ENZYMOLOGY OF ANTIBIOTIC RESISTANCE

在过去的二十年里，人们越来越清楚地认识到，抗生素治疗糖尿病的疗效 由于微生物耐药菌株的常见出现，传染病处于危险之中。 了解抗菌素耐药的机制对于临床和临床有效的病人护理至关重要。 对于制定战略以加强对故意传播病原体的生物防御至关重要。 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都有效 微生物。在引入磷霉素十年后，观察到质粒介导的磷霉素耐药 诊所。由该项目支持的研究证实，抗药性是由一种金属酶引起的。 (FOSA)催化将谷胱甘肽加成到抗生素中，使其失去活性。相似的电阻元件 现已被证明存在于几种致病微生物的基因组中，包括假单胞菌 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 肉毒梭菌。来自该项目的基因组和生化分析表明，有三种截然不同的 金属酶的亚群，称为FOSA、FOSB和FOX，它们通过略有不同的方式赋予抗性 化学机制。本研究项目的目标是鉴定质粒和基因组编码 参与微生物对磷霉素耐药性的蛋白质及其潜在的结构和机制 抗性的酶学。这些目标将通过将酶学、生物物理学和 抗性问题的基因组分析。假单胞菌FOSA的三维结构 铜绿假单胞菌及其近亲FosB和FosX将通过X射线结晶学进行测定。化学物质和 催化机理将通过：(I)检查Mn2+的内部配位球来阐明 在FOSA和FOX中的EPR和Endor谱；(Ii)硫醇选择性的稳态动力学分析 FOSA和FosB，以及(Iii)FosX催化独特水合反应的机理研究。潜力 过渡态缓蚀剂将通过结构、光谱和动力学技术进行研究。这个 底物和抑制剂与酶的相互作用的热力学将通过等温法进行检验。 滴定量热法将特别强调病原菌假单胞菌的酶 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。这样做的目的是 研究的目的是为设计针对这两种质粒的药物建立机制和结构基础。 对磷霉素的遗传和基因编码耐药。

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 biodefence 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 ofglutathione 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 ofmetalloenzymes, 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 and ldnede 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.