ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

抗生素耐药性的酶学

• 批准号: 7006124
• 负责人: RICHARD N ARMSTRONG
• 金额: $ 22.12万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7006124
• 关键词: ENZYMOLOGY; ANTIBIOTIC; RESISTANCE

DESCRIPTION (provided by applicant): In the last two decades it has become increasingly clear that the efficacy of antibiotics for the treatment of infectious diseases is in jeopardy due to the common appearance of drug resistant strains of microorganisms. Understanding the mechanisms of antimicrobial resistance is crucial for effective patient care in the clinic and essential for developing strategies to enhance biodefense against intentionally disseminated of pathogens. Fosfomycin is a potent, broad-spectrum antibiotic effective against both Gram-positive and Gram-negative microorganisms. A decade after its introduction plasmid-mediated resistance to fosfomycin was observed in the clinic. Investigations supported by this project have established that the resistance is due to a metalloenzyme (FosA) that catalyzes the addition of glutathione to the antibiotic, rendering it inactive. Similar resistance elements have now been shown to exist in the genomes of several pathogenic microorganisms including, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus anthrasis, Brucella melitensis, Listeria monocytogenes and Clostridium botulinum. Genomic and biochemical analysis from this project suggest that there are three distinct subgroups of metalloenzymes, termed FosA, FosB and FosX, that confer resistance through somewhat different chemical mechanisms. The objectives of this research project are to identify plasmid and genomically encoded proteins involved in microbial resistance to fosfomycin and to elucidate the underlying structural and mechanistic enzymology of resistance. These objectives will be accomplished by integrating enzymological, biophysical and genomic analyses of the resistance problem. The three-dimensional structures of the FosA from Pseudomonas aeruginosa and its relatives FosB and FosX will be determined by X-ray crystallography. The chemical mechanisms of catalysis will be elucidated by: (i) examination of the inner coordination sphere of Mn 2+ in FosA and FosX by EPR and ENDOR spectroscopy; (ii) a steady state kinetic analysis of the thiol selectivity of FosA and FosB, and (iii) a mechanistic study of the unique hydration reaction catalyzed by FosX. Potential transition state inhibitors will investigated by structural, spectroscopic and kinetic techniques. The thermodynamics of the interaction of substrates and inhibitors with the enzymes will be examined by isothermal titration calorimetry Particular emphasis will be placed on the enzymes from the pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum. The intent of this investigation is to establish the mechanistic and structural bases for the design of drugs to counter both plasmid borne and genomically encoded resistance to fosfomycin.

描述(由申请人提供)： 在过去的二十年里，越来越清楚的是，由于耐药微生物菌株的常见出现，抗生素治疗传染病的疗效正处于危险之中。了解抗菌素耐药的机制对于临床上有效的患者护理至关重要，对于制定加强对病原体故意传播的生物防御的策略也是至关重要的。磷霉素是一种有效的广谱抗生素，对革兰氏阳性和革兰氏阴性细菌都有效。在其引入十年后，在临床上观察到了对磷霉素的质粒介导的耐药性。该项目支持的研究已经确定，耐药性是由于一种金属酶(FOSA)，它催化谷胱甘肽与抗生素的加成，使其失去活性。目前，在铜绿假单胞菌、金黄色葡萄球菌、炭疽杆菌、羊布鲁氏菌、单核细胞增生性李斯特菌和肉毒杆菌等几种病原微生物的基因组中都存在类似的耐药元件。来自该项目的基因组和生化分析表明，有三种不同的金属酶亚群，命名为FOSA、FOSB和FOX，它们通过一些不同的化学机制赋予抗性。本研究的目的是鉴定与微生物对磷霉素耐药有关的质粒和基因编码蛋白，并阐明耐药的潜在结构和机制。这些目标将通过整合抗药性问题的酶学、生物物理和基因组分析来实现。铜绿假单胞菌及其近缘FosB和FosX的FOSA的三维结构将通过X射线结晶学确定。通过EPR和Endor光谱对FOSA和FOSX中Mn2+的内部配位范围进行了考察，对FOSA和FOSB的硫醇选择性进行了稳态动力学分析，并对FOSX催化的独特水合反应进行了机理研究。潜在的过渡态抑制剂将通过结构、光谱和动力学技术进行研究。底物和抑制剂与酶的相互作用的热力学将用等温滴定量热法进行检验，重点将放在铜绿假单胞菌、金黄色葡萄球菌、单核细胞增生性李斯特菌和肉毒杆菌的酶上。这项研究的目的是为药物的设计建立机制和结构基础，以对抗磷霉素携带的和基因编码的耐药性。