Catch-Bond Behavior of Bacteria Binding by Slip Bonds

It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.     

Dynamic force spectroscopy of E. coli P pili

Surface organelles (so-called pili) expressed on the bacterial membrane mediate the adhesion of Escherichia coli causing urinary tract infection. These pili possess some extraordinary elongation properties that are assumed to allow a close bacterium-to-host contact even in the presence of shear forces caused by urine flow. The elongation properties of P pili have therefore been assessed for low elongation speeds (steady-state conditions). This work reports on the behavior of P pili probed by dynamic force spectroscopy. A kinetic model for the unfolding of a helixlike chain structure is derived and verified. It is shown that the unfolding of the quaternary structure of the PapA rod takes place at a constant force that is almost independent of elongation speed for slow elongations (up to approximately 0.4 mum/s), whereas it shows a dynamic response with a logarithmic dependence for fast elongations. The results provide information about the energy landscape and reaction rates. The bond length and thermal bond opening and closure rates for the layer-to-layer bond have been assessed to approximately 0.76 nm, approximately 0.8 Hz, and approximately 8 GHz, respectively. The results also support a previously constructed sticky-chain model for elongation of the PapA rod that until now had been experimentally verified only under steady-state conditions.     

Fast uncoiling kinetics of F1C pili expressed by uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> are revealed on a single pilus level using force-measuring optical tweezers

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S_I to 30 pN for type 1). The corner velocity for dynamic response (1,400 nm/s) was found to be larger than those of the other pili (400–700 nm/s for S and P pili, and 6 nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17 Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.     

Dynamics of Type IV Pili Is Controlled by Switching Between Multiple States

Type IV pili are major bacterial virulence factors supporting adhesion, surface motility, and gene transfer. The polymeric pilus fiber is a highly dynamic molecular machine that switches between elongation and retraction. We used laser tweezers to investigate the dynamics of individual pili of Neisseria gonorrheae at clamped forces between 8 pN and 100 pN and at varying concentration of the retraction ATPase PilT. The elongation probability of individual pili increased with increasing mechanical force. Directional switching occurred on two distinct timescales, and regular stepping was absent on a scale > 3 nm. We found that the retraction velocity is bimodal and that the bimodality depends on force and on the concentration of PilT proteins. We conclude that the pilus motor is a multistate system with at least one polymerization mode and two depolymerization modes with the dynamics fine-tuned by force and PilT concentration.     

Helix-like biopolymers can act as dampers of force for bacteria in flows

Biopolymers are vital structures for many living organisms; for a variety of bacteria, adhesion polymers play a crucial role for the initiation of colonization. Some bacteria express, on their surface, attachment organelles (pili) that comprise subunits formed into stiff helix-like structures that possess unique biomechanical properties. These helix-like structures possess a high degree of flexibility that gives the biopolymers a unique extendibility. This has been considered beneficial for piliated bacteria adhering to host surfaces in the presence of a fluid flow. We show in this work that helix-like pili have the ability to act as efficient dampers of force that can, for a limited time, lower the load on the force-mediating adhesin-receptor bond on the tip of an individual pilus. The model presented is applied to bacteria adhering with a single pilus of either of the two most common types expressed by uropathogenic Escherichia coli, P or type 1 pili, subjected to realistic flows. The results indicate that for moderate flows (~25 mm/s) the force experienced by the adhesin-receptor interaction at the tip of the pilus can be reduced by a factor of ~6 and ~4, respectively. The uncoiling ability provides a bacterium with a "go with the flow" possibility that acts as a damping. It is surmised that this can be an important factor for the initial part of the adhesion process, in particular in turbulent flows, and thereby be of use for bacteria in their striving to survive a natural defense such as fluid rinsing actions.     

The biomechanical properties of E. coli pili for urinary tract attachment reflect the host environment

Uropathogenic Escherichia coli express pili that mediate binding to host tissue cells. We demonstrate with in situ force measuring optical tweezers that the ability of P and type 1 pili to elongate by unfolding under exposure to stress is a shared property with some differences. The unfolding force of the quaternary structures under equilibrium conditions is similar, 28 +/- 2 and 30 +/- 2 pN for P pili and type 1 pili, respectively. However, type 1 pili are found to be more rigid than P pili through their stronger layer-to-layer bonds. It was found that type 1 pili enter a dynamic regime at elongation speeds of 6 nm/s, compared to 400 nm/s for P pili; i.e., it responds faster to an external force. This possibly helps type 1 to withstand the irregular urine flow in the urethra as compared to the more constant urine flow in the upper urinary tract. Also, it was found that type 1 pili refold during retraction at two different levels that possibly could be related to several possible configurations. Our findings highlight functions that are believed to be of importance for the bacterial ability to sustain a basic antimicrobial mechanism of the host and for bacterial colonization.     

