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.

Highlights

  • Type IV pili (Tfp) are bacterial appendages with important biological functions, including motility, DNA transformation, and virulence [1]

  • Measurement of Forces Exerted by type IV pili (Tfp) To measure high Tfp retraction forces from N. gonorrhoeae, we developed a new assay based on elastic micropillars by modifying previously published methods [16,17]

  • A pulling force exerted on a pillar, such as that imposed by an attached pilus fiber, causes a displacement of the pillar tip

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Summary

Introduction

Type IV pili (Tfp) are bacterial appendages with important biological functions, including motility, DNA transformation, and virulence [1]. Interest in the role of Tfp in infection was renewed by recent studies linking Tfp retraction force to the induction of epithelial cell responses [9,10]. The motor protein PilT [11,12] is essential for pilus retraction, and measurements using optical tweezers indicate that retraction of a single Tfp filament by a single PilT motor generates forces of 50–100 piconewtons (pN) [13]. These assays were limited to measuring retraction forces from individual diplococci. We demonstrate the ability of N. gonorrhoeae to impose tremendous forces (nanonewton [nN] range) on its substrate and allow us a more precise and comprehensive view of the physical role of Tfp in N. gonorrhoeae pathogenesis

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