Binding of Phage-Encoded FlaGrab to Motile Campylobacter jejuni Flagella Inhibits Growth, Downregulates Energy Metabolism, and Requires Specific Flagellar Glycans

Sacher, Jessica C; Shajahan, Asif; Butcher, James; Patry, Robert T.; Flint, Annika; Hendrixson, David R.; Stintzi, Alain; Azadi, Parastoo; Szymanski, Christine M.

doi:10.3389/fmicb.2020.00397

Cited by 15 publications

(18 citation statements)

References 71 publications

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“…These new data support and help further explain our previously reported observations that NCTC 12673 phage infection induces upregulation of oxidative stress defense genes in C. jejuni, and that mutation of these genes leads to reduced phage infection efficiency [23]. Lastly, we found that relative to the parent phage, MutC appears to more frequently encode a truncated version of a phase-variable flagellar glycan binding protein, Gp047 (recently renamed FlaGrab, [24]). These results support a model whereby phage expression of FlaGrab during infection may exacerbate oxidative stress in the cell through interaction with flagellar glycan intermediates, suggesting that the phage may have evolved a mechanism of toggling an oxidatively-stressed state in its host, and as a result, its own infection efficiency.…”

Section: Introductionsupporting

confidence: 88%

“…To better understand the possible NCTC 12673 dependence on flagellar motility, and to verify previous results that showed that this phage did not plaque on a "paralyzed" (full-length but non-motile) flagella ΔpflB mutant, we tested NCTC 12673 phage plaquing efficiency on flagellar motor mutants ΔmotA and ΔmotB [21,24]. Interestingly, we found that NCTC 12673 phage infection was essentially abolished on both mutants (Figure 1A).…”

Section: Phage Nctc 12673 Is Unable To Infect Cells In the Absence Of The Flagellar Motor Proteins Mota Or Motbmentioning

confidence: 62%

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Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress

Sacher

Javed

Crippen

et al. 2021

Viruses

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Campylobacter jejuni is a Gram-negative foodborne pathogen that causes diarrheal disease and is associated with severe post-infectious sequelae. Bacteriophages (phages) are a possible means of reducing Campylobacter colonization in poultry to prevent downstream human infections. However, the factors influencing phage-host interactions must be better understood before this strategy can be predictably employed. Most studies have focused on Campylobacter phage binding to the host surface, with all phages classified as either capsule- or flagella-specific. Here we describe the characterization of a C. jejuni phage that requires functional flagellar glycosylation and motor genes for infection, without needing the flagella for adsorption to the cell surface. Through phage infectivity studies of targeted C. jejuni mutants, transcriptomic analysis of phage-resistant mutants, and genotypic and phenotypic analysis of a spontaneous phage variant capable of simultaneously overcoming flagellar gene dependence and sensitivity to oxidative stress, we have uncovered a link between oxidative stress, flagellar motility, and phage infectivity. Taken together, our results underscore the importance of understanding phage-host interactions beyond the cell surface and point to host oxidative stress state as an important and underappreciated consideration for future phage-host interaction studies.

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Section: Introductionsupporting

confidence: 88%

Section: Phage Nctc 12673 Is Unable To Infect Cells In the Absence Of The Flagellar Motor Proteins Mota Or Motbmentioning

confidence: 62%

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress

Sacher

Javed

Crippen

et al. 2021

Viruses

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Section: Discussionmentioning

confidence: 99%

Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage ϕ CbK Predation of Multiflagellin Bacteria

et al. 2021

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Caulobacter crescentus is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, the bacteriophage ϕCbK likely recognizes a common motif among the six known flagellins in C. crescentus with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigations of how phage ϕCbK infects C. crescentus at the molecular level. IMPORTANCE Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in C. crescentus for infection by bacteriophage ϕCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ϕCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ϕCbK attaches to flagellar filaments at the molecular level.

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“…What are the functional consequences of these modifications? It remains to be seen if addition of these post-translational modifications can have an effect on motility as observed in C. difficile(68), or if they are required for phage association and filament assembly like in C. jejuni(69,70).We have established a tractable platform for the isolation of various flagellar filaments from C. crescentus and rapid structure determination via cryo-electron microscopy. We found that both conformational and compositional heterogeneity are potential roadblocks to overcome when determining helical structures.…”

mentioning

confidence: 99%

Flagellar structures from the bacteriumCaulobacter crescentusand implications for phage ϕCbK predation of multi-flagellin bacteria

Eric

Nicoleta

Sanchez

et al. 2020

Preprint

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Caulobacter crescentus is a gram-negative alpha-proteobacterium that commonly lives in oligotrophic fresh and salt-water environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which first adsorb to cells by interaction with the bacterial flagellum. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, bacteriophage ϕCbK likely recognizes a common motif amongst the six known flagellins in C. crescentus with varying degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe post-translational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigation of how phage ϕCbK infects C. crescentus at the molecular level.

show abstract

Binding of Phage-Encoded FlaGrab to Motile Campylobacter jejuni Flagella Inhibits Growth, Downregulates Energy Metabolism, and Requires Specific Flagellar Glycans

Cited by 15 publications

References 71 publications

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress

Flagellar Structures from the Bacterium Caulobacter crescentus and Implications for Phage ϕ CbK Predation of Multiflagellin Bacteria

Flagellar structures from the bacteriumCaulobacter crescentusand implications for phage ϕCbK predation of multi-flagellin bacteria

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