Cucić Stevan, Putzeys Leena, Boon Maarten, Lepp Dion, Lavigne Rob, Khursigara Cezar M, Anany Hany
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
Agriculture and Agri-Food Canada, Guelph Research and Development Centre, Guelph, Ontario, Canada.
mSystems. 2025 Jun 25:e0058725. doi: 10.1128/msystems.00587-25.
is a foodborne pathogenic bacterium that can persist in food-processing environments. Strictly lytic phages have shown promise as biosanitation and biocontrol agents. However, little is known about the molecular progression of phage expression and the host gene expression profile it elicits in . In this work, the P100-like phage CKA15 was characterized using a proteogenomics-based approach to identify virion-associated proteins, Illumina-based RNA-seq to analyze time-resolved host and phage transcript abundance during infection, and ONT-cappable-seq to experimentally determine the operon structure of the phage genome. We detected 29 phage-encoded putative particle-associated proteins. During infection, a progressive decrease in host transcript abundance and an increase in phage transcript abundance are observed. The progression of phage gene expression indicates a switch in functions from hypothetical at 5 min; nucleic acid metabolism at 15; structural proteins at 25; and DNA packaging, tail assembly, and lysis at 40 min post-infection. Using ONT-cappable-seq, we identified 81 phage transcription start sites (TSS) and 66 transcription termination sites (TTS). We used motif analysis to identify two classes of promoters, corresponding to early and late infection stages. Profound changes in the host transcriptome became evident 5 min post-infection. GO enrichment and KEGG pathway analysis indicate a downregulation of host transcription factor expression and an upregulation of translation, cobalamin biosynthesis, and propanediol metabolism. This research contributes to our systems-level understanding of the infection process of a strictly lytic phage infecting an important foodborne pathogen.IMPORTANCE is an important foodborne pathogenic bacterium that contributes to significant mortality worldwide. Since bacteriophages have evolved diverse mechanisms to take over their host bacteria, studying phage interactions with pathogenic bacteria enables researchers to develop novel ways of controlling pathogenic bacteria and tools to study them. Detection of phage particle-associated proteins using mass spectrometry combined with transcriptomic techniques that determine the operon structure of the phage genome, time-resolved transcript abundance of phage, as well as host transcripts, comprises powerful approaches for phage characterization. Moreover, these analyses provide a starting point for hypothesis generation in relation to different aspects of the biology of phages infecting , including phage particle assembly, gene regulation, host takeover, and bacterial response to phage infection.
是一种食源性病原体细菌，可在食品加工环境中持续存在。严格裂解性噬菌体已显示出作为生物卫生和生物防治剂的潜力。然而，关于噬菌体表达的分子进程及其在[具体细菌名称未给出]中引发的宿主基因表达谱，我们知之甚少。在这项工作中，使用基于蛋白质基因组学的方法对P100样噬菌体CKA15进行了表征，以鉴定病毒体相关蛋白，使用基于Illumina的RNA测序分析感染期间时间分辨的宿主和噬菌体转录本丰度，并使用ONT-cappable-seq实验确定噬菌体基因组的操纵子结构。我们检测到29种噬菌体编码的假定颗粒相关蛋白。在感染过程中，观察到宿主转录本丰度逐渐下降，噬菌体转录本丰度增加。噬菌体基因表达的进程表明，在感染后5分钟功能从假设转变为核酸代谢，15分钟为核酸代谢，25分钟为结构蛋白，40分钟为DNA包装、尾部组装和裂解。使用ONT-cappable-seq，我们鉴定出81个噬菌体转录起始位点（TSS）和66个转录终止位点（TTS）。我们使用基序分析鉴定了两类启动子，分别对应感染的早期和晚期阶段。感染后5分钟，宿主转录组发生了明显变化。GO富集和KEGG通路分析表明宿主转录因子表达下调，翻译、钴胺素生物合成和丙二醇代谢上调。这项研究有助于我们从系统层面理解一种严格裂解性噬菌体感染重要食源性病原体的感染过程。重要性[具体细菌名称未给出]是一种重要的食源性病原体细菌，在全球范围内导致大量死亡。由于噬菌体已经进化出多种机制来接管其宿主细菌，研究噬菌体与病原菌的相互作用使研究人员能够开发控制病原菌的新方法和研究它们的工具。使用质谱结合转录组技术检测噬菌体颗粒相关蛋白，该技术可确定噬菌体基因组的操纵子结构、噬菌体的时间分辨转录本丰度以及宿主转录本，是噬菌体表征的有力方法。此外，这些分析为关于感染[具体细菌名称未给出]的噬菌体生物学不同方面的假设生成提供了起点，包括噬菌体颗粒组装、基因调控、宿主接管以及细菌对噬菌体感染的反应。
