Synthetic lethality of bicyclomycin

描述（由申请人提供）：对常用抗生素的耐药性使革兰氏阴性菌引起的许多感染非常难以控制，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，新抗生素的开发变得越来越困难。我们计划的长期目标是恢复一个古老的抗生素类别，双环霉素，用于几种威胁生命的病原体。目前的工作重点是一种新的策略，将双环霉素从一个很大的抑菌剂转化为一个杀菌剂，将严重抑制耐药性的出现。以前的工作与大肠杆菌表明，双环霉素，其目标是Rho转录终止子，防止从DNA中去除的转录延伸复合物时，Rho独立的转录终止子是不存在的。然后复制叉与转录复合物碰撞并产生DNA断裂。尽管DNA断裂具有潜在的致死性，但我们最近发现单独使用双环霉素对几种革兰氏阴性菌几乎没有致死作用。然而，E.大肠杆菌、肺炎克雷伯氏菌和鲍曼不动杆菌与双环霉素加第二种基因表达抑制剂（例如，氯霉素或四环素的抑菌浓度）的组合产生（E. coli）或显著增加（K. pneumoniae、A.鲍曼不动杆菌）双环霉素介导的致死性。我们假设，双环霉素治疗诱导修复功能，严重限制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。我们建议通过测量双环霉素杀死大肠杆菌的能力来鉴定和表征参与双环霉素介导的细胞死亡的基因。在存在和不存在翻译抑制剂的情况下对大肠杆菌突变体进行检测，和2）通过测量双环霉素处理增加由影响双环霉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识别潜在的目标，可用于寻找小分子抑制剂，特异性产生/增强双环霉素介导的致死性。增加双环霉素的其他有利特征的致死性有望恢复对此的兴趣。 作为几种严重的革兰氏阴性菌感染的治疗的独特类别的抗菌剂。

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.