A mobile intron facilitates interference competition between co-infecting viruses

Birkholz, Erica A.; Morgan, Chase J.; Laughlin, Thomas G.; Lau, Rebecca K.; Prichard, Amy; Rangarajan, Sahana; Meza, Gabrielle N.; Lee, Jina; Armbruster, Emily G.; Suslov, Sergey; Pogliano, Kit; Meyer, Justin R.; Villa, Elizabeth; Corbett, Kevin D.; Pogliano, Joe

doi:10.1101/2023.09.30.560319

Cited by 3 publications

(14 citation statements)

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“…2 A ). GFPmut1 can be used to drive PhiKZ nuclear import of proteins that are normally excluded and remain in the host cytoplasm ( 10 , 15 , 16 ). PhiPA3 gp210 is normally excluded from the PhiKZ phage nucleus, but gp210-GFPmut1 is imported, where it becomes toxic by cleaving the PhiKZ genome at a single recognition site and preventing replication ( 15 , 16 ).…”

Section: Resultsmentioning

confidence: 99%

“…GFPmut1 can be used to drive PhiKZ nuclear import of proteins that are normally excluded and remain in the host cytoplasm ( 10 , 15 , 16 ). PhiPA3 gp210 is normally excluded from the PhiKZ phage nucleus, but gp210-GFPmut1 is imported, where it becomes toxic by cleaving the PhiKZ genome at a single recognition site and preventing replication ( 15 , 16 ). We therefore predicted that through selection for phage that can replicate in the presence of gp210-GFPmut1, we could isolate escape mutants that have altered the ability to traffic proteins into the phage nucleus ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Strains expressing gp210-GFPmut1 exhibit a ~10,000-fold reduction in PhiKZ plaquing, and mutants that escape this selection can be isolated ( 16 ). Here, we isolated PhiKZ gp210-GFPmut1 resistant mutants and found they had mutations in one of two genes with approximately equal frequency ( Table 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…Here, we isolated PhiKZ gp210-GFPmut1 resistant mutants and found they had mutations in one of two genes with approximately equal frequency ( Table 1 ). The first is the virion RNA polymerase subunit gp178 gene, which contains the previously identified gp210 nuclease target site ( 16 ). Mutations at this site are expected to reduce or eliminate gp210-mediated cleavage, rendering the mutant resistant to gp210.…”

Section: Resultsmentioning

confidence: 99%

“…Many host proteins are also excluded, including DNA-targeting defense proteins such as Cas9 and restriction endonucleases ( 1 , 4 , 6 , 10 , 12 ), which provides the phage with broad protection against many host defenses. Furthermore, during coinfection of a single cell by two different nucleus-forming phages, the phage nucleus of one species excludes a toxic nuclease produced by the other ( 15 , 16 ). The high degree of specificity seen in protein localization to the phage nucleus has led to the hypothesis that chimalliviruses encode a selective protein trafficking system ( 1 , 2 , 4 , 10 , 15 , 17 ).…”

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confidence: 99%

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An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Morgan,

Enustun,

Armbruster

et al. 2024

Preprint

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View full text Add to dashboard Cite

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

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Phage-phage competition and biofilms reduce the efficacy of a combination of two virulent bacteriophages againstPseudomonas aeruginosa

Bürkle,

Korf,

Lippegaus

et al. 2024

Preprint

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Combined use of virulent bacteriophages (phages) and antibiotics reduces the severity of difficult-to-treatPseudomonas aeruginosainfections in many patients.In vitromethods that attempt to reproduce more than one physiological state of the pathogens can provide a valuable assessment of antibacterials like phages. Here, by measuring bacterial killing kinetics and individual replication in different growth conditions, including biofilms and a human lung epithelial cell line, we elucidated factors influencing the efficacy of two virulent phages againstP. aeruginosaPAO1. A single administration of phages effectively reduced theP. aeruginosaviability in planktonic conditions and infected human lung cell cultures, however, the emergence of phage-resistant variants occurred subsequently. In static biofilms, the phage combination displayed initial inhibition of biofilm dispersal, but sustained control was achieved only by combining phages and meropenem. In contrast, surface-attached biofilms exhibited tolerance to phage and/or meropenem, suggesting a spatiotemporal variation in the antibacterial effect. Moreover, the phage with the shorter lysis time lysedP. aeruginosamore rapidly and selected a specific nucleotide polymorphism that conferred a competitive disadvantage and cross-resistance to the second phage of the combination. The sequential addition of phages resulted in their unimpeded replication with no increase in bacteriolytic activity. These findings highlight biofilm developmental stages, phage-phage competition, and phage resistance as factors restricting thein vitroefficacy of a two-phage combination. Our findings provide a framework for selecting and optimizing phage combinations for enhanced efficacy againstP. aeruginosa, a metabolically flexible pathogen that undergoes specific adaptation within the infected lung.

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A mobile intron facilitates interference competition between co-infecting viruses

Cited by 3 publications

References 86 publications

An essential and highly selective protein import pathway encoded by nucleus-forming phage

An essential and highly selective protein import pathway encoded by nucleus-forming phage

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Phage-phage competition and biofilms reduce the efficacy of a combination of two virulent bacteriophages againstPseudomonas aeruginosa

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