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. 2019 May;4(5):774-780.
doi: 10.1038/s41564-019-0378-9. Epub 2019 Feb 25.

Pseudomonas Aeruginosa Orchestrates Twitching Motility by Sequential Control of Type IV Pili Movements

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Free PMC article

Pseudomonas Aeruginosa Orchestrates Twitching Motility by Sequential Control of Type IV Pili Movements

Lorenzo Talà et al. Nat Microbiol. .
Free PMC article

Abstract

Prokaryotes have the ability to walk on surfaces using type IV pili (TFP), a motility mechanism known as twitching1,2. Molecular motors drive TFP extension and retraction, but whether and how these movements are coordinated is unknown3. Here, we reveal how the pathogen Pseudomonas aeruginosa coordinates the motorized activity of TFP to power efficient surface motility. To do this, we dynamically visualized TFP extension, attachment and retraction events at high resolution in four dimensions using label-free interferometric scattering microscopy (iSCAT)4. By measuring TFP dynamics, we found that the retraction motor PilT was sufficient to generate tension and power motility in free solution, while its partner ATPase PilU may improve retraction only in high-friction environments. Using precise timing of successive attachment and retraction, we show that P. aeruginosa engages PilT motors very rapidly and almost only when TFP encounter the surface, suggesting contact sensing. Finally, measurements of TFP dwell times on surfaces show that tension reinforced the adhesion strength to the surface of individual pili, thereby increasing effective pulling time during retraction. The successive control of TFP extension, attachment, retraction and detachment suggests that sequential control of motility machinery is a conserved strategy for optimized locomotion across domains of life.

Conflict of interest statement

Competing interests: Authors declare no competing interests.

