Mechanotaxis directs Pseudomonas aeruginosa twitching motility

Abstract

The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by retractile extracellular filaments called type IV pili (T4P). Single cells twitch by sequential T4P extension, attachment, and retraction. How single cells coordinate T4P to efficiently navigate surfaces remains unclear. We demonstrate that P. aeruginosa actively directs twitching in the direction of mechanical input from T4P in a process called mechanotaxis. The Chp chemotaxis-like system controls the balance of forward and reverse twitching migration of single cells in response to the mechanical signal. Collisions between twitching cells stimulate reversals, but Chp mutants either always or never reverse. As a result, while wild-type cells colonize surfaces uniformly, collision-blind Chp mutants jam, demonstrating a function for mechanosensing in regulating group behavior. On surfaces, Chp senses T4P attachment at one pole, thereby sensing a spatially resolved signal. As a result, the Chp response regulators PilG and PilH control the polarization of the extension motor PilB. PilG stimulates polarization favoring forward migration, while PilH inhibits polarization, inducing reversal. Subcellular segregation of PilG and PilH efficiently orchestrates their antagonistic functions, ultimately enabling rapid reversals upon perturbations. The distinct localization of response regulators establishes a signaling landscape known as local excitation-global inhibition in higher-order organisms, identifying a conserved strategy to transduce spatially resolved signals.

Keywords: chemotaxis; mechanosensing; motility; twitching; type IV pili.

Fig. 1.

The Chp system regulates the twitching trajectories of individual P. aeruginosa cells. (A) Schematic representation of the major components of the T4P and Chp system. (B) Phase contrast snapshots of forward and reverse migration of twitching cells. $\overrightarrow{t}$ is a unit vector oriented along the cell body in the initial direction of motion. $\overrightarrow{d}$ is the unit displacement vector. δ is the dot product $\overrightarrow{d}.\overrightarrow{t}$, which quantifies displacements relative to the initial direction of motility. The red triangle indicates the initial leading pole of the bacterium. (Scale bar, 2 µm.) (C) Graphs of cumulative net displacement as a function of time, highlighting the forward and reverse twitching behavior of Chp mutants. Each curve corresponds to an individual cell trajectory. Tracks of reversing WT cells are highlighted in black. At any given time, a curve oriented toward the top right corresponds to a cell moving forward, while a curve oriented toward top left corresponds to reverse movement (cf. Inset). ΔpilGcpdA constantly reverses twitching direction, while ΔpilH cells persistently move forward.

Fig. 2.

The Chp system controls reversals of twitching P. aeruginosa cells. (A) Quantification of reversal rates in Chp and cAMP mutants. ΔpilGcpdA has highest reversal frequency. ΔpilH has a twofold lower reversal frequency than ΔcpdA. Circles correspond to biological replicates, and black bars represent their mean. (B) Snapshots of WT reversing upon collision with another cell (Left). The same sequence for a ΔpilH cell, failing to reverse upon collision (Right). The red triangle indicates the initial leading pole of the bacterium. (Scale bar, 2 µm.) (C) Fraction of cells reversing upon collision with another cell. About half of WT cells reverse after collision, ΔpilH almost never reserves after collision, and ΔpilGcpdA almost always reverses. Circles correspond to biological replicates, and black bars represent their mean. (D) Phase contrast image sequence of WT cells reversing upon collision with glass microfibers and other cells. The dashed lines indicate the position of the fiber. (Scale bar, 2 µm.) (E) Fraction of WT cells reversing upon collision with another cell and with a glass microfiber. A total of 60% of cells reverse after collision, irrespective of the type of obstacle. Circles correspond to biological replicates, and black bars represent their mean. (F) While WT is able to move efficiently at high density, the reduced ability of ΔpilH to reverse upon collision leads to cell jamming and clustering. (Scale bar, 50 µm.) Background strain: PAO1 ΔfliC.

Fig. 3.

