Mechanisms of Antibiotic Efflux in Campylobacter

描述（由申请方提供）：细菌抗生素外排转运蛋白是赋予抗菌剂内在和获得性耐药性的重要参与者。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原因，也是NIAID B类优先病原体列表中的病原体，含有不同家族的多种抗生素外排转运蛋白。在之前的应用期间，我们确定了弯曲杆菌中的抗性-结瘤-分裂（RND）家族的两个外排泵（CmeABC和CmeDEF）的功能和调节。我们的研究结果表明，外排系统不仅有助于抗菌药物的耐药性，但也有重要的生理功能，促进弯曲杆菌在动物肠道定植。我们还发现，CmeR，一种转录因子，抑制cmeABC和胆汁盐（通常存在于肠道）诱导cmeABC的表达，通过抑制CmeR的cmeABC的启动子的结合。我们最近的初步研究也强烈表明，CmeR是一个全球性的调节剂，并调节MF（主要促进剂）和MATE（多药和有毒化合物挤出）转运蛋白以及C4-二羧酸转运蛋白的表达，这些转运蛋白可能参与弯曲杆菌适应氧气有限的环境，这些环境存在于弯曲杆菌在动物宿主中占据的生态位中。这些发现清楚地表明，抗生素外排系统的调节表达在抗菌素耐药性和促进弯曲杆菌适应环境变化中起着重要作用。尽管这些最新的进展，大多数的CmeR调节外排转运蛋白在弯曲杆菌的功能尚未得到表征，和控制的转运蛋白的表达和CmeR调节的结构基础的分子机制仍有待确定。为了弥补我们对弯曲杆菌中主动外排系统的理解中的这些重要差距，我们计划在本申请中追求3个具体目标：1）确定弯曲杆菌中MF和MATE转运蛋白的调节机制和功能。空肠的CmeR调节的C4-二羧酸转运系统的作用，在促进弯曲杆菌适应氧气有限的环境，和3）阐明CmeR调节的结构基础和胆汁盐的诱导机制，使用X-射线晶体学。拟议的研究利用了我们实验室的独特资源，并利用当代分子，遗传和生化方法以及已建立的动物模型。一旦完成，拟议的工作连同在前一个申请期进行的研究将揭示细菌中抗生素外排转运蛋白的功能和调控机制的新信息。这些发现也将有助于确定控制和治疗抗生素耐药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.