Measurement of Forces Exerted by 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]. Our force sensor consists of an array of evenly spaced micropillars made of an elastic hydrogel (Figure 1A and 1B, and Video S1, to see a video of the device). A pulling force exerted on a pillar, such as that imposed by an attached pilus fiber, causes a displacement of the pillar tip. This displacement can be correlated to the force applied on the pillar after proper calibrations. Modifying the elastic properties of the gel, we were able to vary the stiffness of the pillars from 100 to 500 pN/μm. This allowed us to record forces in the pN to nanonewton (nN) range. Bacteria were seeded on these force sensors in Dulbecco's modified Eagle's medium (DMEM) tissue culture medium, allowed to interact with each other as in an infection assay, and the displacement of the pillars adjacent to the bacteria was recorded. The motion of the pillars revealed two types of pulling behavior from the bacteria. As reported previously [13], we observed transient retraction events—or pulls—lasting anywhere from a few tenths of a second to several seconds, with a magnitude <100 pN (1, trace β in Figure 1C). We also recorded much longer retraction events lasting from a few seconds to several hours, which exerted much higher forces of between 200 pN and 1 nN (2, trace β in Figure 1C). Until now, retraction events of this magnitude have never been seen in N. gonorrhoeae. In all cases, only a few pillars were observed to be moving at a given time relative to stationary neighboring pillars (trace α in Figure 1C), showing that these retraction events were specific to particular pillars. Tfp retraction forces under those conditions were up to 10 times higher than those recorded for a single Tfp filament.

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larger image TIFF original image Download: 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). https://doi.org/10.1371/journal.pbio.0060087.g001

We next asked if there was a regular pattern associated with these high-amplitude forces. We used two methods for recording the forces exerted on a pillar (see Figure S1 for details). We used either video rate (30 Hz) imaging to capture the motion of a pillar's tip (dynamic studies), or recording the deflection of the pillars by taking a picture at the base and at the tip of the pillar in fixed samples (static studies). Dynamic studies allowed us to monitor accurately short-lived pulling events, whereas the static studies allowed us to view long-term pulling events. Histograms of both types of recordings revealed a low force peak that was characteristic of single Tfp retraction events (70 ± 20 pN for static studies, and 40 ± 20 pN for dynamic studies) (Figure 1D and 1F, respectively). The two types of force recordings differed in the percentage of higher force measurements as, for instance, the maximum force (∼1 nN) events represent ∼1% of the measurements in the static studies and only 0.1% in the dynamical studies (Figure 1D and 1F, respectively). In the force histograms from the fixed samples, we observed a number of peaks with values that were roughly multiples of a single filament retraction force (Figure 1D). These peaks indicated that higher force retractions may involve the simultaneous pulling of 2, 3, 4 or more Tfp. A careful examination of rare dynamic events lends additional support to this possibility (Figure S2). In most cases, force increased in a step-wise fashion, commonly at increments of 70–100 pN.

Why have such high forces (nN) not been recorded before? One possible explanation is that previous assays were conducted in medium containing a high concentration of bovine serum albumin (BSA; 1 mg/ml) [7,13]. In many cases, BSA has the potential to prevent nonspecific interactions between proteins, and this could apply to proteinaceous assemblies such as the Tfp. To test the effect of BSA on Tfp retraction, we conducted our measurements in the presence of BSA. Under those conditions, we obtained very different histograms (Figure 1E). We observed a primary peak in the histogram that was much higher than the primary peak recorded in the absence of BSA, and we recorded subsequent peaks that were much smaller than those measured in the absence of BSA. Thus, low-force retraction events were more numerous in the presence of BSA. Furthermore, the maximum forces measured in the presence of BSA never reached the levels observed in the medium without BSA either in static or dynamic measurements (Figure 1F and 1G). Thus, the addition of BSA to the assay medium inhibits bacteria from pulling with high forces.