Synthetic lethality of bicyclomycin

描述（由申请人提供）：对常用抗生素的耐药性使得革兰氏阴性菌引起的许多感染很难控制，有时感染无法治愈。迫切需要新的抗生素来控制这些感染；然而，开发新的抗生素正变得越来越困难。我们项目的长期目标是复兴一种古老的抗生素，双环霉素，用于治疗几种危及生命的病原体。目前的工作重点是将双环霉素从主要的抑菌剂转化为杀菌剂的新策略，这将严重限制耐药性的出现。先前对大肠杆菌的研究表明，当Rho转录终止子不存在时，以Rho转录终止子为靶点的双环霉素可以阻止转录延伸复合物从DNA上移除。然后复制叉与转录复合体碰撞，产生DNA断裂。虽然DNA断裂具有潜在的致命性，但我们最近发现单独使用双环霉素治疗对几种革兰氏阴性菌几乎没有致死作用。然而，用双环霉素加第二种基因表达抑制剂（如氯霉素或四环素抑菌浓度）联合治疗大肠杆菌、肺炎克雷伯菌和鲍曼不动杆菌，要么产生（大肠杆菌），要么显著增加（肺炎克雷伯菌、鲍曼不动杆菌）双环霉素介导的致死率。我们假设双环霉素治疗诱导的修复功能严重限制了双环霉素介导的致死活性；当翻译抑制剂也存在时，双环霉素诱导的修复被阻断，双环霉素变得致命。我们建议鉴定和表征与双环霉素介导的细胞死亡相关的基因：1)通过测量双环霉素在存在和不存在翻译抑制剂的情况下杀死大肠杆菌突变体的能力；2)通过测量双环霉素处理增加影响双环霉素致死率的基因编码蛋白丰度的能力。这项工作的主要结果将是对双环霉素介导的细胞死亡机制的遗传理解。一个重要的应用将是确定潜在的靶标，这些靶标可用于寻找小分子抑制剂，以特异性地产生/增强双环霉素介导的致死率。加上致命的其他有利特性的双环霉素预计将恢复对这方面的兴趣

DESCRIPTION (provided by applicant): Resistance to commonly used antibiotics has rendered many infections caused by Gram-negative bacteria very difficult to control, and sometimes the infections are untreatable. New antibiotics are urgently needed to control these infections; however, new antibiotics are becoming increasingly difficult to develop. The long-term goal of our program is to revive an old antibiotic class, the bicyclomycins, for several life-threatening pathogens. The present work focuses on a novel strategy for converting bicyclomycin from a largely bacteriostatic agent into a bactericidal one that will severely restric emergence of resistance. Previous work with Escherichia coli indicated that bicyclomycin, which targets the Rho transcription terminator, prevents removal of transcription elongation complexes from DNA when Rho-independent transcriptional terminators are absent. Replication forks then collide with the transcription complexes and generate DNA breaks. Although DNA breaks are potentially lethal, we recently found that treatment with bicyclomycin alone has little lethal effet on several Gram-negative bacteria. However, co-treatment of E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii with bicyclomycin plus a second inhibitor of gene expression (e.g. bacteriostatic concentrations of chloramphenicol or tetracycline) either generates (E. coli) or dramatically increases (K. pneumoniae, A. baumannii) bicyclomycin-mediated lethality. We hypothesize that bicyclomycin treatment induces repair functions that severely limit bicyclomycin-mediated lethal activity; when inhibitors of translation are also present, bicyclomycin-induced repair is blocked, and bicyclomycin becomes lethal. We propose to identify and characterize genes involved in bicyclomycin-mediated cell death 1) by measuring the ability of bicyclomycin to kill E. coli mutants in the presence and absence of translation inhibitors and 2) by measuring the ability of bicyclomycin treatment to increase the abundance of proteins encoded by genes that affect bicyclomycin lethality. The primary outcome of the work will be a genetic understanding of the mechanism underlying bicyclomycin-mediated cell death. An important application will be identification of potential targets that can be used to fin small-molecule inhibitors for specifically generating/enhancing bicyclomycin-mediated lethality. Adding lethality to other favorable features of bicyclomycin is expected to revive interest in this distinct class of antimicrobial as a treatment for several serious Gram-negative infections.