Using genetics and multi-scale imaging to understand the mechanisms underlying mycobacteriophage host choice

项目摘要 人类和细菌病原体之间的军备竞赛正在加速。显然需要 开发新的工具和战略来应对抗生素耐药性。近年来，噬菌体又回来了， 当抗生素失效的时候，它可能会成为救生艇。最近的成功，包括治疗一种扩散的药物- 抗结核分枝杆菌（Mab）感染与个性化的噬菌体混合物2，激发了持续的 这种创新疗法的改进。噬菌体工程的进展使修饰噬菌体成为可能。 因此它们是组成性杀菌的并且具有潜在的可调宿主范围3。这是吸引人的临床 M.噬菌体复合体具有可变的噬菌体易感性谱-最有可能是因为它们是 基因非常多样化，有着超大的附属基因组此外，噬菌体防御的众多 在模式生物耻垢分枝杆菌中鉴定的系统表明， 非结核分枝杆菌（NTMs）。为了能够为遗传多样性NTMs指定噬菌体宿主选择， 需要鉴定用于宿主识别的共享噬菌体受体，以及保守的噬菌体防御系统， 需要被定性。 抗生素是细菌感染的一线治疗药物。但噬菌体疗法可能能够补充 目前的护理标准。许多研究已经描述了细菌对抗生素的敏感性增加， 已经获得了对噬菌体的抵抗力。联合抗生素/噬菌体疗法利用了这一现象， 但是为了形成更有效的组合，需要更好地理解药物之间的相互作用， 和噬菌体药代动力学/药效学以及驱动抗性的潜在遗传关系， 易感性 对分枝杆菌噬菌体特异性决定簇知之甚少。更神秘的是， 宿主因子调节并直接作为噬菌体的受体发挥作用。该项目提出了一个实验性的 鉴定和表征Mab临床分离株中噬菌体耐药机制的工作流程， 噬菌体防御系统2）自发突变和3）附着抑制。在第一个目标中，向前遗传 将采用筛选来鉴定感兴趣的基因。在第二个目标中，功能和机制将是 在第三个目标中，将确定抗性的获得是否与以下因素有关： 抗生素敏感性的变化。

PROJECT SUMMARY The arms race between humans and bacterial pathogens is accelerating. There is a clear need for the development of new tools and strategies to confront antibiotic resistance. In recent years, phage have returned as a possible lifeboat when antibiotics fail. Recent successes, including the treatment of a disseminated drug- resistant Mycobacterium abscessus (Mab) infection with a personalized phage cocktail2, motivate the continued refinement of this innovative therapy. Advances in phage engineering have made it possible to modify phages so that they are constitutively bactericidal and with potentially tunable host ranges3. This is appealing as clinical isolates of the M. abscessus complex have variable phage susceptibility profiles – most likely because they are genetically very diverse, with oversized accessory genomes. Furthermore, the multitude of phage defense systems identified in the model organism Mycobacterium smegmatis suggests that orthogonal systems exist in non-tuberculous mycobacteria (NTMs). To be able to specify phage host choice for genetically diverse NTMs, shared phage receptors used for host recognition need to be identified, and conserved phage defense systems need to be characterized. Antibiotics are first-line therapies for bacterial infections. But phage therapies may be able to complement the current standard of care. Many studies have described increased sensitivities to antibiotics in bacteria that have acquired resistance to phage. Combination antibiotic/phage therapies take advantage of this phenomenon, but to formulate more effective combinations, a better understanding is needed of the interplay between drug and phage pharmacokinetics/pharmacodynamics and the underlying genetic relationships driving resistance and susceptibility. Very little is known about the mycobacteriophage determinants of specificity. Even more mysterious, are the host factors that regulate, and function directly as receptors for phage. This project presents an experimental workflow to identify and characterize phage resistance mechanisms in Mab clinical isolates due to 1) dedicated phage defense systems 2) spontaneous mutations and 3) attachment inhibition. In the first aim, forward genetic screens will be employed to identify genes of interest. In the second aim, function and mechanism will be characterized, and in the third aim it will be determined whether acquisition of resistance is associated with changes in antibiotic sensitivity.