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

Abstract

Many bacterial pathogens display glycosylated surface structures that contribute to virulence, and targeting these structures is a viable strategy for pathogen control. The foodborne pathogen Campylobacter jejuni expresses a vast diversity of flagellar glycans, and flagellar glycosylation is essential for its virulence. Little is known about why C. jejuni encodes such a diverse set of flagellar glycans, but it has been hypothesized that evolutionary pressure from bacteriophages (phages) may have contributed to this diversity. However, interactions between Campylobacter phages and host flagellar glycans have not been characterized in detail. Previously, we observed that Gp047 (now renamed FlaGrab), a conserved Campylobacter phage protein, binds to C. jejuni flagella displaying the nine-carbon monosaccharide 7-acetamidino-pseudaminic acid, and that this binding partially inhibits cell growth. However, the mechanism of this growth inhibition, as well as how C. jejuni might resist this activity, are not well-understood. Here we use RNA-Seq to show that FlaGrab exposure leads C. jejuni 11168 cells to downregulate expression of energy metabolism genes, and that FlaGrab-induced growth inhibition is dependent on motile flagella. Our results are consistent with a model whereby FlaGrab binding transmits a signal through flagella that leads to retarded cell growth. To evaluate mechanisms of FlaGrab resistance in C. jejuni, we characterized the flagellar glycans and flagellar glycosylation loci of two C. jejuni strains naturally resistant to FlaGrab binding. Our results point toward flagellar glycan diversity as the mechanism of resistance to FlaGrab. Overall, we have further characterized the interaction between this phage-encoded flagellar glycan-binding protein and C. jejuni, both in terms of mechanism of action and mechanism of resistance. Our results suggest that C. jejuni encodes as-yet unidentified mechanisms for generating flagellar glycan diversity, and point to phage proteins as exciting lenses through which to study bacterial surface glycans.

Keywords: Campylobacter jejuni; bacterial surface structures; bacteriophages; flagella; mass spectrometry; motility; protein glycosylation; pseudaminic acid.

Figure 1

Addition of CC-FlaGrab to C. jejuni 11168 cells leads to changes in gene expression. For all transcribed C. jejuni 11168 genes, the negative log of the FDR-adjusted p-value is plotted against the log₂ fold change in transcription following CC-FlaGrab treatment compared with buffer-treated controls.

Figure 2

C. jejuni 11168 wild type, ΔmotA mutant, and ΔmotB mutant cells are similarly bound by CC-FlaGrab, as shown by immunogold labeling with FlaGrab-specific antibodies and transmission electron microscopy. (Inset) Only wild type cell growth is cleared by CC-FlaGrab, as shown by digital images of overnight growth on soft agar overlay plates following spotting with equal concentrations of CC-FlaGrab. All images are representative of experiments done in duplicate. Scale bars represent 800 nm.

Figure 3

Transmission electron microscopy with immunogold labeling using CC-FlaGrab followed by anti-FlaGrab antibodies and then immunogold-conjugated secondary antibodies to probe C. jejuni strains. From top left: strains C. jejuni 11168, 12567, 12660, 12664, 12661-1, and 12661-2. Strain 12661 is depicted twice to show the two phenotypes that were observed with this strain. Scale bars represent 800 nm.

Figure 4

Schematic depicting the flagellar glycosylation loci of strains C. jejuni 12567, 12660, 12661 and 12664, as compared to C. jejuni 11168. Strain names indicated in red refer to strains that either variably (12661) or consistently (12664) showed a lack of FlaGrab binding by immunogold/TEM. Asterisks/bolded genes contain a phase-variable poly-G tract. Colors represent genes involved in the same biological pathway (green: pse biosynthesis, yellow: leg biosynthesis, black: flagellar filament biosynthesis) or genes within the same family according to predicted function (purple: DUF2920 domain-containing genes, pink: maf genes). Other genes are indicated in gray. (A)–(I) are used to indicate regions of heterogeneity in gene content between strains, which are referred to in the text. Arrow direction indicates direction of transcription. Gene lengths are not to scale. Pseudogenes are not depicted.

Figure 5

Strain 12661 harbors a poly-G tract-containing insertion in pseD that is not present in strains 11168, 12567, 12660, or 12664. Nucleotide sequence alignment of an internal region of pseD for all strains showing the 25 inserted nucleotides in strain 12661, nine of which make up a poly-G tract.

Figure 6

LC/MS-MS fragmentation spectra of trypsin-digested C. jejuni 11168 flagellar glycopeptide ¹⁸⁰ISTSGEVQFTLK¹⁹¹. (A) ¹⁸⁰ISTSGEVQFTLK¹⁹¹ + Pse5Ac7Ac (oxonium ion m/z = 317.13); (B) ¹⁸⁰ISTSGEVQFTLK¹⁹¹ + Pse5Ac7Am (oxonium ion m/z = 316.15); (C), ¹⁸⁰ISTSGEVQFTLK¹⁹¹ + DMGA-Pse5Ac7Ac (oxonium ion m/z = 391.17); (D) ¹⁸⁰ISTSGEVQFTLK¹⁹¹ + DMGA-Pse5Ac7Am (oxonium ion m/z=390.19). Oxonium ions are circled, and proposed structures of monosaccharides are shown.

Figure 7

Percent relative abundance of flagellar glycans presented by C. jejuni 11168, 12661, and 12664 for four detected C. jejuni FlaA/FlaB peptides as determined by LC-MS/MS. In 12664, the peptide ⁴⁶⁴TTAFGVK⁴⁷⁰ was not detected, and thus no bar is depicted.

Figure 8

LC/MS-MS fragmentation spectra of trypsin-digested C. jejuni 12661 flagellar glycopeptide ¹⁸⁰ISSSGEVQFTLK¹⁹¹. (A) MS² spectrum of ¹⁸⁰ISSSGEVQFTLK¹⁹¹ + a glycan of oxonium ion m/z = 317.13 (C₁₃H₂₁O₇${\text{N}}_2^{+}$), (B) MS² spectrum of ¹⁸⁰ISSSGEVQFTLK¹⁹¹ + a glycan of oxonium ion m/z = 332.15 (C₁₃H₂₂O₇${\text{N}}_3^{+}$). Oxonium ions are circled, and proposed structures of monosaccharides are shown.

Figure 9

MS² fragmentation spectra of trypsin-digested C. jejuni 12664 flagellar glycopeptide ¹⁸⁰ISSSGEVQFTLK¹⁹¹. (A) MS² spectrum of ¹⁸⁰ISSSGEVQFTLK¹⁹¹ + a glycan of oxonium ion m/z = 316.15 (C₁₃H₂₂O₆${\text{N}}_3^{+}$); (B) MS² spectrum of ¹⁸⁰ISSSGEVQFTLK¹⁹¹ + a glycan of oxonium ion m/z = 331.16 (C₁₃H₂₃O₆${\text{N}}_4^{+}$). Oxonium ions are circled, and proposed structures of monosaccharides are shown.

Figure 10

Transmission electron micrographs of FlaGrab-labeled C. jejuni 11168 and 11168Δcj1295. Images are representative of experiments done in duplicate. Scale bars represent 800 nm.