Campylobacter jejuni motility integrates specialized cell shape, flagellar filament, and motor, to coordinate action of its opposed flagella

Abstract

Campylobacter jejuni rotates a flagellum at each pole to swim through the viscous mucosa of its hosts' gastrointestinal tracts. Despite their importance for host colonization, however, how C. jejuni coordinates rotation of these two opposing flagella is unclear. As well as their polar placement, C. jejuni's flagella deviate from the norm of Enterobacteriaceae in other ways: their flagellar motors produce much higher torque and their flagellar filament is made of two different zones of two different flagellins. To understand how C. jejuni's opposed motors coordinate, and what contribution these factors play in C. jejuni motility, we developed strains with flagella that could be fluorescently labeled, and observed them by high-speed video microscopy. We found that C. jejuni coordinates its dual flagella by wrapping the leading filament around the cell body during swimming in high-viscosity media and that its differentiated flagellar filament and helical body have evolved to facilitate this wrapped-mode swimming.

Fig 1. Campylobacter jejuni wraps the leading flagellum and swims faster in viscous media.

As the viscosity of the media increases, the proportion of cells with wrapped leading flagellar filaments also increases (A and B). In addition to the increase in wrapping, both the swim-halo diameter in soft agar and single-cell swimming velocity increase substantially in the presence of methylcellulose. (C and D). In MH broth without methylcellulose added, unwrapped and wrapped cells swim at comparable velocities, while wrapped cells outperform unwrapped cells as velocity increased (E). Doubly-flagellated wrapped cells swim faster than singly-flagellated (i.e. ΔfliI) unwrapped or wrapped cells (F). In B, 100–150 cells in each methylcellulose concentration were used to determine the proportion of wrapped to unwrapped cells in the population. In D, E and F, red bars represent average velocity ± SEM.

Fig 2. Changing swimming trajectory involves a switch in wrapped filament polarity.

Wrapped cells change swimming direction, or tumble, by switching which filament is wrapped. The cell comes to a stop when motor rotation switches from CCW to CW, which also causes the wrapped filament to unwrap. The once lagging, unwrapped filament then wraps around the cell body while the previously wrapped filament becomes the trailing, unwrapped filament. Scale bars equal to 1.5 μm.

Fig 3. The cell body and flagellar filament have opposite handedness.

TIRFM revealed that the cell body is a right-handed (RH) helix, while the filament assumes a left-handed (LH) helical conformation (A and B), and that the filament remains a LH helix whether it is wrapped or unwrapped. The pitch of both the flagellar filament and cell body are approximately identical at ~40° (C).

Fig 4. Unwrapping efficiency and chemotactic ability are impaired in straight cells.

Switching of motor rotation from CCW to CW usually results in filament unwrapping in the WT, but not in straight cells. During reversals, the wrapped filament of Δpgp1 cells will often peel away from the cell body, but fail to fully unwrap (red arrow) (A). In the straight cell background, filament unwrapping upon switching only occurs 26% of the time, compared to 69% in helical cells (B). An aerotaxis competition (C) between WT and Δpgp1 cells revealed that, in addition to reaching the O₂-rich border (double-sided tape) first, the WT also formed a high density, persisting swarm at the border that left few stragglers behind (D and E). Scale bars in A equal to 2 μm. Fluorescence intensity signals in E are the average of three replicate experiments for each mutant.

Fig 5. FlaB forms a more rigid filament than FlaA.

To determine whether the FlaA portion of the filament is less rigid than the FlaB portion, a flow chamber was constructed that allowed us to flow buffer over labeled, immobilized cells (A). When buffer was flowed past WT (i.e. flaA^S397C flaB⁺) or all-FlaA filaments, they stretched to become ~30% longer than their relaxed length. In contrast, all-FlaB filaments stretched only ~10% under the same flow conditions (B and C). For C, 16 all-FlaA filaments and 20 all-FlaB filaments were measured for elongation under flow, error bars represent the SEM for each.