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

利用遗传学和多尺度成像来了解分枝杆菌噬菌体宿主选择的机制

• 批准号: 10308509
• 负责人: Eric J. Rubin
• 金额: $ 19.72万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308509
• 关键词: Animal Model; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Automobile Driving; Bacteria; Bacterial Infections; Bacteriophages; Binding; Biological Assay; CRISPR/Cas technology; Cell Wall; Clinical; Combined Antibiotics; Complement; Complex; Development; Drug resistance; Ecosystem; Engineering; Fluorescence-Activated Cell Sorting; Fluorescent Probes; Genes; Genetic; Genetic Screening; Genome; Health; Human; Image; Imaging Device; Incentives; Infection; Innovative Therapy; Insertion Mutation; Integration Host Factors; Libraries; Life Cycle Stages; Medical; Mutate; Mutation; Mycobacteriophages; Mycobacterium Infections; Mycobacterium abscessus; Mycobacterium smegmatis; Phage Receptors; Pharmaceutical Preparations; Phase; Predisposition; Resistance; Resistance development; Specificity; System; Therapeutic; Therapy Clinical Trials; To specify; arms race; bacterial resistance; bactericide; clinically relevant; design; genetic regulatory protein; human pathogen; innovation; insight; interest; knock-down; microbial; mutant; mycobacterial; non-tuberculosis mycobacteria; novel therapeutics; particle; pathogen; pathogenic bacteria; pharmacokinetics and pharmacodynamics; resistance gene; resistance mechanism; standard of care; success; tool; transcription factor; transposon sequencing

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.

项目摘要 人类与细菌病原体之间的武器竞赛正在加速。显然需要 开发面对抗生素耐药性的新工具和策略。近年来，噬菌体返回 当抗生素失败时，可能是救生艇。最近的成功，包括治疗传播药物 具有个性化噬菌体鸡尾酒2的抗性分枝杆菌（MAB）感染 这种创新疗法的完善。噬菌体工程的进步使修改噬菌体成为可能 因此它们始终是杀菌性的，并且可能具有可调的宿主范围3。这是临床的 脓肿分枝杆菌复合物的分离株具有可变的噬菌体敏感性曲线 - 很可能是因为它们是 遗传上非常多样化，具有超大的附件基因组。此外，庞大的噬菌体防御 模型生物分枝杆菌中确定的系统表明，正交系统存在于 非结核分枝杆菌（NTMS）。为了能够为遗传学的NTM指定噬菌体宿主选择， 需要识别用于宿主识别的共享噬菌体受体，并构成噬菌体防御系统 需要表征。 抗生素是细菌感染的一线疗法。但是噬菌体疗法可能能够完成 当前的护理标准。许多研究描述了细菌对抗生素的敏感性提高 已经获得了对噬菌体的抵抗力。组合抗生素/噬菌体疗法利用这种现象， 但是，要制定更有效的组合，需要更好地了解药物之间的相互作用 和噬菌体药代动力学/药效学以及潜在的遗传关系驱动抗药性和 敏感性。 关于分枝杆菌的确定特异性知之甚少。更神秘的是 调节并直接作为噬菌体接收器的宿主因子。该项目提出了一个实验性 识别和表征MAB临床分离株中噬菌体抗性机制的工作流程，这是1）专用的 噬菌体防御系统2）赞助突变和3）附着抑制。在第一个目标中，前向遗传 将雇用屏幕来识别感兴趣的基因。在第二个目标中，功能和机制将是 表征，在第三个目标中，将确定抵抗的获取是否与 抗生素灵敏度的变化。