Physical properties of Escherichia coli P pili measured by optical tweezers

The mechanical behavior of individual P pili of uropathogenic Escherichia coli has been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of approximately 10(3) PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. They are particularly important in the pathogenesis of E. coli colonizing the upper urinary tract and kidneys. A biological model system has been established for in situ measurements of the forces that occur during mechanical stretching of pili. A mathematical model of the force-versus-elongation behavior of an individual pilus has been developed. Three elongation regions of pili were identified. In region I, P pili stretch elastically, up to a relative elongation of 16 +/- 3%. The product of elasticity modulus and area of a P pilus, EA, was assessed to 154 +/- 20 pN (n=6). In region II, the quaternary structure of the PapA rod unfolds under a constant force of 27 +/- 2 pN (n approximately 100) by a sequential breaking of the interactions between adjacent layers of PapA subunits. This unfolding can elongate the pilus up to 7 +/- 2 times. In region III, pili elongate in a nonlinear manner as a result of stretching until the bond ruptures.     

Observation of Bacterial Type I Pili Extension and Contraction under Fluid Flow

Type I pili are proteinaceous tethers that mediate bacterial adhesion of uropathogenic Escherichia coli to surfaces and are thought to help bacteria resist drag forces imparted by fluid flow via uncoiling of their quaternary structure. Uncoiling and recoiling have been observed in force spectroscopy experiments, but it is not clear if and how this process occurs under fluid flow. Here we developed an assay to study the mechanical properties of pili in a parallel plate flow chamber. We show that pili extend when attached E. coli bacteria are exposed to increasing shear stresses, that pili can help bacteria move against moderate fluid flows, and characterize two dynamic regimes of this displacement. The first regime is consistent with entropic contraction as modeled by a freely jointed chain, and the second with coiling of the quaternary structure of pili. These results confirm that coiling and uncoiling happen under flow but the observed dynamics are different from those reported previously. Using these results and those from previous studies, we review the mechanical properties of pili in the context of other elastic proteins such as the byssal threads of mussels. It has been proposed that the high extensibility of pili may help recruit more pili into tension and lower the force acting on each one by damping changes in force due to fluid flow. Our analysis of the mechanical properties suggests additional functions of pili; in particular, their extensibility may reduce tension by aligning pili with the direction of flow, and the uncoiled state of pili may complement uncoiling in regulating the force of the terminal adhesin.     

Using laser tweezers to measure twitching motility in Neisseria

Dynamic properties of type IV pili are essential for their function in bacterial infection, twitching motility and gene transfer. Laser tweezers are versatile tools to study the molecular mechanism underlying pilus dynamics at the single molecule level. Recently, these optical tweezers have been used to monitor pilus elongation and retraction in vivo at a resolution of several nanometers. The force generated by type IV pili exceeds 100 pN making pili the strongest linear motors characterized to date. The study of pilus dynamics at the single molecule level sheds light on kinetics, force generation, switching and mechanics of the Neisseria gonorrhoeae pilus motor.     

Nanoscale Pulling of Type IV Pili Reveals Their Flexibility and Adhesion to Surfaces over Extended Lengths of the Pili

Type IV pili (T4P) are very thin protein filaments that extend from and retract into bacterial cells, allowing them to interact with and colonize a broad array of chemically diverse surfaces. The physical aspects that allow T4P to mediate adherence to many different surfaces remain unclear. Atomic force microscopy (AFM) nanoscale pulling experiments were used to measure the mechanical properties of T4P of a mutant strain of Pseudomonas aeruginosa PAO1 unable to retract its T4P. After adhering bacteria to the end of an AFM cantilever and approaching surfaces of mica, gold, or polystyrene, we observed adhesion of the T4P to all of the surfaces. Pulling of single and multiple T4P on retraction of the cantilever from the surfaces could be described using the worm-like chain (WLC) model. Distinct peaks in the measured distributions of the best-fit values of the persistence length L_p on two different surfaces provide strong evidence for close-packed bundling of very flexible T4P. In addition, we observed force plateaus indicating that adhesion of the T4P to both hydrophilic and hydrophobic surfaces occurs along extended lengths of the T4P. These data shed new light, to our knowledge, on T4P flexibility and support a low-affinity, high-avidity adhesion mechanism that mediates bacteria-surface interactions.     

