Cooperative retraction of bundled type IV pili enables nanonewton force generation

Abstract

The causative agent of gonorrhea, Neisseria gonorrhoeae, bears retractable filamentous appendages called type IV pili (Tfp). Tfp are used by many pathogenic and nonpathogenic bacteria to carry out a number of vital functions, including DNA uptake, twitching motility (crawling over surfaces), and attachment to host cells. In N. gonorrhoeae, Tfp binding to epithelial cells and the mechanical forces associated with this binding stimulate signaling cascades and gene expression that enhance infection. Retraction of a single Tfp filament generates forces of 50-100 piconewtons, but nothing is known, thus far, on the retraction force ability of multiple Tfp filaments, even though each bacterium expresses multiple Tfp and multiple bacteria interact during infection. We designed a micropillar assay system to measure Tfp retraction forces. This system consists of an array of force sensors made of elastic pillars that allow quantification of retraction forces from adherent N. gonorrhoeae bacteria. Electron microscopy and fluorescence microscopy were used in combination with this novel assay to assess the structures of Tfp. We show that Tfp can form bundles, which contain up to 8-10 Tfp filaments, that act as coordinated retractable units with forces up to 10 times greater than single filament retraction forces. Furthermore, single filament retraction forces are transient, whereas bundled filaments produce retraction forces that can be sustained. Alterations of noncovalent protein-protein interactions between Tfp can inhibit both bundle formation and high-amplitude retraction forces. Retraction forces build over time through the recruitment and bundling of multiple Tfp that pull cooperatively to generate forces in the nanonewton range. We propose that Tfp retraction can be synchronized through bundling, that Tfp bundle retraction can generate forces in the nanonewton range in vivo, and that such high forces could affect infection.

Figure 1. N. gonorrhoeae Tfp Can Exert Forces in the nN Range

(A) Schematic of the force measurement assay used in this study. (B) SEM micrograph of a microcolony with Tfp pulling on pillars (here the pillars are molded in polydimethylsiloxane, a widely used, silicon-based polymer, to enable SEM). Scale bar = 2 μm. (C) Time course of the force exerted on a pillar. (1, trace β): short-lived, low-force pulling event. (2, trace β): long-lived, high-force event. Trace α shows a neighboring pillar that did not move. (D and E), Histograms of the forces recorded in static studies for WT MS11 in DMEM (D) or in DMEM + BSA (E). Inserts are differential interference contrast images of representative microlonies in the samples. Scale bar=5 μm. (F and G), Histograms of the forces recorded in dynamic studies of WT MS11 in DMEM (F) or DMEM + BSA (G). Inserts represent close-ups of the tail of each force distribution curve (forces between 200 pN and 1000 pN).

Figure 2. Tfp from WT MS11 and MS11PilT in Different States of Bundling

WT: MS11 not exposed to BSA; WT + BSA: MS11 incubated with BSA; PilT: MS11PilT not exposed to BSA; WT + polylysine: MS11 incubated with polylysine. (A) SEM micrographs. Scale bar = 500 nm. (B) Fluorescent images of microcolonies immunostained with monoclonal anti-Tfp antibody (anti-SM1). Scale bar = 2 μm. (C) Thin-section EM image of Tfp and Tfp bundles. Insert in each panel represents a higher magnification image of representative Tfp in the sample. Note the bundles in the WT and WT+polylysine samples that are not present in the WT+BSA and pilT samples. Scale bar = 500nm. Scale bar = 50 nm in the inserts.

Figure 3. Structure of Tfp Bundles

(A) SEM picture of a collection of bundles in a polylysine-treated sample. Scale bar = 500 nm. (B) Thin-section microscopy image of the cross-section of a collection of bundles in a polylysine-treated sample (here composed of two bundles). Scale bar = 50 nm. (C) Thin-section microscopy image of the long axis of a collection of bundles in a polylysine-treated sample (here composed of three bundles). Scale bar = 50 nm. (D) Thin-section microscopy image of a bundle attached to a bacterium in a sample incubated with DMEM only. Scale bar = 50 nm.

Figure 4. Model for Tfp Bundling and Retraction

(A) Progressive rise of force applied by a bacterium on one pillar over time (more than 30 min total time). (B) Cartoon showing the successive association (bundling) of Tfp filaments with each other and their retraction. Timeline begins with top left panel and is read from left to right, top to bottom.