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Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

ABSTRACT: Host-pathogen interactions (HPIs) are pivotal in regulating establishment, progression, and outcome of an infection. Affinity-purification mass spectrometry has become instrumental for the characterization of HPIs, however the targeted nature of exogenously expressing individual viral proteins has limited its utility to the analysis of relatively small pathogens. Here we present the use of co-fractionation mass spectrometry (SEC-MS) for the high-throughput analysis of HPIs from native viral infections of two Jumbophages (phiKZ and phiPA3) in Pseudomonas aeruginosa. This enabled the detection >6000 unique host-pathogen and >200 pathogen-pathogen interactions for each phage, encompassing >50% of the phage proteome. Interactome-wide comparison across phages showed similar perturbed protein interactions suggesting fundamentally conserved mechanisms of phage predation within the KZ-like phage family. Prediction of novel ORFs revealed a phiPA3 complex showing strong structural and sequence similarity to phiKZ nvRNAp, suggesting phiPA3 also possesses two RNA polymerases acting at different stages of the infection cycle. We further expanded our understanding on the molecular organization of the virion packaged and injected proteome by identifying 23 novel virion components and 5 novel injected proteins, as well as providing the first evidence for interactions between KZ-like phage proteins and the host ribosome. To enable accessibility to this data, we developed PhageMAP, an online resource for network query, visualization, and interaction prediction (https://phagemap.ucsf.edu/). We anticipate this study will lay the foundation for the application of co-fractionation mass spectrometry for the scalable profiling of hostpathogen interactomes and protein complex dynamics upon infection.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Pseudomonas Virus Phikz (ncbitaxon:169683) Pseudomonas Aeruginosa (ncbitaxon:287)

SUBMITTER: Danielle Swaney

PROVIDER: MSV000091715 | MassIVE | Fri Apr 14 07:53:00 BST 2023

REPOSITORIES: MassIVE

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Project description:We used microarray analysis to investigate whole genome transcriptome dynamics of the marine cyanobacterium Prochlorococcus sp. strain MED4 and the T7-like podovirus P-SSP7 over a time course during the 8 hour latent period of lytic infection prior to cell lysis. Manuscript Summary: Interactions between bacterial hosts and their viruses (phages) lead to reciprocal genome evolution through a dynamic co-evolutionary process1-5. Phage-mediated transfer of host genes – often located in genome islands – has had a major impact on microbial evolution1, 4, 6. Furthermore, phage genomes have clearly been shaped by the acquisition of genes from their hosts2, 3, 5. Here we investigate whole-genome expression of a host and phage, the marine cyanobacterium Prochlorococcus and a T7-like cyanophage during lytic infection, to gain insight into these co-evolutionary processes. While most of the phage genome was linearly transcribed over the course of infection, 4 phage-encoded bacterial metabolism genes were part of the same expression cluster, even though they are physically separated on the genome. These genes — encoding photosystem II D1 (psbA), high-light inducible protein (hli), transaldolase (talC) and ribonucleotide reductase (nrd) — are transcribed together with phage DNA replication genes and appear to make up a functional unit involved in energy and deoxynucleotide production needed for phage replication in resource-poor oceans. Also unique to this system was the upregulation of numerous genes in the host during infection. These may be host stress response genes, and/or genes induced by the phage. Many of these host genes are located in genome islands and have homologues in cyanophage genomes. We hypothesize that phage have evolved to utilize upregulated host genes, leading to their stable incorporation into phage genomes and their subsequent transfer back to hosts in genome islands. Thus activation of host genes during infection may be directing the co-evolution of gene content in both host and phage genomes. Keywords: time course, viral infection, marine cyanobacteria, podovirus, bacteriophage, stress response

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Next-generation interaction proteomics for quantitative Jumbophage-bacteria interaction mapping

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