Aminoglycoside Resistance in Bacteria

描述(申请人提供)：双功能氨基糖苷修饰酶aac(6‘)-ie-aph(2“)-Ia是革兰氏阳性球菌中氨基糖苷类抗生素最重要的耐药酶，因为它单独对几乎所有氨基糖苷类抗生素产生耐药性，并在临床肠球菌和葡萄球菌分离株中广泛传播。在革兰氏阴性杆菌中出现密切相关的单功能APH(2”)-Ib磷酸转移酶和AAC(6’)-Im乙酰转移酶，预示着这些扩大光谱的酶有可能在多种临床重要病原体中传播。缺乏有关这些酶的动力学和结构信息是设计新型氨基糖苷类抗生素和氨基糖苷类修饰酶抑制剂的主要障碍。其长期目标是阐明氨基糖苷类修饰酶与底物相互作用的动力学和结构机制，为合理设计新的有效抗生素提供有力的依据。本申请的目的是对APH(2“)-Ib、AAc(6‘)-Im和Aac(6’)-Ie-APH(2”)-Ia酶进行详细的研究，以追求三个特定的目标：1.对AAc(6‘)-Im、APH(2“)-Ib和Aac(6’)-Ie-APH(2”)-Ia氨基糖苷类修饰酶进行动力学和结构研究；2)阐明氨基糖苷磷酸转移酶选择性核苷酸三磷酸的分子机制；3)评价AAc(6‘)-Ie-APH(2“)-Ia氨基糖苷类修饰酶的进化潜力。多学科的方法，包括分子生物学，蛋白质化学，详细的酶学，核磁共振和质谱学对反应产物的纯化和结构分析，以及天然酶及其与产物、底物和底物类似物的X射线结晶学，将被用来阐明这些临床上重要的抗生素耐药酶的机制。对AAc(6‘)-Im、APH(2“)-Ib和AAc(6’)-Ie-APH(2”)-Ia酶的详细结构和动力学研究，以及对氨基糖苷磷酸转移酶识别核苷酸三磷酸(NTPs)的分子机制的阐明，将使我们深入了解它们独特的广泛的活性范围。这些研究也将有助于我们了解这些酶的作用机制，并为合理设计新型氨基糖苷类抗生素耐受氨基糖苷类修饰酶和针对氨基糖苷磷酸转移酶核苷酸结合部位的抑制剂提供坚实的基础。评估AAC(6‘)-Ie-APH(2“)-Ia双功能酶在拓宽氨基糖苷类抗生素耐药谱方面的进化潜力将为临床上重要的氨基糖苷类抗生素的未来应用提供重要的指导。最终，这些知识将为设计新的氨基糖苷类抗生素和氨基糖苷类修饰酶的抑制剂提供基础，用于治疗危及生命的感染。 公共卫生相关性：拟议的研究与公共健康相关，因为氨基糖苷类修饰酶的分子和结构机制的阐明最终有望导致新的氨基糖苷类抗生素和抑制剂的开发。这种新型抗菌剂的设计将成为治疗危及生命的传染病的突破口。

DESCRIPTION (provided by applicant): The bifunctional aminoglycoside-modifying enzyme AAC(6')-Ie-APH(2")-Ia is the single most important resistance enzyme for aminoglycoside antibiotics in Gram-positive cocci as it is singlehandedly produces resistance to virtually all aminoglycoside antibiotics and broadly disseminated among clinical enterococcal and staphylococcal isolates. Appearance of the closely-related monofunctional APH(2")-Ib phosphotransferase and AAC(6')-Im acetyltransferase in Gram-negative bacteria heralding the potential for spread of these expanded- spectrum enzymes among a wide variety of clinically important pathogens. Lack of kinetic and structural information regarding these enzymes constitutes the major impediment for the design of novel aminoglycoside antibiotics and inhibitors of aminoglycoside-modifying enzymes. The long-term goal is to delineate kinetic and structural mechanisms of interaction of aminoglycoside-modifying enzymes with their substrates to provide strong basis for the rational design of new effective antibiotics. The objectives in this particular application is to conduct detailed studies of the APH(2")-Ib, AAC(6')-Im, and AAC(6')-Ie-APH(2")-Ia enzymes by pursuing three specific aims: 1. Perform kinetics and structural studies of the AAC(6')-Im, APH(2")-Ib, and AAC(6')-Ie- APH(2")-Ia aminoglycoside-modifying enzymes; 2) Elucidate molecular mechanisms for the nucleotide triphosphate selectivity by the aminoglycoside phosphotransferases; and 3) Evaluate evolutionary potential of the bifunctional AAC(6')-Ie-APH(2")-Ia aminoglycoside-modifying enzyme. Multidisciplinary approach that include molecular biology, protein chemistry, detailed enzymology, purification and structural analyses of the products of the reactions by NMR and mass spectrometry, and X-ray crystallography of the native enzymes and their complexes with products, substrates and substrate analogues will be utilized to elucidate mechanistic aspects of these clinically important antibiotic-resistance enzymes. The detailed structural and kinetic studies of the AAC(6')-Im, APH(2")-Ib, and AAC(6')-Ie-APH(2")-Ia enzymes and elucidation of the molecular mechanism of recognition of nucleotide triphosphates (NTPs) by aminoglycoside phosphotransferases disclosed in this grant proposal will provide insight into their unique over-encompassing breadth of activity. These studies will also contribute to our understanding of the mechanisms of these enzymes, and provide solid basis for the rational design of novel aminoglycoside antibiotics resistant to modification by the aminoglycoside-modifying enzymes and inhibitors targeting the nucleotide binding site of aminoglycoside phosphotransferases. Evaluation of the evolutionary potential of the bifunctional AAC(6')-Ie-APH(2")-Ia enzyme towards broadening the spectrum of conferred resistance to aminoglycoside antibiotics would provide an important guidance for the future utility of clinically important aminoglycosides. Ultimately, such knowledge will provide the basis for the design of new aminoglycoside antibiotics and inhibitors of the aminoglycoside- modifying enzymes for treatment of life-threatening infections. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because elucidation of molecular and structural mechanisms of aminoglycoside-modifying enzymes is ultimately expected to result in development of novel aminoglycoside antibiotics and inhibitors. Design of such novel antibacterial agents will be a breakthrough for treatment of life-threatening infectious diseases.