The localization of the extension motor PilB sets the direction of twitching and the polarization of T4P activity. (A) Snapshot of chromosomal fluorescent protein fusions to the extension motor PilB, its regulator FimX, and the retraction motors PilT and PilU. (Scale bars, 5 µm.) (B) Simultaneous imaging of PilB-mNG and T4P by correlative iSCAT fluorescence. White arrowheads indicate T4P. (Scale bar, 5 µm.) (C) Fraction of cells with more T4P at bright versus dim fluorescent pole. Most cells have more T4P at the bright PilB-mNG pole. We could not distinguish a T4P depletion at the bright retraction motor poles. Each circle is the mean fraction for one biological replicate. Black bars correspond to their mean across replicates. (D) Comparison of the symmetry of polar fluorescence between moving and nonmoving cells. PilB and FimX signal is more asymmetric in moving cells, which is not the case for PilT and PilU. (E) Fraction of cells twitching in the direction of their brightest pole. Circles correspond the fraction of each biological replicate, and black bars represent their mean.

Fig. 4.

Mechanical input signal from T4P controls the polarization of FimX, the activator of the extension motor PilB. (A) Kymograph of mNG-FimX fluorescence in a nonmoving cell 10 min after surface contact. The bright fluorescent focus sequentially disappears from one pole to appear at the opposite to establish oscillations. (B) Fraction of cells that showed pole to pole oscillations in WT and ΔpilA. The proportion of oscillating WT reduces as they remain on the surface, conversely increasing the proportion of stably polarized cells. (C) Kymograph of mNG-FimX fluorescence in a ΔpilA background 60 min after surface contact. (Scale bar, 5 µm.) (D) Most ΔpilA cells maintain oscillatory fluctuations in mNG-FimX polar localization.

Fig. 5.

PilG and PilH control the polarization of T4P extension machinery. Snapshots of PilB-mNG (A) and mNG-FimX (B) fluorescence in WT, ΔpilG, ΔpilH, and ΔcpdA background. (Scale bar, 5 µm.) (C and D) Normalized fluorescence profiles along the major cell axis of the motor protein PilB and its activator FimX (SI Appendix, Fig. S8A). Solid lines represent the mean normalized fluorescence profiles across biological replicates. The shaded area represents SD across biological replicates. (E and G) Polar localization index of PilB-mNG and mNG-FimX respectively, quantifying the extent of polar signal compared with a diffused configuration (SI Appendix, Fig. S8B). An index of 0 and 1 respectively correspond to completely diffuse and polar signals. Relative to WT and ΔcpdA, polar localization is higher in ΔpilH and lower in in ΔpilG. (F and H) Symmetry index of PilB-mNG and mNG-FimX, respectively, representing the ratio of the brightest pole fluorescence to the total polar fluorescence. The values 0.5 and 1 respectively correspond to a symmetric bipolar and a unipolar localization. ΔpilH has higher symmetry index than WT and ΔcpdA. Circles represent the median of each biological replicate. Black bars represent (vertical) mean and (horizontal) SD across biological replicates.

Fig. 6.

PilG and PilH dynamic localization establish a local-excitation, global-inhibition signaling landscape. (A) Snapshots of mNG-PilG and mNG-PilH fluorescence. (Scale bar, 5 µm.) (B) Comparison of mNG-PilG and mNG-PilH normalized mean fluorescent profiles. (C) The polar localization index of mNG-PilG is relatively large showing PilG is mostly polar. In contrast, mNG-PilH has a low polar localization index and is thus mostly cytoplasmic. Circles represent the median of each biological replicate. Black bars represent the (vertical) mean and (horizontal) SD across biological replicates. (D) Protein polarization relative to the twitching direction. Cells predominantly move toward the brighter mNG-PilG pole. The fraction for mNG-PilH is close to 50%, corresponding to a random polarization relative to the direction of motion. Black bars represent the mean across biological replicates. (E) Comparison of the symmetry of the polar fluorescent foci of moving cells with nonmoving cells for mNG-PilG and mNG-PilH fusion proteins. There is an enrichment for mNG-PilG polar asymmetry in moving cells but no differences in mNG-PilH. Black bars represent the mean across biological replicates. (F) Fraction of cells with more T4P at bright versus dim mNG-PilG fluorescent pole. Most cells have more T4P at the bright mNG-PilG pole, similar to PilB (data from Fig. 3C in gray as reference). Each circle is the mean fraction for one biological replicate. Black bars correspond to their mean across replicates.