A hybrid receptor binding protein enables phage F341 infection of Campylobacter by binding to flagella and lipooligosaccharides

Abstract

Flagellotropic bacteriophages are interesting candidates as therapeutics against pathogenic bacteria dependent on flagellar motility for colonization and causing disease. Yet, phage resistance other than loss of motility has been scarcely studied. Here we developed a soft agar assay to study flagellotropic phage F341 resistance in motile Campylobacter jejuni. We found that phage adsorption was prevented by diverse genetic mutations in the lipooligosaccharides forming the secondary receptor of phage F341. Genome sequencing showed phage F341 belongs to the Fletchervirus genus otherwise comprising capsular-dependent C. jejuni phages. Interestingly, phage F341 encodes a hybrid receptor binding protein (RBP) predicted as a short tail fiber showing partial similarity to RBP1 encoded by capsular-dependent Fletchervirus, but with a receptor binding domain similar to tail fiber protein H of C. jejuni CJIE1 prophages. Thus, C. jejuni prophages may represent a genetic pool from where lytic Fletchervirus phages can acquire new traits like recognition of new receptors.

Keywords: Campylobacter; Fletchervirus; flagella; flagellotropic phage; phage; phage receptor; phage resistance; receptor binding protein.

Figure 1

Motile phage F341 resistant C. jejuni NCTC12658 variants (LJ strains) isolated in vitro demonstrate lack of phage binding. (A) Soft agar (SA) assay with C. jejuni NCTC12658, NCTC12568∆motA and NCTC12658 exposed to phage F341 demonstrating different levels of swarming and non-swarming colonies. Examples of swarming colonies spreading out on the agar plate by flagellar motility are indicated by arrows. (B) Phage F341 infection and motility profiles of isolated swarming NCTC12658 colonies exposed to phage F341 (LJ strains). (C) Phage F341 adsorption assay of phage resistant LJ strains. t equals the timepoint in minutes where samples were collected, and the number of free phages determined. Pfu/ml of free phages at time zero was accounted as 100%. The images represent two independent experiments; however, the LJ colonies were only isolated from plates from one of the independent experiments. Error bars and ± indicated mean standard deviations.

Figure 2

Phage F341 uses lipooligosaccharides as a secondary receptor for successful infection of its host C. jejuni NCTC12658. (A) Schematic representation of the lipooligosaccharides (LOS) putatively produced by C. jejuni NCTC12658, the LJ phage F341 resistant NCTC12658 variants and the C. jejuni NCTC12658ΔLOS (C. jejuni NCTC12658Δ05515-05525) mutant. (B) Standard plaque assays of phage F341 on its propagation host C. jejuni NCTC12658, the non-motile C. jejuni NCTC12658ΔmotA mutant and C. jejuni NCTC12658ΔLOS. (C) Mean F341 log10 PFU/mL values from standard plaque assays on C. jejuni NCTC12658, C. jejuni NCTC12658ΔmotA and C. jejuni NCTC12658ΔLOS. 3 × 10 μL of undiluted phage F341 stock and tenfold serial dilutions up to 10⁻⁷ are spotted clockwise from the top of all plaque assay plates. The images represent 2–4 independent experiments and error bars indicated standard deviations. Gal, galactose; GalNAc, N-acetylgalactoseamine; Neu5Ac, N-acetylneuraminic acid (sialic acid); Hep, heptose; Glu, glucose; Kdo, ketodeoxytonic acid. See also Supplementary Figure S2.

Figure 3

Flagellotropic phage F341 belongs to the Fletchervirus genus but shows limited sequence similarity to related phages in the RBP region. (A) Comparative genomics of phage F341 and 18 Fletchervirus phages. Whole genome alignment visualized as a heat map for average nucleotide identity. Closest relatives are further indicated by the trees. (B) Alignment of the receptor binding protein region in Fletchervirus phages dependent on the capsular polysaccharide for infection and phage F341. Phage F358 is used as a representative of capsular-dependent Fletchervirus phages. (C) Protein alignment of the RBP of phage F341 and the tail fiber protein H of the CJIE1 prophage in C. jejuni strain HF5-4A-4. Comparative genomics was performed using CLC main workbench version 20.0.4 using the whole genome alignment 20.1 tool with default settings. See also Supplementary Figures S3, S4.

Figure 4

Phage F341 encodes and expresses a unique hybrid RBP with a short tail fiber morphology. (A) Standard and anti-RBP serum plaque assays of phage F341 on its host C. jejuni NCTC12658. (B) Mean F341 log10 pfu/ml values from standard and anti-RBP serum plaque assays on C. jejuni NCTC12658. Anti-RBP1 is serum produced against RBP1 from capsular-dependent Fletchervirus phage F358 (C) Transmission electron microscopy of negatively stained phage F341 and when mixed with immunogold labeled anti-RBP serum. Labeled antibodies bound to phage F341 tail fibers are indicated by arrows. (D) AlphaFold2 predictions of homotrimers of RBP1 from capsular-dependent Fletchervirus phage F358, the RBP from flagellotropic Fletchervirus phage F341 and the tail fiber protein H of the CJIE1 prophage in C. jejuni strain HF5-4A-4. The pectin lyase domain in RBP1 is indicated in cyan blue, whereas the C-terminal region demonstrating sequence similarity between F341_RBP and the tail fiber protein H is indicated in light pink. 3 × 10 μL of undiluted phage F341 stock and tenfold serial dilutions up to 10⁻⁷ are spotted clockwise from the top of all plaque assay plates. Anti-RBP and anti-RBP1 serum was added at a 1:10 ratio to the phage F341 stock for 1 h at room temperature before the serial dilutions were prepared and spotted on the NCTC12658 lawn. The images represent 2–4 independent experiments and error bars indicated standard deviations. See also Supplementary Figure S5.