Mechanisms of antibiotic efflux in Campylobacter

描述（由申请人提供）：空肠弯曲杆菌是一种引起人类胃肠炎的重要食源性病原体，已经进化出多种机制来对抗各种抗生素的作用，这对公众健康构成了严重威胁。许多这些耐药机制，如gyrA突变和β -内酰胺酶的产生，赋予弯曲杆菌对特定抗生素的耐药性。然而，主动外排系统从细菌细胞中挤出结构多样的抗生素，有助于对多种药物的内在和获得性耐药。虽然先前的研究表明空肠梭菌可能存在功能性外排系统，但这种病原体的抗生素外排机制尚未明确。

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.