Global Proteomic Profiling of Salmonella Infection by a Giant Phage - PubMed (original) (raw)

. 2019 Feb 19;93(5):e01833-18.

doi: 10.1128/JVI.01833-18. Print 2019 Mar 1.

Nurul Humaira Mohd Redzuan 2, Melissa K Barton 2, Nur Amira Md Amin 2, Maxim I Desmond 2, Lily E Adams 2, Bazla Ali 2, Sammy Pardo 1, Dana Molleur 1, Weimin Wu 3, William W Newcomb 3, Michael V Osier 2, Lindsay W Black 4, Alasdair C Steven 3, Julie A Thomas 5

Affiliations

Global Proteomic Profiling of Salmonella Infection by a Giant Phage

Susan T Weintraub et al. J Virol. 2019.

Abstract

The 240-kb Salmonella phage SPN3US genome encodes 264 gene products, many of which are functionally uncharacterized. We have previously used mass spectrometry to define the proteomes of wild-type and mutant forms of the SPN3US virion. In this study, we sought to determine whether this technique was suitable for the characterization of the SPN3US proteome during liquid infection. Mass spectrometry of SPN3US-infected cells identified 232 SPN3US and 1,994 Salmonella proteins. SPN3US proteins with related functions, such as proteins with roles in DNA replication, transcription, and virion formation, were coordinately expressed in a temporal manner. Mass spectral counts showed the four most abundant SPN3US proteins to be the major capsid protein, two head ejection proteins, and the functionally unassigned protein gp22. This high abundance of gp22 in infected bacteria contrasted with its absence from mature virions, suggesting that it might be the scaffold protein, an essential head morphogenesis protein yet to be identified in giant phages. We identified homologs to SPN3US gp22 in 45 related giant phages, including ϕKZ, whose counterpart is also abundant in infected bacteria but absent in the virion. We determined the ϕKZ counterpart to be cleaved in vitro by its prohead protease, an event that has been observed to promote head maturation of some other phages. Our findings are consistent with a scaffold protein assignment for SPN3US gp22, although direct evidence is required for its confirmation. These studies demonstrate the power of mass spectral analyses for facilitating the acquisition of new knowledge into the molecular events of viral infection.IMPORTANCE "Giant" phages with genomes >200 kb are being isolated in increasing numbers from a range of environments. With hosts such as Salmonella enterica, Pseudomonas aeruginosa, and Erwinia amylovora, these phages are of interest for phage therapy of multidrug-resistant pathogens. However, our understanding of how these complex phages interact with their hosts is impeded by the proportion (∼80%) of their gene products that are functionally uncharacterized. To develop the repertoire of techniques for analysis of phages, we analyzed a liquid infection of Salmonella phage SPN3US (240-kb genome) using third-generation mass spectrometry. We observed the temporal production of phage proteins whose genes collectively represent 96% of the SPN3US genome. These findings demonstrate the sensitivity of mass spectrometry for global proteomic profiling of virus-infected cells, and the identification of a candidate for a major head morphogenesis protein will facilitate further studies into giant phage head assembly.

Keywords: Salmonella; bacteriophage assembly; giant phage; mass spectrometry.

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Figures

FIG 1

FIG 1

Major genes required to form the virion of a myovirus (phage with a contractile tail). (A) Scheme of the ordering of genes required to make the head (or capsid) of most tailed phages. Red question marks indicate genes that have yet to be identified in SPN3US. (B) Linear representation of the SPN3US genome showing the locations of known head and tail morphogenesis genes from panel A. (C) Representation of the major genes required to make a contractile tail. Capsid and tail images are by W. Wu (work in progress).

FIG 2

FIG 2

Production of SPN3US proteins during liquid infection of Salmonella enterica serovar Typhimurium LT2. Black inner ring peaks represent an estimate of relative abundance (SC/M, spectral count divided by molecular mass) of each protein at Time-60. The relative numbers of mass spectral counts at each time point adjusted by the highest number of spectra detected for each protein is indicated. The exterior ring depicts the SPN3US genome map with virion protein genes shaded blue and nonvirion protein genes shaded black. Highly abundant proteins, including major virion proteins, the major capsid protein (MCP), the tail sheath, and tube proteins are labeled.

FIG 3

FIG 3

Identification of Salmonella proteins during infection by SPN3US, and in an uninfected control. (A) Major outer membrane proteins. (B and C) Major ribosomal proteins and proteins with roles relating to protein expression and folding. (D) Major RNA polymerase and DNA polymerase subunits.

FIG 4

FIG 4

Mass spectral identification of major SPN3US major head proteins and the scaffold protein candidate, gp22, during infection.

FIG 5

FIG 5

The gene locale of SPN3US scaffold candidate, gp22, is conserved in related phages.

FIG 6

FIG 6

Assay of ϕKZ gp54 with the prohead protease gp175. (A) SDS-PAGE gel of assay of recombinant ϕKZ proteins gp54 with gp175 in a lysate of E. coli proteins. A control of gp54 only was included. The amounts of each sample loaded are indicated. Gel slices processed for MS analyses are indicated by a red square. (B) Semitryptic and tryptic mass spectra for gp54 identified by MS in the gel slices indicated in panel A.

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