Periplasmic Zinc Management and Homeostasis in Paracoccus denitrificans

摘要 病原菌对抗生素耐药性的普遍存在已成为人类健康的一大威胁， 这引起了人们的关注，并刺激了对新抗生素靶点的研究。一个特别有希望的目标是 细菌ATP结合盒（ABC）转运蛋白超家族，其偶联水解 ATP对各种溶质跨细胞膜的运输起着重要作用。细菌ABC 转运蛋白与高亲和力溶质结合蛋白（SBP）协同工作， 结合底物并将其递送至转运蛋白。在沙门氏菌和链球菌中 其中，破坏编码ABC转运蛋白和特异于肺炎链球菌的SBP的基因， 锌显着减弱动物模型中的毒力，凸显这些系统是有效的药物 目标的我们已经在脱氮副球菌中鉴定了两个锌特异性ABC转运蛋白操纵子， ZnuABC和AztABCD，并且已经表征了迄今为止假设的蛋白质（AztD），其充当 Zn分子伴侣，直接将Zn转移到该系统的SBP（AztC）。该项目将利用 P. dentrificans作为人类病原体中高度同源系统的模型， 碳青霉烯耐药肠杆菌（CRE）。这些生物体与广泛的- 是潜在致命的医院感染的病原体 感染.我们将确定AztC的金属结合和转移的精确机制， 使用结构和分子生物学方法， 生物物理技术Azt和Znu系统的生理作用将由以下决定： 使这些基因在P. acrficans中的基因敲除，并表征生长缺陷 锌限制培养基中的表型。高分辨率结构信息结合体内 功能将产生新的洞察过渡金属进口的机制在细菌和 可能为合理设计金属摄取抑制剂作为多药耐药的抗生素提供依据。 耐药病原体。

ABSTRACT The prevalence of antibiotic resistance among pathogenic bacteria has become a major health concern and has spurred the search for novel antibiotic targets. A particularly promising target is the superfamily of bacterial ATP-binding cassette (ABC) transporters, which couple the hydrolysis of ATP to the transport of a wide variety of solutes across the cell membrane. Bacterial ABC transporters work in conjunction with a high affinity solute binding protein (SBP) that specifically binds substrate and delivers it to the transporter. In Salmonella enterica and Streptococcus pneumoniae among others, disruption of genes encoding ABC transporters and SBPs specific for Zn dramatically attenuates virulence in animal models, highlighting these systems as potent drug targets. We have identified two Zn-specific ABC transporter operons in Paracoccus denitrificans, ZnuABC and AztABCD, and have characterized a hitherto hypothetical protein (AztD) that acts as a Zn chaperone, directly transferring Zn to the SBP of that system (AztC). This project will utilize P. dentrificans as a model for highly homologous systems in human pathogens belonging to the carbapenem-resistant Enterobacteriacaea (CRE). These organisms are associated with broad- spectrum antimicrobial resistance and are the causative agents of potentially deadly nosocomial infections. We will determine the precise mechanism of metal binding and transfer for AztC and AztD proteins from P. denitrificans and the CRE pathogen Citrobacter koseri using structural and biophysical techniques. The physiological roles of the Azt and Znu systems will be determined by making genetic knockouts of these genes in P. denitrficans and characterizing growth deficient phenotypes in Zn-limited medium. High-resolution structural information combined with in vivo functionality will yield new insight into the mechanisms of transition metal import in bacteria and potentially provide a basis for the rational design of metal uptake inhibitors as antibiotics for multi- drug resistant pathogens.