as well as the C4-dicarboxylate transporters potentially involved in Campylobacter adaptation to oxygen-limited environments, which exist in the niches occupied by Campylobacter in animal hosts. These findings clearly indicate that the modulated expression of the antibiotic efflux system plays important roles in antimicrobial resistance and in facilitating Campylobacter adaptation to environmental changes. Despite these recent advances, the majority of the CmeR-regulated efflux transporters in Campylobacter have not been functionally characterized, and the molecular mechanisms governing the expression of the transporters and the structural basis of CmeR regulation remain to be determined. To close these important gaps in our understanding of the active efflux system in Campylobacter, we plan to pursue 3 specific aims in this application to 1) determine the regulatory mechanisms and functions of the MF and MATE transporters in C. jejuni, 2) define the roles of the CmeR-regulated C4-dicarboxylate transport system in facilitating Campylobacter adaptation to oxygen-limited environments, and 3) elucidate the structural basis of CmeR regulation and the induction mechanisms of bile salts using X-ray crystallography. The proposed studies take advantage of unique resources available in our laboratory and utilize contemporary molecular, genetic, and biochemical approaches as well as an established animal model. Once completed, the proposed work together with the studies conducted in the previous application period will reveal novel information on the functions and regulatory mechanisms of antibiotic efflux transporters in bacteria. The findings will also help to identify potential molecular targets for the control and treatment of antibiotic resistant Campylobacter.