BETA LACTAMASE MUTATIONS IN ANTIBIOTIC RESISTANCE

这项拟议的研究的目标是在 分子水平上突变是如何改变底物轮廓的 临床重要的TEM-1β-内酰胺酶。这些具体的问题 将解决以下问题： 1.哪些β-内酰胺酶突变会导致对 β-内酰胺类抗生素？透射电子显微镜-1中的每个氨基酸位置 β-内酰胺酶将被随机抽取所有可能的氨基酸样本 使用一种新的诱变技术进行替换。所有的 然后将对随机替换进行筛选，以确定这些氨基酸 导致β-内酰胺酶活性增加的酸替代 一组临床相关的β-内酰胺类抗生素。 2.突变如何改变底物光谱 β-内酰胺酶？将使用生化方法进一步 特异度表征新的水解力 变种人。这些研究将包括对突变体的纯化。 蛋白质和动力学参数的测定。这个 突变蛋白的构象稳定性也将 由多种方法测定。 3.哪些β-内酰胺酶突变会导致抗生素增加 临床分离株的耐药性？获取的信息来自 上述研究将被用于设计寡核苷酸探针， 将专门检测编码特异性突变体的基因 临床分离的含超广谱β-内酰胺酶细菌。这些研究将 检验随机替代实验的预测能力 并提供有关β-内酰胺作用机制的具体信息 抗生素耐药性。 总而言之，这些研究将系统地评估 TEM-1β-内酰胺酶通过突变扩大其底物谱。 从这些研究中获得的信息将是即时的 经验值在预测给定磁化率中的应用 对β-内酰胺酶突变的抗生素。在漫长的岁月里 我们想用这些数据来预测β-内酰胺酶 会对抗生素治疗的选择性压力做出反应。

The goal of the proposed research is to characterize at the molecular level how mutations can alter the substrate profile of the clinically important TEM-1 beta-lactamase. These specific questions will be addressed: 1. What beta-lactamase mutations cause an increase in resistance to beta-lactam antibiotics? Every amino acid position in TEM-1 beta-lactamase will be randomized to sample all possible amino acid substitutions using a novel mutagenesis technique. All of the random substitutions will then be screened to identify those amino acid substitutions that result in increased beta-lactamase activity towards a set of clinically relevant beta-lactam antibiotics. 2. How do mutations change the substrate spectrum of beta-lactamase? Biochemical methods will be used to further characterize the new hydrolytic capacities of the specificity mutants. These studies will include purification of the mutant proteins and determination of kinetic parameters. The conformational stability of the mutant proteins also will be determined by a variety of methods. 3. What beta-lactamase mutations result in increase antibiotic resistance in clinical isolates? The information obtained from the above studies will be used to design oligonucleotide probes that will specifically detect genes encoding specificity mutants among TEM beta-lactamase-containing clinical isolates. The studies will test the predictive capacity of the random substitution experiments and provide specific information about the mechanisms of beta-lactam antibiotic resistance. In total, these studies will assess systematically the ability of TEM-1 beta-lactamase to expand its substrate spectrum by mutation. The information gained in these studies will be of immediate empirical value in predicting the susceptibility of a given antibiotic to mutational changes in the beta-lactamase. In the long term we would like to use the data to predict how beta-lactamase will respond to the selective pressure of antibiotic therapy.