Mechanisms of antibiotic efflux in Campylobacter

描述（由申请人提供）：空肠弯曲菌是引起人类胃肠炎的重要食源性病原体，已进化出多种机制来抵消各种抗生素的作用，这对公共卫生构成了严重威胁。其中许多耐药机制，如gyrA突变和β-内酰胺酶产生，赋予弯曲菌对特定抗生素的耐药性。然而，主动外排系统将结构多样的抗生素挤出细菌细胞，有助于对多种药物的内在和获得性耐药性。虽然先前的研究表明可能存在功能性外排系统，但mC。空肠，在这种病原体中的抗生素外排机制尚未确定。 利用转座子诱变与其他方法相结合，我们最近已经确定了一个三基因操纵子（命名为cmeABC）编码的三方抗生素外排泵，有助于C。空肠对结构无关的抗生素、重金属、胆盐和其他有毒化合物的耐药性。我们的初步数据和C. jejuni NCTC 11168的研究表明，存在另外的抗生素外排系统（名为cmeDEF），并且cmeABC和cmeDEF可能受到转录抑制因子的调节。基于这些观察结果和细菌抗生素外排系统的已知特征，我们假设CmeDEF与CmeABC结合在挤出各种药剂中起重要作用，并且调节蛋白对外排泵的调节表达显著有助于弯曲杆菌对多种抗生素的内在和获得性抗性。为了验证我们的假设，我们计划i）确定CmeDEF的作用及其与CmeABC在介导弯曲杆菌对多种药物的耐药性中的相互作用，以及ii）鉴定和表征调节抗生素外排系统表达的转录阻遏物。各种遗传和生物化学方法，包括随机和位点特异性诱变，重组蛋白，底物积累试验，和DNA结合试验将被用来定义外排系统的功能和它们与调控蛋白的相互作用。预计这些研究将填补我们对C.空肠弯曲菌，并可能开辟新的途径，设计有效的手段，以预防和治疗抗生素耐药弯曲杆菌。

DESCRIPTION (provided by applicant): Campylobacter jejuni, an important foodborne pathogen causing gastroenteritis in humans, has evolved multiple mechanisms to counteract the action of various antibiotics, which has posed a serious threat to public health, Many of these resistance mechanisms, such as gyrA mutations and beta-lactamase production, confer Campylobacter resistance to specific antibiotics. However, the active efflux systems, which extrude structurally diverse antibiotics out of bacterial cells, contribute to the intrinsic and acquired resistance to multiple drugs. Although previous studies suggested the possible presence of functional efflux systems m C. jejuni, the antibiotic efflux machinery in this pathogen has not been defined. Using transposon mutagenesis in conjunction with other approaches, we have recently characterized a three-gene operon (named cmeABC) encoding a tripartite antibiotic efflux pump that contributes to C. jejuni resistance to structurally unrelated antibiotics, heavy metals, bile salts, and other toxic compounds. Our preliminary data and the genomic sequence of C. jejuni NCTC 11168 suggested the presence of an additional antibiotic efflux system (name cmeDEF) and the possible regulation of cmeABC and cmeDEF by transcriptional repressors. Based on these observations and the known features of bacterial antibiotic efflux systems, we hypothesize that CmeDEF in conjunction with CmeABC plays an important role in extruding various agents, and the modulated expression of the efflux pumps by regulatory proteins contributes significantly to the intrinsic and acquired resistance of Campylobacter to multiple antimicrobials. To test our hypothesis, we plan to i) determine the role of CmeDEF and its interplay with CmeABC in mediating Campylobacter resistance to multiple drugs and ii) to identify and characterize the transcriptional repressors that modulate the expression of the antibiotic efflux systems. Various genetic and biochemical approaches, including random and site-specific mutagenesis, recombinant proteins, substrate accumulation assay, and DNA binding assays will be utilized to define the functions of the efflux systems and their interplay with regulatory proteins. It is anticipated that the proposed studies will close a major gap in our understanding of the antibiotic resistance mechanisms in C. jejuni and may open new avenues for the design of effective means to prevent and treat antibiotics-resistant Campylobacter.