P pili are protein filaments expressed by uropathogenic Escherichia coli that mediate binding to glycolipids on epithelial cell surfaces, which is a prerequisite for bacterial infection. When a bacterium, attached to a cell surface, is exposed to external forces, the pili, which are composed of ∼103 PapA protein subunits arranged in a helical conformation, can elongate by unfolding to a linear conformation. This property is considered important for the ability of a bacterium to withstand shear forces caused by urine flow. It has hitherto been assumed that this elongation is plastic, thus constituting a permanent conformational deformation. We demonstrate, using optical tweezers, that this is not the case; the unfolding of the helical structure to a linear conformation is fully reversible. It is surmised that this reversibility helps the bacteria regain close contact to the host cells after exposure to significant shear forces, which is believed to facilitate their colonization.
Uropathogenic Escherichia coli are implicated in 75%–80% of uncomplicated urinary tract infections and in severe pyelonephritis (Hultgren et al, 1993). Such bacteria constitute a model system for how Gram‐negative bacteria in general attach to host tissue by pili structures on their surface (Sauer et al, 2000b). However, it has remained largely unknown as to how the physical properties of pili influence the ability of a bacterium to withstand shear forces, such as those from the rinsing action of urine. Bacterial binding is commonly mediated by multiple pili. To understand the interactions from multi‐pili binding, one needs to assess the physical properties of an individual pilus. Such measurements have hitherto been nontrivial to perform. However, by access to modern techniques for force measurements in biological systems, and in particular optical tweezers (Fällman et al, 2004), important properties of individual pili, such as their force‐versus‐elongation response, can now be assessed. Examples of entities that have been studied by such techniques are the adhesion forces of type 1 pili (Liang et al, 2000), the twitching motility of Neisseria gonorrhoeae mediated by type IV pili retraction (Merz et al, 2000) and elongation properties of P pili (Jass et al, 2004).
As illustrated in Fig 1A, the main part of a P pilus, which is expressed predominantly by isolates from the upper urinary tract (Johnson & Russo, 2002; Russo & Johnson, 2003), constitutes a thin micron‐long helical rod (Bullitt et al, 1992; Sauer et al, 2000a), composed of ∼103 PapA subunits of molecular mass 16.5 kDa, coupled to their nearest neighbours by a β‐strand complementation (Choudhury et al, 1999; Sauer et al, 1999, 2002; Zavialov et al, 2003) and combined in a right‐handed helical arrangement with 3.28 subunits per turn (Gong & Makowski, 1992; Jacob‐Dubuisson et al, 1993; Bullitt & Makowski, 1995; Bullitt et al, 1996). Most of the elongation properties of a P pilus are therefore governed by the PapA rod. Recent studies have shown that a single P pilus elongates by a passage through three elongation regions when exposed to mechanical stress (Jass et al, 2004). Region I (for elongations of up to ∼20% of its relaxed length; Bullitt & Makowski, 1995; Jass et al, 2004) is characterized by a linear force‐versus‐elongation response and is considered to originate from an elastic stretching of the quaternary structure of the PapA rod (Jass et al, 2004; Fig 1B). Region II (for elongation up to a few times its relaxed length; Bullitt & Makowski, 1995; Jass et al, 2004) is characterized by an elongation under constant force and originates from the unfolding of the helical rod (Jass et al, 2004; Fig 1C). Region III, finally, is characterized by a nonlinear force‐versus‐elongation response and corresponds to a linearization of the head‐to‐tail interaction between the subunits (Jass et al, 2004; Fig 1D).
The ability of a uropathogenic E. coli to resist the rinsing action of urine flow and to subsequently colonize tissue depends, however, not only on the static or elongation properties of the pili but also on their ability to contract. We have therefore, in this work, investigated and assessed the contraction properties of P pili.
A typical force‐versus‐elongation response from a piliated E. coli HB101/pPAP5 bacterium that binds to a surface, measured by optical tweezers, is shown in the supplementary information online (including supplementary Fig S1 online). On the basis of this knowledge, and in particular of the elongation‐independent unfolding force of the helix of a single P pilus, Fuf, of 27±2 pN (Jass et al, 2004), it is possible to investigate the contraction properties of a single P pilus under controlled conditions.
