Characterization of long-circulating phages isolated from in vivo mouse studies

从小鼠体内研究中分离出的长循环噬菌体的表征

• 批准号: 10308532
• 负责人: CAROLYN M TESCHKE
• 金额: $ 20.13万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 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 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308532
• 关键词: Aftercare; Amino Acid Substitution; Bacteriophage P22; Bacteriophage lambda; Bacteriophages; Biochemical; Biological Assay; Biophysics; Blood; Blood Circulation; Capsid; Capsid Proteins; Cardiovascular system; Cell Culture Techniques; Cell Line; Cells; Charge; Chemicals; Cryoelectron Microscopy; Engineering; Escherichia coli; Exhibits; Future; Genetic; Genome; Hour; Human; Immune; In Vitro; Learning; Longevity; Modeling; Modification; Molecular; Mus; Mutate; Mutation; Outcome; Pathogenicity; Patients; Peptide Hydrolases; Phenotype; Positioning Attribute; Renaissance; Resolution; Salmonella; Structure; Surface; Testing; Time; Wild Type Mouse; Work; base; design; ds-DNA; experimental study; in vitro Assay; in vivo; insight; macrophage; mouse model; mutant; particle; pathogenic bacteria; stability testing; time use; translational impact

PROJECT SUMMARY The recent renaissance in bacteriophage therapy has led to a number of successful cases in human patients. Various studies in mice and humans have shown that bacteriophages are rapidly cleared, meaning the bacteriophages cannot be observed in patient blood after the treatment. Thus, one of the main challenges facing the expanded use of bacteriophage therapy is the creation of bacteriophages that are not cleared rapidly from the patient. The argument for this is clear -- the longer infectious bacteriophages can stay in the body, the greater their chance of finding their pathogenic bacteria host. The longevity of circulating bacteriophages has been investigated several times. These studies showed that longer circulating bacteriophages can be isolated. Unfortunately, often the genomes of these bacteriophages were not sequenced nor were the bacteriophages characterized, leaving a gap in our understanding of how these bacteriophages were altered to survive in the body for extended times. The objective of this proposal is to understand the mechanisms leading to longer bacteriophage survival in the circulatory system. Our work is designed to provide important translational insights for making synthetic bacteriophages able to circulate longer inside patients and thereby increasing the efficacy of bacteriophage therapy. Our central hypothesis is that enhanced circulation times are due to amino acid substitution(s) in the major capsid protein that either stabilize the bacteriophage or allow the bacteriophage to evade capture by immune cells. A classic study of longer-circulating lambda bacteriophages revealed that a single mutation in the capsid protein was responsible for the phenotype, but the mechanism for this phenotype was not investigated. We propose to focus on bacteriophages P22 and Sf6 to understand the mechanism for in vivo stabilization in these two different but tractable and well-characterized bacteriophages, with the following specific aims: Aim 1) Hypothesis: the circulation time is increased by amino acid substitutions in the major capsid protein. Aim1a) Isolate and characterize bacteriophages with enhanced circulation times. Aim1b) Determine if the mutated residues resulting in increased circulation time is a generalizable feature. Aim 2) Hypothesis: the long circulating mutant bacteriophages are not degraded by macrophages.

项目总结 最近噬菌体疗法的复兴导致了一些在人类患者中成功的病例。 在老鼠和人类身上进行的各种研究表明，噬菌体可以迅速清除，这意味着 治疗后，患者血液中不能观察到噬菌体。因此，面临的主要挑战之一 噬菌体疗法的扩展用途是创造出不能迅速清除的噬菌体。 病人。关于这一点的论点是明确的--传染性噬菌体在体内停留的时间越长，就越大 它们找到致病细菌宿主的机会。循环中的噬菌体的寿命已经被 调查了好几次。这些研究表明，可以分离出循环时间更长的噬菌体。 不幸的是，这些噬菌体的基因组通常没有测序，噬菌体也没有测序 在我们对这些噬菌体是如何被改变以在 身体的时间延长了。这项建议的目标是了解导致更长时间 噬菌体在循环系统中的存活。我们的工作旨在提供重要的翻译见解 使合成的噬菌体能够在患者体内循环更长时间，从而提高疗效 噬菌体疗法的最新进展。 我们的中心假设是，循环时间的增加是由于主要的氨基酸替代(S)。 稳定噬菌体或允许噬菌体逃避免疫捕获的衣壳蛋白 细胞。一项对循环时间更长的Lambda噬菌体的经典研究表明，衣壳中的单一突变 蛋白质是导致这种表型的原因，但这种表型的机制还没有被研究。我们 建议将重点放在噬菌体P22和SF6上，以了解它们在体内稳定的机制 两个不同但易驯服且特性良好的噬菌体，具体目标如下： 目的1)假说：主要衣壳蛋白中的氨基酸取代增加了循环时间。 Aim1a)分离和鉴定循环次数增加的噬菌体。 Aim1b)确定突变残基导致循环时间延长是否是可概括的特征。 目的2)假设：长循环突变噬菌体不被巨噬细胞降解。