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.

Author Summary

Type IV pili are filamentous appendages borne by a large number of pathogenic and nonpathogenic bacteria. They play crucial roles in basic microbial processes such as surface motility, virulence, and DNA exchange. Neisseria gonorrhoeae, the causative agent of gonorrhea, can extend and retract these long, thin threads—around 6 nm in diameter and up to 30 μm long—to explore and pull on the environment. The retraction of one N. gonorrhoeae pilus filament can exert forces of 50–100 piconewtons, or roughly 10,000 times the bacterium's bodyweight. The bacteria can exert those forces on human cells that they infect, and force has been shown to be an important parameter in their infectivity. We use a micropillar assay system to show that N. gonorrhoeae cells can exert even higher forces by forming bundles of 8–10 filaments that act as coordinated retractable units. The bacteria can thus achieve forces in the nanonewton range (or 100,000 times their bodyweight) making them the strongest microscale elements known to date. This study demonstrates the power and cooperativity of pilus nanomotors and opens new territories for the exploration of force-mediated bacteria–host-cell interactions.

Abstract

To make its way into cells, Neisseria gonorrhoeae, the pathogen that causes gonorrhea, may exert nanonewton forces through cooperative bundling of pilus fibers.