Mechanisms of antibiotic efflux in Campylobacter

描述(申请人提供)：空肠弯曲菌，一种重要的食源性致病菌，在人类中引起胃肠炎，已经进化出多种机制来抵消各种抗生素的作用，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，使弯曲杆菌对特定的抗生素产生抗药性。然而，主动外排系统将结构多样的抗生素从细菌细胞中排出，导致对多种药物的内在和获得性耐药性。虽然以前的研究表明空肠弯曲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。 结合转座子突变和其他方法，我们最近鉴定了一个编码三部分抗生素外排泵的三基因操纵子(命名为cmeABC)，它有助于空肠弯曲菌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。我们的初步数据和空肠弯曲菌NCTC 11168的基因组序列表明，存在一个额外的抗生素外排系统(名为cmeDEF)，并可能通过转录抑制因子调节cmeABC和cmeDEF。基于这些观察和细菌抗生素外排系统的已知特征，我们推测CmeDEF和CmeABC在挤出各种药物方面起着重要作用，调节蛋白对外排泵的调节表达对弯曲杆菌对多种抗菌素的内在和获得性耐药性有重要贡献。为了验证我们的假设，我们计划i)确定CmeDEF及其与CmeABC的相互作用在调节弯曲杆菌对多种药物的耐药性中的作用，以及ii)鉴定和表征调节抗生素外排系统表达的转录抑制物。不同的遗传和生化方法，包括随机和定点突变、重组蛋白、底物积累试验和DNA结合分析，将被用来确定外排系统的功能及其与调节蛋白的相互作用。预计拟议的研究将弥合我们对空肠弯曲菌耐药机制的认识上的一个重大空白，并可能为设计有效的预防和治疗耐药弯曲杆菌的方法开辟新的途径。

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.