Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly

Abstract

Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a “missing” β′ subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution.IMPORTANCE In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ϕKZ.

Global Proteomic Profiling of Salmonella Infection by a Giant Phage

“…This problem is exacerbated in "giant" or "jumbo" phages with long double-stranded DNA (dsDNA) genomes (Ͼ200 kb) (1,2), such as that of Pseudomonas aeruginosa phage KZ (280 kb), whose genome was the first giant phage genome to be published (3). Recent studies have demonstrated that phages related to KZ infect a range of clinically and agriculturally important gammaproteobacteria, including Salmonella enterica (e.g., SPN3US), Cronobacter sakazakii (e.g., CR5), and Erwinia amylovora (e.g., PhiEaH2) (3)(4)(5)(6)(7)(8)(9)(10). These giant phages are of interest for biocontrol purposes (e.g., phage therapy) and the fact that they have many unusual characteristics compared to other phage types.…”

“…In KZ, the E proteins form a large (15 to 20 MDa) proteinaceous structure-the inner body (IB)-wrapped within the packaged DNA (16). We recently determined that the SPN3US head contains 50 different proteins, the most abundant being the major capsid protein (MCP), which forms the outer shell, and the E proteins gp53 and gp54 (10,15). Giant phage E proteins are assumed to enter the host cell with the phage genome because the KZ IB disappears upon ejection of the genome (17).…”

Radiation Mitigating Properties of Intranasally Administered KL4Surfactant in a Murine Model of Radiation-Induced Lung Damage

“…Similarly, robust lung fibrosis is observed as early as 16–18 weeks postirradiation (24, 52), which is likely due to the use of younger mice (6–8 weeks old) instead of slightly older mice (10–12 weeks old) in the thorax irradiations. Importantly, thoracic exposure models provide useful and much needed information on partial exposure scenarios where lung only is exposed or when radionuclides are inhaled.…”

“…Bronchoalveolar lavage fluid (BALF) cytokine concentrations were determined, as previously described (24), using the Mouse Cytokine 20-Plex Panel (Invitrogen ™ , Carlsbad, CA), according to the manufacturer’s protocol. This multiplex panel permits simultaneous quantification of multiple cytokines in solution by capturing them onto antibody-coated spectrally distinct fluorescent microspheres and measuring fluorescence intensity using the BioPlex 200 (Bio-Rad ® Laboratories Inc., Hercules, CA) system.…”

“…Therefore, we have been exploring these mechanisms of action associated with KL 4 surfactant as they relate to mitigation of radiation-induced lung damage. In our current study, we evaluated the effectiveness of KL 4 surfactant as a novel approach to mitigate both early radiation-induced pneumonitis and the delayed manifestations of lung injury (lung fibrosis) using the well-characterized C57BL/6 thoracic-irradiation mouse model (24–27). Our underlying premise is that KL 4 surfactant, via its immune-modulatory properties (20, 21, 28), should prevent activation of, and/or downregulate the cascade of multiple inflammatory mediators that have been implicated in pathophysiology of radiation-induced lung injury.…”