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.

项目总结 人类与细菌病原体之间的军备竞赛正在加速。很明显，有必要对 开发应对抗生素耐药性的新工具和战略。近年来，噬菌体又回来了 作为抗生素失效时的救生艇。最近的成功，包括对一种传播的药物的治疗- 耐药脓肿分枝杆菌(Mab)感染与个性化噬菌体鸡尾酒2，激励继续 这一创新疗法的改进。噬菌体工程的进展使修饰噬菌体成为可能 因此它们在构成上是杀菌的，并且具有潜在的可调节的宿主范围。这在临床上很有吸引力 脓肿分支杆菌复合体的分离株具有不同的噬菌体敏感性图谱--很可能是因为它们是 基因非常多样化，带有超大的附属基因组。此外，大量的噬菌体防御 在模式生物污垢分枝杆菌中发现的系统表明，在 非结核分枝杆菌(NTMS)。为了能够为遗传多样性的NTM指定噬菌体宿主选择， 需要确定用于识别宿主的共享噬菌体受体，并保守噬菌体防御系统 需要被定性。 抗生素是细菌感染的一线治疗方法。但噬菌体疗法或许能够补充 目前的护理标准。许多研究都描述了细菌对抗生素的敏感性增加 对噬菌体产生了抵抗力。抗生素/噬菌体联合疗法利用了这一现象， 但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/药效学和潜在的遗传关系驱动耐药性和 敏感度。 对分枝杆菌噬菌体的特异性决定因素知之甚少。更神秘的是 这些寄主因子直接作为噬菌体的受体来调节和发挥作用。这个项目展示了一种试验性的 鉴定和表征Mab临床分离株中噬菌体抗性机制的工作流程 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 筛查将被用于识别感兴趣的基因。在第二个目标中，将功能和机制 在第三个目标中，将确定获得抵抗力是否与 抗生素敏感性的变化。