Periplasmic Zinc Management and Homeostasis in Paracoccus denitrificans

摘要 病原菌中抗生素耐药性的流行已成为一大健康问题 这引起了人们的关注，并促使人们寻找新的抗生素靶标。一个特别有希望的目标是 细菌三磷酸腺苷结合盒(ABC)转运蛋白的超家族，它连接着水解酶 三磷酸腺苷对多种溶质在细胞膜上的运输有重要作用。细菌ABC 转运蛋白与高亲和力的溶质结合蛋白(SBP)共同工作，这种蛋白具有特异性 结合底物并将其传递给运输器。肠道沙门氏菌和链球菌 肺炎等，编码ABC转运蛋白和SBPs的基因中断 锌显著降低动物模型的毒性，强调这些系统是有效的药物 目标。我们已经在脱氮副球藻中鉴定出两个锌特异的ABC转运子操纵子， ZnuABC和AztABCD，并鉴定了一种迄今假设的蛋白质(AztD)，它起到了 一种锌的伴侣，直接将锌转移到该体系的SBP(AZTC)。该项目将利用 牙根假单胞菌作为人类病原体高度同源系统的模型 碳青霉烯类耐药肠杆菌(CRE)。这些生物体与广泛的- 抗菌谱耐药性是潜在致命医院感染的病原菌 感染。我们将确定AZTC和AZTC金属结合和转移的精确机制 脱氮假单胞菌和Citrobacter koseri的AztD蛋白的结构和结构分析 生物物理技术。AZT和ZNU系统的生理作用将由以下因素决定 脱氮假单胞菌中这些基因的基因敲除及其生长缺陷的特征 缺锌条件下的表型。结合体内的高分辨率结构信息 功能性将为细菌和细菌中过渡金属输入的机制提供新的见解 为合理设计金属摄取抑制剂作为抗菌药物提供了依据。 抗药性病原体。

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.