A typical elongation and contraction study of the binding mediated by P pili is shown in Fig 2. Fig 2A and 2B, respectively, represents two consecutive cycles of elongation and contraction of pili from the same bacterium. Bacterium‐to‐bead distances of some specific features are indicated.
The first elongation cycle (blue curve in Fig 2A) gave rise to, for the first 0.1 μm, a linear increase of the binding force with distance, up to ∼170 pN, originating from a number of pili either loosely bound to the bead or mediating the force by a short pili length (either because the pili are short or because the pili mediate the force by only a fraction of their length), all elongated in their linear region. The successive roll‐off (between 0.1 and 0.3 μm) originated from both a nonlinearity of the detection of the position of the bead in the trap and pili that sequentially entered their unfolding regions. At 0.3 and 0.4 μm, a number of pili detached from the bead, showing up as sudden decreases in force (from 200 to 100 pN) together with an instantaneous elongation of the bond.
The increase of the force that took place for bacterium‐to‐bead lengths between 0.85 and 1.05 μm was caused by one or a few short pili entering their third, nonlinear elongation region. At 1.05 μm, these pili detached, which brought the force down to ∼50 pN and the bacterium‐to‐bead distance up to 1.4 μm.
The binding was, after this detachment, mediated solely by pili being in their unfolding region, as indicated by the elongation‐independent force response. On the basis of the force required for unfolding of the helix of a single P pilus, Fuf (Jass et al, 2004), it could be concluded that this binding was mediated by two pili. The simultaneous unfolding of two pili continued until the bacterium‐to‐bead distance was 2.37 μm, where one of the two pili detached, resulting in a binding mediated by only one pilus (at a force level of ∼27 pN).
To investigate the contraction properties of an individual pilus in its unfolding region, the elongation of the binding was stopped and reversed as soon as just one pilus mediated the binding, but before it entered its third elongation region (at 2.55 μm). If the unfolding of the helical structure of the PapA rod was plastic (irreversible), as previously suggested in the literature (Bullitt & Makowski, 1995), the contraction force would have decreased well below the level of the unfolding force of a single pilus as the bacterium‐to‐bead distance was decreased. The black curve in Fig 2A shows, however, that the force was maintained at the same level during contraction as during extension, at ∼27 pN. This evidence indicates that the unfolding of the quaternary, helical structure of the PapA rod in P pili is reversible and thus not plastic.
Fig 2A further shows that the contraction of the single pilus continued at a virtually constant force down to a bacterium‐to‐bead distance of about 0.47 μm. At this point, the PapA rod had completely regained its helical conformation, and the pilus returned to its first elongation region. This shows that the pilus contraction is reversible over the entire region II.
Moreover, the force decreased linearly with contraction through region I until it reached the zero force level at a bacterium‐to‐bead distance of 0.40 μm. This provides evidence that the contraction in the linear region is also fully elastic (and thus also fully reversible).
To investigate the reproducibility of P pili binding, as well as contraction behaviour in region III, a second elongation‐and‐contraction cycle was performed with the same bacterium. It was found that the second elongation‐and‐contraction cycle, displayed by the red and green curves in Fig 2B, significantly resembles the first one. The fact that the first parts of the two force–elongation curves largely resemble each other indicates that pili bind in a similar manner for repeated interactions at the same position of the bacterium. The remaining parts of the binding behaviour also have a high degree of similarity between the two elongation cycles.
This time, the elongation of the binding was pursued until the remaining pilus was well into its third elongation region, in which the helix had fully unfolded and extended to an elongated linear conformation (which it entered at ∼2.6 μm). The elongation was halted at a bacterium‐to‐bead distance of 3.47 μm, before the remaining pilus detached.
The distance between the bead and the bacterium was finally decreased a second time to investigate the contraction behaviour of the pilus in its third elongation region as well as the reproducibility of the entire contracting process. The contraction of the pilus, which fully retraced the elongation in the third region, indicates that stretching in this region is elastic, and thereby fully reversible.
