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噬菌体发现与筛选平台:通过共培养靶向发现噬菌体

Phage DisCo: targeted discovery of bacteriophages by co-culture.

作者信息

Rand Eleanor A, Quinones-Olvera Natalia, Jean Kesther D C, Hernandez-Perez Carmen, Owen Siân V, Baym Michael

机构信息

Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA.

Laboratory of Systems Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.

出版信息

mSystems. 2025 Jun 17;10(6):e0164424. doi: 10.1128/msystems.01644-24. Epub 2025 May 28.

Abstract

Phages interact with many components of bacterial physiology from the surface to the cytoplasm. Although there are methods to determine the receptors and intracellular systems a specified phage interacts with retroactively, finding a phage that interacts with a chosen piece of bacterial physiology is very challenging. Variation in phage plaque morphology does not to reliably distinguish distinct phages, and therefore many potentially redundant phages may need to be isolated, purified, and individually characterized to find phages of interest. Here, we present a method in which multiple bacterial strains are co-cultured on the same screening plate to add an extra dimension to plaque morphology data. In this method, covery by -culture (Phage DisCo), strains are isogenic except for fluorescent tags and one perturbation expected to impact phage infection. Differential plaquing on the strains is easily detectable by fluorescent signal and implies that the perturbation made to the surviving strain in a plaque prevents phage infection. We validate the Phage DisCo method by showing that characterized phages have the expected plaque morphology on Phage DisCo plates and demonstrate the power of Phage DisCo for multiple targeted discovery applications, from receptors to phage defense systems.IMPORTANCEIn this work, we describe a targeted phage discovery method that allows immediate isolation of phages with specific traits. Currently, to find a phage with specific properties, huge libraries of phages must be collected and screened retroactively. This assay, Phage Discovery by Co-culture (Phage DisCo), works by co-culture of host strains that are identical except for one perturbation that may interfere with phage infection and a unique fluorescent marker. These strains are co-cultured with an environmental sample of interest in traditional plaque assay format, making phage characteristics easily identifiable by fluorescent signal after imaging of the screening plate. We validate that Phage DisCo can identify phages with specific properties, even when these phages are rare in samples. This approach allows rapid exploration of the diversity within phage samples with vastly streamlined processes, and we anticipate it will be widely adopted within the phage discovery field.

摘要

噬菌体与细菌生理的许多成分相互作用,从表面到细胞质。虽然有方法可以追溯性地确定特定噬菌体相互作用的受体和细胞内系统,但找到与选定的细菌生理部分相互作用的噬菌体极具挑战性。噬菌体噬菌斑形态的变化并不能可靠地区分不同的噬菌体,因此可能需要分离、纯化并单独表征许多潜在冗余的噬菌体,以找到感兴趣的噬菌体。在这里,我们提出了一种方法,将多种细菌菌株在同一筛选平板上共培养,为噬菌斑形态数据增添一个额外维度。在这种共培养噬菌体发现法(Phage DisCo)中,除了荧光标签和一个预期会影响噬菌体感染的干扰因素外,菌株是同基因的。通过荧光信号很容易检测到菌株上的差异噬菌斑形成,这意味着噬菌斑中存活菌株所受到的干扰阻止了噬菌体感染。我们通过证明已表征的噬菌体在Phage DisCo平板上具有预期的噬菌斑形态来验证Phage DisCo方法,并展示了Phage DisCo在从受体到噬菌体防御系统等多种靶向发现应用中的作用。

重要性

在这项工作中,我们描述了一种靶向噬菌体发现方法,可立即分离具有特定特性的噬菌体。目前,为了找到具有特定特性的噬菌体,必须收集大量噬菌体文库并进行追溯性筛选。这种共培养噬菌体发现法(Phage DisCo)的工作原理是将除一个可能干扰噬菌体感染的干扰因素和一个独特荧光标记外完全相同的宿主菌株进行共培养。这些菌株与感兴趣的环境样本以传统噬菌斑测定形式进行共培养,在对筛选平板成像后,通过荧光信号很容易识别噬菌体特征。我们验证了Phage DisCo即使在样本中这些噬菌体很罕见时也能识别具有特定特性的噬菌体。这种方法允许通过大幅简化的流程快速探索噬菌体样本中的多样性,我们预计它将在噬菌体发现领域得到广泛采用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/db6b/12172434/213fbf7c866f/msystems.01644-24.f001.jpg

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