Multi-omics characterization of a lytic phage targeting <i>Listeria monocytogenes</i>

ABSTRACT Listeria monocytogenes is a foodborne pathogenic bacterium that can persist in food-processing environments. Strictly lytic Listeria 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 Listeria . 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 Listeria monocytogenes 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 L. monocytogenes , including phage particle assembly, gene regulation, host takeover, and bacterial response to phage infection.

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