However, when the pilus had been elongated into its third region, the force‐versus‐distance response shows hysteresis when the pilus returned from region III to region II during contraction. The pilus acted with a lower contraction force (∼12 pN) for a short distance (for bacterium‐to‐bead distances from 2.7 to 2.5 μm) before the contraction force again returned to its normal value (∼27 pN). For further contraction, the PapA rod continued to fold at a contraction force of ∼27 pN until the entire helix was recovered (at ∼0.47 μm). Beyond this, the pilus again contracted elastically in its region I until it had been fully relieved of its tension, in a similar way to the first contraction cycle. The remaining negative force at the end of the retraction has been seen in some measurements, and it is believed to originate from either a drift of the system during the time the measurement was performed (in this case 23 min, because of a shift of the height of the bead in the trap) or a repelling force from pili protruding from the bacterium.
The retracting behaviour shown in Fig 2, which shows an elastic response in elongation regions I and III, a reversible unfolding behaviour in region II, including a lower contraction force when the pilus returns from region III to region II, was found in all elongation‐and‐contraction studies (∼100), and is therefore considered to constitute a ‘typical’ contraction behaviour of an elongated P pilus.
This work has shown that a PapA rod, in general, contracts (that is, refolds its helical structure) spontaneously after elongation. This implies that unfolding of the quaternary structure of the PapA rod does not constitute a permanent plastic deformation, as has been proposed (Bullitt & Makowski, 1995); instead, it represents a reversible conformation‐changing elongation.
This work has also shown that the PapA rod refolds its helical structure at a force, Ff, that is, within the measurement errors, equal to the unfolding force, Fuf, given by 27±2 pN (Jass et al, 2004). The contraction force of ∼27 pN is considered to originate from a sequential ‘adjacent‐turn‐interaction’ that transforms the thin fibrillar conformation of the PapA polymer into a right‐handed helical rod by connecting the first unfolded PapA subunit in the linear conformation (the nth subunit) to the (n−3)th and/or (n−4)th subunits in the folded part of the PapA rod (Bullitt et al, 1996; Soto et al, 1998; Fig 1C). When the nth (unfolded) subunit has been docked, the subsequent subunit (the (n+1)th) will automatically be dragged closer to the folded part of the helical rod so that its complementary surface can interact with the corresponding surface(s) on the (n−2)th and/or (n−3)th units. This adjacent‐turn‐interaction folding proceeds in a serial manner until the entire PapA rod has been refolded. This conclusion is supported by the fact that the contraction is independent of elongation. These results thus suggest that the binding force between the complementary surfaces in adjacent turns of the helix is equal to the unfolding force, Fuf, and thus that the unfolding of the pili is fully reversible.
This sequential folding can, however, only take place when at least one turn of the rod has been formed. When the entire rod has been extended, as takes place when a pilus has been elongated into its third elongation region, there are no PapA units that are close enough for the complementary surfaces at one turn to mediate any force to the adjacent turn (Fig 1D). This implies that the nth subunit to (n−3)th and/or (n−4)th subunit folding can only take place when at least some part of the rod is folded. The only force that can contract the fully extended pilus under these conditions is the ‘head‐to‐tail interaction’ between subsequent subunits, between the (n−1)th and the nth subunits (Bullitt et al, 1996; Soto et al, 1998). The force corresponding to this interaction, Fhti, which is weaker than that between adjacent turns of the helix, can be identified in the data above as the force with which the contraction takes place when the pilus returns from region III to region II during contraction. This is shown in the lower part of the elongation‐and‐contraction curve in Fig 2B, which shows hysteresis, that is, for a bacterium‐to‐bead separation of 2.5–2.7 μm. Our measurements thereby suggest that the contraction force that corresponds to the head‐to‐tail interaction for P pili, Fhti, is ∼12 pN.
While contraction takes place by this head‐to‐tail interaction, no subunits are rolled up; the contraction only takes place as a consequence of a slight decrease in binding angle between the various head‐to‐tail interactions linking the adjacent subunits. However, once a few subunits regain the helical conformation of one turn, a tightly coiled helix follows by the adjacent‐turn‐interaction in a serial manner, at a force equal to the folding force, Ff.
