Synthetic lethality of bicyclomycin

描述(申请人提供)：对常用抗生素的抗药性使许多由革兰氏阴性菌引起的感染很难控制，有时感染无法治疗。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。我们计划的长期目标是重振一种古老的抗生素类别，双环素类药物，用于治疗几种威胁生命的病原体。目前的工作集中在一种新的策略，将双环素从主要的抑菌剂转变为杀菌剂，将严重逆转耐药性的出现。以前对大肠杆菌的研究表明，针对Rho转录终止子的双环霉素，在缺乏Rho非独立转录终止子的情况下，可以阻止转录延伸复合体从DNA中移除。然后，复制叉子与转录复合体碰撞，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，仅用双环霉素治疗对几种革兰氏阴性菌几乎没有致命作用。然而，双环素和另一种基因表达抑制剂(如氯霉素或四环素的抑菌浓度)与双环素联合作用于大肠杆菌、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)双环素介导的致死率。我们假设，双环素处理诱导的修复功能严重限制了双环素介导的致死活性；当翻译抑制物也存在时，双环素诱导的修复被阻断，双环霉素变得致命。我们建议鉴定和鉴定与双环素介导的细胞死亡相关的基因，1)通过测量在翻译抑制剂存在和不存在的情况下双环素杀死大肠杆菌突变株的能力，以及2)通过测量双环霉素处理增加影响双环素致死性的基因编码的蛋白质的丰度的能力。这项工作的主要成果将是对双环素介导的细胞死亡机制的遗传学理解。一个重要的应用将是识别潜在的靶点，这些靶点可以用来寻找小分子抑制剂，以特定地产生/增强双环素介导的致命性。在双环素的其他有利特性上增加杀伤力，预计将重新引起人们对这一问题的兴趣 一种独特的抗菌剂，用于治疗几种严重的革兰氏阴性感染。

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.