Dynamics of type IV pili is controlled by switching between multiple states

Abstract

Type IV pili are major bacterial virulence factors supporting adhesion, surface motility, and gene transfer. The polymeric pilus fiber is a highly dynamic molecular machine that switches between elongation and retraction. We used laser tweezers to investigate the dynamics of individual pili of Neisseria gonorrheae at clamped forces between 8 pN and 100 pN and at varying concentration of the retraction ATPase PilT. The elongation probability of individual pili increased with increasing mechanical force. Directional switching occurred on two distinct timescales, and regular stepping was absent on a scale > 3 nm. We found that the retraction velocity is bimodal and that the bimodality depends on force and on the concentration of PilT proteins. We conclude that the pilus motor is a multistate system with at least one polymerization mode and two depolymerization modes with the dynamics fine-tuned by force and PilT concentration.

Figure 1

Experimental setup. (a) Sketch of the setup. A bacterium was immobilized on a polystyrene-coated cover glass. When a pilus was bound to the bead in the laser trap and retracted, the bead was displaced by a distance d from the center of the trap. At a predefined distance d corresponding to a force F, a feedback was triggered that moved the piezo table to keep d constant. (b) Typical length change x of a single pilus of MS11_{pilT ind} as a function of time at 60 pN; (c and d) MS11_{pilT oe} at 100 pN. The curves b to d were broken down into retraction intervals (black), elongation intervals (green), and pauses (red).

Figure 2

Pairwise displacements of pilus retraction. This example shows the normalized probability p of pairwise displacements Δx from a pilus retraction event of MS11_{pilT oe} at 100 pN with an average velocity of 251 nm/s (solid line) and a test in which the microscope stage moved a stuck bead on a cover glass with a velocity of 283 nm/s (dotted line).

Figure 3

Randomness parameter r normalized to step size: (gray) MS11; (white) MS11_{pilT ind}; and (black) MS11_{pilT oe}. Error bars represent SD.

Figure 4

External force enhances the probability for pilus elongation. (a) Length change of a single pilus at 15 and (b) at 100 pN. Negative length change corresponds to pilus retraction. (c–e) Probability for pilus elongation (white), pausing (gray), and pilus retraction (black) for (c) MS11, (d) MS11_{pilT oe} (increased PilT), and (e) MS11_{pilT ind} (decreased PilT). The corresponding data set contains 465 retraction curves. Error bars represent SD obtained from statistical resampling.

Figure 5

Different timescales of pilus elongation and retraction intervals. (a) Cumulative histogram of pilus retraction intervals (MS11_{pilT ind}, 60 pN; circles). (dashed line) Best fit to a single exponential function and (solid line) best fit to a double exponential function with τ_ret1 = 9 ± 1 ms, τ_ret2 = 118 ± 2 ms. (b) Cumulative histogram of pilus elongation intervals (MS11_{pilT ind}, 60 pN; circles). (dashed line) Best fit to a single exponential function and (solid line) best fit to a double exponential function with τ_ext1 = 11.5 ± 0.5 ms, τ_ext2 = 134 ± 3 ms. (c) Pilus retraction intervals obtained from the double exponential fit of cumulative histogram (MS11_{pilT ind}) of pilus retraction intervals. (d) Pilus elongation intervals obtained from the double exponential fit of cumulative histogram of pilus elongation intervals.

Figure 6

Velocity of pilus retraction is bimodal. (a) Histogram of retraction velocities at varying force (MS11) of in total 149 events from 29 bacteria (b) Histogram of retraction speeds at varying PilT concentrations at 30 pN. (c) Average velocity of the low velocity mode: (gray) MS11 and (black) MS11_{pilT oe}. (d) Average velocity of the high velocity mode: (gray) MS11 and (black) MS11_{pilT oe}.

Figure 7

Velocity switching events during a single pilus retraction event. (a) Example of length change of the pilus of a MS11_{pilT oe} bacterium at 100 pN over time with distinct velocities modes. (b) Corresponding histogram of the retraction speed of a.

Figure 8

Hypothetical kinetic model describing the experimental data. Switching between different energy landscapes may explain different velocity modes and different timescales of switching. (a) Free energy of depolymerized state is energetically lower than polymerized state. Thus, the retraction rate k₋ is higher than the elongation rate k₊. (b) With increasing external force, the free energy of the polymerized state decreases compared to the depolymerized state. The switching rate k between the landscapes depends on force. (c) The high velocity mode may be described by an energy landscape in which the free energy difference between polymerized and the depolymerized states increases compared to a.