Force-dependent polymorphism in type IV pili reveals hidden epitopes

Abstract

Through evolution, nature has produced exquisite nanometric structures, with features unrealized in the most advanced man-made devices. Type IV pili (Tfp) represent such a structure: 6-nm-wide retractable filamentous appendages found in many bacteria, including human pathogens. Whereas the structure of Neisseria gonorrhoeae Tfp has been defined by conventional structural techniques, it remains difficult to explain the wide spectrum of functions associated with Tfp. Here we uncover a previously undescribed force-induced quaternary structure of the N. gonorrhoeae Tfp. By using a combination of optical and magnetic tweezers, atomic force microscopy, and molecular combing to apply forces on purified Tfp, we demonstrate that Tfp subjected to approximately 100 pN of force will transition into a new conformation. The new structure is roughly 3 times longer and 40% narrower than the original structure. Upon release of the force, the Tfp fiber regains its original form, indicating a reversible transition. Equally important, we show that the force-induced conformation exposes hidden epitopes previously buried in the Tfp fiber. We postulate that this transition provides a means for N. gonorrhoeae to maintain attachment to its host while withstanding intermittent forces encountered in the environment. Our findings demonstrate the need to reassess our understanding of Tfp dynamics and functions. They could also explain the structural diversity of other helical polymers while presenting a unique mechanism for polymer elongation and exemplifying the extreme structural plasticity of biological polymers.

Fig. 1.

N. gonorrhoea Tfp undergoes reversible force-induced polymorphism. (A) Schematic of the optical tweezers experimental design. (B) Movie frames illustrating the position of the elastic pillar and silica bead before and after pilus transition. (Scale bar: 1 μm.) (C) Time course of the distance between the center of the bead and the center of the pillar and of the deflection of the pillar (time 0 = time of the Tfp transition). The displacement of the pillar represents a force of 45 pN. (D) Schematic of the magnetic tweezers experimental design. (E) Successive fluorescent images of a fluorescently labeled Tfp before transition (Left), in transition (Center), and after recovery (Right). (Scale bar: 1 μm.)

Fig. 2.

AFM characterization of the force-induced Tfp transition. (A) Schematic of the AFM experimental design. (B) Typical example of force-extension curves showing the transition occurring at ∼100 pN. Insets are zooms on the force peaks for four curves. (C) Force histogram of the force associated with the Tfp transition event averaging 100 ± 20 pN (standard deviation, n = 1,210). (D) The reversibility of the force-induced transition is demonstrated by the observation of multiple transition events in two consecutive trajectories separated by a time Δt = 3 min.

Fig. 3.

Extended Tfp conformation reveals hidden epitopes. (A) A schematic of the molecular combing experimental design. (B) Close-up of fluorescent images of stretched or unstretched TAMRA-prestained purified Tfp. The green signal indicates the SM1 epitope; the red signal (TAMRA) labels the exterior of the Tfp fiber. (Scale bar: 1 μm.) (C) Close-up of fluorescent images of stretched or unstretched purified Tfp. The green signal indicates the SM1 epitope; the red signal (Pan127 antibody) labels the exterior of the Tfp fiber. (Scale bar: 1 μm.) (D) Fluorescent images of TAMRA-prestained purified Tfp processed by molecular combing. The red signal is against an exposed region of Tfp (TAMRA), whereas the green signal indicates the SM1 epitope. (Scale bar: 5 μm.) (E) Dual fluorescent images of the SM1 epitope (Green) and the exposed epitope Pan127 (Red) of purified Tfp processed by molecular combing. (Scale bar: 1 μm.)

Fig. 4.

Stretched Tfp are 40% narrower than unstretched Tfp. (A) Electron micrograph of purified Tfp processed by molecular combing. Note the smaller diameter of the stretched Tfp. Direction of stretch indicated by the double-headed arrow. (Scale bar: 50 nm.) (B) Electron micrograph of purified Tfp processed by molecular combing. Note the smaller diameter of the stretched Tfp. Direction of stretch indicated by the double-headed arrow. (Scale bar: 50 nm.) (C) Plots of averaged intensities across a line drawn perpendicularly to the direction of a stretched Tfp at the level of either the star or the circle in B. Those plots, along with B, present an example of partially transitioned Tfp.