The ‘s’‐shaped form of the force‐versus‐distance curves in region III has a certain resemblance to those from studies of other protein polymers, for example, from titin (Rief et al, 1997). Such an ‘s’‐shaped form has been suggested to originate from an unravelling of the tertiary Ig‐like structure domains. This is, however, not a likely explanation in the present case, as an unravelling of the tertiary Ig‐like structure domains would result in the disintegration of the β‐strand complementation between subunits, causing an irreversible breakdown of the PapA structure. This, in turn, would presumably not give rise to a fully reversible behaviour, as was found in our study, or even a complete breakage of the subunit–subunit complexes.
The fact that consecutive force–elongation curves have such a large degree of similarity (as can be seen from a comparison of Fig 2A,B) indicates not only that pili bind in a similar manner for repeated interactions at the same position of the bacterium, but also that the pili neither break nor get detached by a breakage of the anchorage when the force becomes large. Instead, the data suggest that pili primarily detach from the bead when the force becomes large (under the conditions prevalent in these studies).
Moreover, the fact that the PapA rod has the ability to regain its original shape quickly after exposure to forces that cause various degrees of elongation is believed to assist the bacteria in regaining close contact with the host cells even after exposure to large external forces. In turn, this is believed to facilitate their colonization and subsequent infection. It is also possible that the ability of the PapA rod to refold may constitute a target for development of new antibacterial agents.
As the elongation and contraction forces of region II of ∼27 pN have been identified as the unfolding and folding forces of the quaternary structure of the PapA rod, respectively, it is also possible to speculate whether the folding force is the driving force when the pilus is dragged through the usher in the membrane during the biogenesis of the pilus (Jones et al, 1992; Thanassi et al, 1998).
The ability to study the unfolding–contraction mechanism of pili structures in real time has led to an improved understanding of the mechanisms behind pathogenic fibre formation–deformation and their functional features. This should aid in the development of new ways to understand, prevent and treat a variety of serious human diseases. Future studies should aim at elucidating the dynamic properties of P pili and the role of individual amino acids in the subunit–subunit interaction in the quaternary structure of the P pilus as well as other pili structures on Gram‐negative bacteria.
Bacterial strains and growth conditions. To avoid possible interference of other similar (that is, fimbrial) surface structures, the P pili were expressed in the otherwise afimbriated E. coli strain HB101 from the plasmid pPAP5. The E. coli strain HB101/pPAP5 carries the entire pap gene cluster on the vector pBR322 and expresses normal P pili (Lindberg et al, 1984). The bacteria were cultured on trypticase soy agar at 37°C and the expression of functional pili was assessed by haemagglutination assays (Jass et al, 2004).
Bead‐based contraction assay. Samples were prepared directly on a silanized hydrophobic coverslip by sequentially adding 25 μl of freshly resuspended E. coli in PBS, 5 μl of activated polystyrene beads of 9.6 μm and 5 μl of small beads in PBS (pH 7.4; Jass et al, 2004). Bacteria and beads were prepared according to the procedures described previously (Fällman et al, 2004). The mounting and measurement procedure is described in supplementary information online (including supplementary Fig S2 online).
Optical tweezers force‐measuring system. Force measurements were performed by the use of an optical tweezers system, which is described in detail elsewhere (Fällman & Axner, 1997; Leitz et al, 2002; Fällman et al, 2004; Jass et al, 2004). The instrumentation was carefully calibrated with respect to force using two separate calibration techniques on the basis of brownian motion and Stokes drag force (Fällman et al, 2004). The stiffness of the optical trap was ∼0.2 pN/nm. The velocity of the cover glass movement was 0.01 μm/s. This corresponds to a loading rate of ∼2 pN/s.
Supplementary information is available at EMBO reports online (http://www.nature.com/embor/journal/v6/n1/extref/7400310‐s1.pdf).
This work was supported by the Swedish Research Council. Economical support for the construction of a force‐measuring optical tweezers system from the Kempe Foundation is acknowledged.
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