Figures

Fig. 1
Fig. 1. iSCAT reveals extracellular bacterial filaments.
(a) Brightfield (left) and iSCAT (right) images of a single WT P. aeruginosa cell. The brightfield image only shows the rod-shaped body of the bacterium, while multiple extracellular structures are visible in iSCAT. Flagellum (black arrowhead) and type IV pili with constant (black arrow) or spatially varying contrast (white arrow). (b) A deletion mutant of the pilin gene pilA displayed no extracellular slender structures, while (c) a mutant in the flagellin subunit gene fliC had no polar filaments with alternating contrast. Inserted illustrations in iSCAT images show the position of cell and filaments relative to the coverslip surface. (d) Quantification of mean TFP per cell with iSCAT. WT has about one pilus per cell in average, (number of cells from the same culture n = 20), pilA- has none (n = 19) but WT levels are restored in the complemented strain (n = 22). fliC- mutant cells have more TFP than WT (n = 15), which decreases to WT level in the complemented strain (n = 18). We attribute the hyperpiliation of fliC- to a selection effect during sample loading (see Materials and Methods). Small circles are individual measurements, large circles are means of bootstrap medians, error bars represent bootstrap 95% confidence interval. (e) Attached TFP length distribution in WT (combined number of pili from cells imaged in at least three biological replicates n = 27) and fliC- (n = 54). The flagellum-less mutant has no defect in TFP length. Small circles represent individual measurements, large circles are bootstrap medians and error bars bootstrap 95% confidence interval. (a, b, c) Scale bars: 2 µm.
Fig. 2
Fig. 2. Visualization of TFP position and retraction in three dimensions.
(a) Images of three representative TFP positions and orientations visualized by iSCAT, with corresponding iSCAT intensity values along the length of the fiber (below). Changes in iSCAT contrast allows us to infer pilus position in 3D. TFP that have constant contrast are flat on the surface (left panel), the ones with alternating contrast form a finite angle with the coverslip (middle panel), and curved fibers with oscillating and irregular contrast are defined as “floppy” (right panel). Below each image, a plot of the pixel grey value along the pilus allows us to determine if the pili lay flat, at an angle or are fluctuating. The illustration represents the putative 3D orientation of the pilus and the cell body. (b) TFP of fliC- deletion mutant cells exhibit all three morphologies (Supplementary video 3). (c) Cells lacking both retraction motor genes pilT and pilU only show floppy TFP, demonstrating retraction and tension force generates straight TFP morphology (Supplementary video 4). We encountered these features throughout all our visualizations. Tensed, flat or at an angle (black arrow), and floppy (white arrows) pili were observed with similar results in all our retractile strains (WT, fliC-, and pilU- fliC-). Whereas only the floppy pili were observed in our pilT- fliC-. (a, b, c) Scale bars: 2 µm. a.u., arbitrary units.
Fig. 3
Fig. 3. PilU does not affect TFP dynamics in free solution.
(a) iSCAT images of pilT- fliC- and pilU- fliC- mutants. pilT- fliC- cells never undergo retraction on the timescale of our movies. The pilU- fliC- mutant picture shows a retraction of one TFP. The black arrow indicates a tensed pilus; the white arrows indicate floppy pili. Scale bar: 2 µm. (b) Number of TFP in motor mutants and their corresponding complementation strains. These have similar numbers of surface pili except pilT- fliC- (number of cells from the same culture n = 11), which had more (fliC- n = 15, pilT- fliC- complemented n = 15, pilU- fliC- n = 18, pilU- fliC- complemented n = 17). Large circles are means of bootstrap medians and error bars bootstrap 95% confidence intervals. Small circles are individual measurements. (c) The average lengths of TFP that attached on the glass surface in fliC- (combined number of pili from cells imaged in at least three biological replicates n = 54), pilU- fliC- (n = 47) and pilT- fliC- (n = 45). (d) Retraction frequencies for pilU- mutant (combined number of pili from cells imaged in at least three biological replicates n = 23,) compared to fliC- (n = 23). The pilU- mutant show a slight decrease in retraction frequencies compared to fliC-. (e) Average displacement per retraction for fliC- (number of tracks n = 13) and pilU- fliC- (n = 15). Motility does not strongly differ between these mutans. (c,d,e) Small circles correspond to individual measurements, large circles are medians of bootstrap medians and error bars are bootstrap 95% confidence interval. A difference between two groups is defined as statistically significant when their 95% confidence intervals don’t overlap.
Fig. 4
Fig. 4. Coordination of TFP retraction motors.
(a) Illustration of the optimal sequence of events for TFP function: extension, attachment, tension and release from the surface. (b) Successive events from (a) visualized with iSCAT. Attached pilus tip appears as a stationary dark signal at 0 ms (black arrow). The whole fiber transitions into lower intensity values at τt = 87 ms, before detaching at τd = 1.4 s. Scale bar: 2 µm (c) Close up view of attachment and tension from (b) with corresponding intensity profile along the pilus. A dip in intensity is observed at the tip at 0 ms (black arrow on image and graph), transitioning to a uniform low value at τt. (d) Measurements of dwell and tension times in WT (total number of pili from at least three biological replicates n = 27), fliC- (n = 54) and pilU- fliC- (n = 47) and pilT- fliC- (n = 45). Comparing retraction-capable to retraction-deficient mutants show that TFP tension increases dwell time. The tension times in the retraction-capable cells are close to the dwell time of pilT- fliC-, showing that motors engage rapidly to initiate retraction. There is no defect in tension time in pilU- fliC- indicating that PilT is sufficient to initiate this rapid response. (e) TFP retraction frequencies for attached and unattached TFP in fliC- (number of cells from at least three biological replicates n = 30). TFP retract almost only after their tips touch the surface, indicating attachment stimulates retraction. (d and e) Small circles correspond to individual measurements, large circles to median and error bars to bootstrap 95% confidence intervals. A difference between two groups is defined as statistically significant when their 95% confidence intervals don’t overlap. (f) Proposed model for sequential control of TFP motion. During spatial fluctuations (i), attachment of TFP tip to the surface generates a signal activating PilT (ii). This causes pilus retraction and tension, reinforcing attachment and resulting in longer dwell times and cell displacement (iii). PilU engages to power cell displacement under strong loads, for example in environments with increased friction on the cell body (iv).

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