噬菌体的定向进化:受挫于多产的噬菌体。

IF 3.9 2区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Applied and Environmental Microbiology Pub Date : 2024-11-20 Epub Date: 2024-10-30 DOI:10.1128/aem.00884-24
Tracey Lee Peters, Jacob Schow, Emma Spencer, James T Van Leuven, Holly Wichman, Craig Miller
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引用次数: 0

摘要

目前存在各种定向进化方法,旨在获得宿主范围更广的噬菌体,通常以噬菌体抗性或非抗性细菌宿主为目标。这些方法的一般前提是在多个细菌宿主上繁殖噬菌体,汇集裂解物,并重复这一过程,直到噬菌体能在目标宿主上形成斑块。从理论上讲,这将产生含有输入噬菌体及其进化后的噬菌体后代的裂解液。然而,在实践中,这种裂解液也可能包括来自细菌宿主的噬菌体。在这里,我们介绍了我们在铜绿假单胞菌噬菌体-宿主系统中研究噬菌体进化时采用的一种定向进化方法--阿佩尔曼斯方案的经验,我们观察到鸡尾酒噬菌体的宿主范围迅速扩大。进一步的实验和测序发现,所观察到的宿主范围扩大是由于一种来自溶原宿主的卡萨达班病毒噬菌体造成的,而这种噬菌体只包含在前三轮实验中。在最初使用的 8 个细菌宿主中,该噬菌体可以感染其中的 5 个,使其能够持续增殖,直到实验结束。在从阿佩尔曼斯实验中分离出来的噬菌体测序样本中,有一半含有这种噬菌体,但尽管在定向进化条件下,它似乎并没有进化。这项工作凸显了噬菌体在定向进化实验中的影响,以及对产出噬菌体进行基因验证的重要性,特别是对于那些试图获得用于噬菌体疗法的噬菌体的人来说。本研究还指出了细菌宿主菌株之间的种内拮抗试验对于建立抑制活性基线和确定噬菌体是否存在的作用。重要意义 定向进化是噬菌体进化的一种常见策略,它通常以致病菌株为目标,扩大宿主范围。在这项研究中,我们利用铜绿假单胞菌系统中的定向进化研究了噬菌体宿主范围的扩展。我们发现,噬菌体是定向进化中的积极参与者,并有助于观察宿主范围的扩展。由于噬菌体普遍存在于细菌宿主中,尤其是致病菌株,而所有定向进化方法都涉及在一个或多个细菌宿主上反复繁殖噬菌体,因此噬菌体制备物中噬菌体的存在是实验设计和结果解释中需要考虑的一个因素。这些结果凸显了在选择细菌菌株时通过基因或种内拮抗试验筛选噬菌体的重要性,并将有助于改进未来定向进化研究的实验设计。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Directed evolution of bacteriophages: thwarted by prolific prophage.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage.IMPORTANCEDirected evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies.

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来源期刊
Applied and Environmental Microbiology
Applied and Environmental Microbiology 生物-生物工程与应用微生物
CiteScore
7.70
自引率
2.30%
发文量
730
审稿时长
1.9 months
期刊介绍: Applied and Environmental Microbiology (AEM) publishes papers that make significant contributions to (a) applied microbiology, including biotechnology, protein engineering, bioremediation, and food microbiology, (b) microbial ecology, including environmental, organismic, and genomic microbiology, and (c) interdisciplinary microbiology, including invertebrate microbiology, plant microbiology, aquatic microbiology, and geomicrobiology.
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