Bacterial cell killing by topoisomerase I mediated DNA lesion

拓扑异构酶 I 介导的 DNA 损伤杀死细菌细胞

基本信息

批准号：
7333269
负责人：
Yuk-Ching Tse-Dinh
金额：
$ 37.15万
依托单位：
NEW YORK MEDICAL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7333269
关键词：
Anti-Bacterial Agents Antibiotics Bacteria Bacterial DNA Topoisomerase I Bacterial Typing Biological Assay Cells Class Cleaved cell Collaborations Community Hospitals Complex Condition DNA DNA Fingerprinting DNA Topoisomerases DNA biosynthesis DNA lesion Development Emerging Communicable Diseases Enzymes Escherichia coli Face Future General Population Genes Genetic Recombination Genetic Transcription Genomic Library Goals Gram-Negative Bacteria Growth In Vitro Infection Laboratories Lead Lesion Libraries Malignant Neoplasms Measurement Mediating Modeling Monitor Mutagenesis Mutation Pathway interactions Personal Satisfaction Plasmids Predisposition Prevention Protein Biosynthesis Proteins Public Health Pulsed-Field Gel Electrophoresis Recombinants Research Research Personnel Resistance Role Screening procedure Therapeutic Agents Topoisomerase Type I DNA Topoisomerases Yersinia pestis bactericide biodefense cell killing cellular targeting desire follow-up genetic strain human TOP1 protein killings medical schools mutant novel pathogen pathogenic bacteria programs recombinational repair response small molecule

项目摘要

DMA topoisomerases are ubiquitous enzymes involved in DNA replication, transcription and recombination. It is well known that trapping of covalent complexes formed between cleaved DNA and type II or type IB DNA topoisomerases by therapeutic agents leads to the killing of cancer or bacterial cells. The class of type IA DNA topoisomerases is a promising target for novel therapetuic agents, but molecules targeting type IA DNA topoisomerases have not been discovered. We have identified a mutant of Yersinia pestis topoisomerase I that can result in extensive killing when expressed in E. coli cells due to the stabilization of the covalent complex with cleaved DNA. This is the first demonstration of bacterial cell killing from accumlated covalent complex formed by a type IA DNA topoisomerase. The specific aims of the project are: 1. A high-through-put assay will be utilized at the NSRB facility at Harvard Medical School to screen the 150,000 compounds available for small molecules that will result in accumulation of covalent complex formed by recombinant Yersinia pestis topoisomerase I. Hits will be characterized by in vitro topoisomerase assay and bacterial cell killing and further developed in collaboration with medicinal chemists at NSRB. 2. To investigate the mechanism of bacterial cell killing by stabilized type IA DNA topoisomerase covalent complex, E. coli expressing the mutant Y. pestis topoisomerase I that forms the stablized covalent complex will be studied. Sensitivity to topoisomerase I induced DNAlesion will be compared under different growth conditions to determine the role of DNA replication and protein synthesis in the cell killing mechanism. 3. To identify other factors influencing the suscepbility of bacteria to killing by trapped type IAtopoisomerase cleaved complex, E. coli strains with mutations in recombination and repair pathways will be studied. An E. coli genomic library in a multi-copy plasmid will be used to identify proteins that when expressed in a higher level, can confer resistance to topoisomerase I mediated cell killing. Transposon mutagenesis will be carried out to screen for mutants with increased sensitivity or resistance to the cell killing. The emergence of pathogenic bacteria resistant to all common antibiotics represent a critical challenge in public health. Future terrorist attacks employing bacterial pathogens could involve agents resistant to current antibiotics. This research has the potential to lead to the discovery of a novel class of antibiotics.

DMA 拓扑异构酶是参与 DNA 复制、转录和重组的普遍存在的酶。众所周知，捕获切割的 DNA 与 II 型或 IB 型之间形成的共价复合物治疗剂的 DNA 拓扑异构酶可杀死癌细胞或细菌细胞。类型类别 IA DNA 拓扑异构酶是新型治疗剂的一个有前景的靶标，但靶向 IA 型的分子 DNA拓扑异构酶尚未被发现。我们已经鉴定出鼠疫耶尔森氏菌的突变体拓扑异构酶 I 在大肠杆菌细胞中表达时，由于其稳定性，可导致广泛杀伤与切割的 DNA 的共价复合物。这是首次证明杀灭细菌细胞由 IA 型 DNA 拓扑异构酶形成的累积共价复合物。该项目的具体目标是： 1. 哈佛医学院 NSRB 设施将采用高通量检测来筛选 150,000 种小分子化合物可导致共价复合物的积累由重组鼠疫耶尔森菌拓扑异构酶 I 形成。命中将通过体外拓扑异构酶进行表征检测和细菌细胞杀灭，并与 NSRB 的药物化学家合作进一步开发。 2. 探讨稳定型IA DNA拓扑异构酶共价杀伤细菌细胞的机制复合物，表达鼠疫耶尔森氏菌拓扑异构酶 I 突变体的大肠杆菌，形成稳定的共价复合物将被研究。将比较不同生长条件下对拓扑异构酶 I 诱导的 DNA 损伤的敏感性确定 DNA 复制和蛋白质合成在细胞杀伤机制中的作用的条件。 3. 确定影响细菌对 IA 型拓扑异构酶杀灭敏感性的其他因素将研究具有重组和修复途径突变的裂解复合体大肠杆菌菌株。安·E。多拷贝质粒中的大肠杆菌基因组文库将用于鉴定在更高表达水平下表达的蛋白质水平，可以赋予对拓扑异构酶 I 介导的细胞杀伤的抵抗力。将进行转座子诱变筛选出对细胞杀伤具有更高敏感性或抵抗力的突变体。对所有常见抗生素产生耐药性的病原菌的出现是一个严峻的挑战公共卫生。未来使用细菌病原体的恐怖袭击可能涉及对细菌具有抵抗力的病原体目前的抗生素。这项研究有可能发现一类新型抗生素。