Impairment of the biomechanical compliance of P pili: a novel means of inhibiting uropathogenic bacterial infections?

Gram-negative bacteria often initiate their colonization by use of extended attachment organelles, so called pili. When exposed to force, the rod of helix-like pili has been found to be highly extendable, mainly attributed to uncoiling and recoiling of its quaternary structure. This provides the bacteria with the ability to redistribute an external force among a multitude of pili, which enables them to withstand strong rinsing flows, which, in turn, facilitates adherence and colonization processes critical to virulence. Thus, pili fibers are possible targets for novel antibacterial agents. By use of a substance that compromises compliance of the pili, the ability of bacteria to redistribute external forces can be impaired, so they will no longer be able to resist strong urine flow and thus be removed from the host. It is possible such a substance can serve as an alternative to existing antibiotics in the future or be a part of a multi-drug. In this work we investigated whether it is possible to achieve this by targeting the recoiling process. The test substance was purified PapD. The effect of PapD on the compliance of P pili was assessed at the single organelle level by use of force-measuring optical tweezers. We showed that the recoiling process, and thus the biomechanical compliance, in particular the recoiling process, can be impaired by the presence of PapD. This leads to a new concept in the search for novel drug candidates combating uropathogenic bacterial infections—“coilicides”, targeting the subunits of which the pilus rod is composed.     

Type-IV Pilus Deformation Can Explain Retraction Behavior

Polymeric filament like type IV Pilus (TFP) can transfer forces in excess of 100 pN during their retraction before stalling, powering surface translocation(twitching). Single TFP level experiments have shown remarkable nonlinearity in the retraction behavior influenced by the external load as well as levels of PilT molecular motor protein. This includes reversal of motion near stall forces when the concentration of the PilT protein is loweblack significantly. In order to explain this behavior, we analyze the coupling of TFP elasticity and interfacial behavior with PilT kinetics. We model retraction as reaction controlled and elongation as transport controlled process. The reaction rates vary with TFP deformation which is modeled as a compound elastic body consisting of multiple helical strands under axial load. Elongation is controlled by monomer transport which suffer entrapment due to excess PilT in the cell periplasm. Our analysis shows excellent agreement with a host of experimental observations and we present a possible biophysical relevance of model parameters through a mechano-chemical stall force map.     

Unveiling molecular interactions that stabilize bacterial adhesion pili

Adhesion pili assembled by the chaperone-usher pathway are superelastic helical filaments on the surface of bacteria, optimized for attachment to target cells. Here, we investigate the biophysical function and structural interactions that stabilize P pili from uropathogenic bacteria. Using optical tweezers, we measure P pilus subunit-subunit interaction dynamics and show that pilus compliance is contour-length dependent. Atomic details of subunit-subunit interactions of pili under tension are shown using steered molecular dynamics (sMD) simulations. sMD results also indicate that the N-terminal “staple” region of P pili, which provides interactions with pilins that are four and five subunits away, significantly stabilizes the helical filament structure. These data are consistent with previous structural data, and suggest that more layer-to-layer interactions could compensate for the lack of a staple in type 1 pili. This study informs our understanding of essential structural and dynamic features of adhesion pili, supporting the hypothesis that the function of pili is critically dependent on their structure and biophysical properties.     

Unraveling the Role of Surface Mucus-Binding Protein and Pili in Muco-Adhesion of Lactococcus lactis

Adhesion of bacteria to mucus may favor their persistence within the gut and their beneficial effects to the host. Interactions between pig gastric mucin (PGM) and a natural isolate of Lactococcus lactis (TIL448) were measured at the single-cell scale and under static conditions, using atomic force microscopy (AFM). In parallel, these interactions were monitored at the bacterial population level and under shear flow. AFM experiments with a L. lactis cell-probe and a PGM-coated surface revealed a high proportion of specific adhesive events (60%) and a low level of non-adhesive ones (2%). The strain muco-adhesive properties were confirmed by the weak detachment of bacteria from the PGM-coated surface under shear flow. In AFM, rupture events were detected at short (100−200 nm) and long distances (up to 600−800 nm). AFM measurements on pili and mucus-binding protein defective mutants demonstrated the comparable role played by these two surface proteinaceous components in adhesion to PGM under static conditions. Under shear flow, a more important contribution of the mucus-binding protein than the pili one was observed. Both methods differ by the way of probing the adhesion force, i.e. negative force contact vs. sedimentation and normal-to-substratum retraction vs. tangential detachment conditions, using AFM and flow chamber, respectively. AFM blocking assays with free PGM or O-glycan fractions purified from PGM demonstrated that neutral oligosaccharides played a major role in adhesion of L. lactis TIL448 to PGM. This study dissects L. lactis muco-adhesive phenotype, in relation with the nature of the bacterial surface determinants.     

Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity

Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.     

The mechanical properties of E. coli type 1 pili measured by atomic force microscopy techniques

The first step in the encounter between a host and a pathogen is attachment to the host epithelium. For uropathogenic Escherichia coli, these interactions are mediated by type 1 and P adhesive pili, which are long (approximately 1 microm) rods composed of more than 1000 protein subunits arranged in a helical structure. Here we used single-molecule atomic force microscopy to study the mechanical properties of type 1 pili. We found that type 1 pili readily extend under an applied force and that this extensibility is the result of unwinding the pilus rod's helical quaternary structure. The forced unraveling is also reversible, with helical rewinding taking place under considerable forces (approximately 60 pN). These data are similar to those obtained on P pili using optical tweezers, indicating that these are conserved properties of uropathogenic E. coli pili. We also show that our data can readily be reproduced using Monte Carlo simulation techniques based on a two-state kinetic model. This model provides a simple way to extrapolate the mechanical behavior of pili under a wide range of forces. We propose that type 1 pilus unraveling is an essential mechanism for absorbing physiological shear forces encountered during urinary tract infections and probably essential for adhesion and colonization of the bladder epithelium.     

Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S_II) and believed to play a role in ascending urinary tract infections (S_I). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S_II and S_I pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S_II pili and 21 pN for S_I pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX _II) indicated that absence of the sfaX _II gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S_I are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.     

Steered Molecular Dynamics Simulations of a Type IV Pilus Probe Initial Stages of a Force-Induced Conformational Transition

Type IV pili are long, protein filaments built from a repeating subunit that protrudes from the surface of a wide variety of infectious bacteria. They are implicated in a vast array of functions, ranging from bacterial motility to microcolony formation to infection. One of the most well-studied type IV filaments is the gonococcal type IV pilus (GC-T4P) from Neisseria gonorrhoeae, the causative agent of gonorrhea. Cryo-electron microscopy has been used to construct a model of this filament, offering insights into the structure of type IV pili. In addition, experiments have demonstrated that GC-T4P can withstand very large tension forces, and transition to a force-induced conformation. However, the details of force-generation, and the atomic-level characteristics of the force-induced conformation, are unknown. Here, steered molecular dynamics (SMD) simulation was used to exert a force in silico on an 18 subunit segment of GC-T4P to address questions regarding the nature of the interactions that lead to the extraordinary strength of bacterial pili. SMD simulations revealed that the buried pilin α1 domains maintain hydrophobic contacts with one another within the core of the filament, leading to GC-T4P''s structural stability. At the filament surface, gaps between pilin globular head domains in both the native and pulled states provide water accessible routes between the external environment and the interior of the filament, allowing water to access the pilin α1 domains as reported for VC-T4P in deuterium exchange experiments. Results were also compared to the experimentally observed force-induced conformation. In particular, an exposed amino acid sequence in the experimentally stretched filament was also found to become exposed during the SMD simulations, suggesting that initial stages of the force induced transition are well captured. Furthermore, a second sequence was shown to be initially hidden in the native filament and became exposed upon stretching.     

Physical properties of the specific PapG–galabiose binding in <Emphasis Type="Italic">E. coli</Emphasis> P pili-mediated adhesion

Detailed analyses of the mechanisms that mediate binding of the uropathogenic Escherichia coli to host cells are essential, as attachment is a prerequisite for the subsequent infection process. We explore, by means of force measuring optical tweezers, the interaction between the galabiose receptor and the adhesin PapG expressed by P pili on single bacterial cells. Two variants of dynamic force spectroscopy were applied based on constant and non-linear loading force. The specific PapG-galabiose binding showed typical slip-bond behaviour in the force interval (30-100 pN) set by the pilus intrinsic biomechanical properties. Moreover, it was found that the bond has a thermodynamic off-rate and a bond length of 2.6 x 10(-3) s(-1) and 5.0 A, respectively. Consequently, the PapG-galabiose complex is significantly stronger than the internal bonds in the P pilus structure that stabilizes the helical chain-like macromolecule. This finding suggests that the specific binding is strong enough to enable the P pili rod to unfold when subjected to strong shear forces in the urinary tract. The unfolding process of the P pili rod promotes the formation of strong multipili interaction, which is important for the bacterium to maintain attachment to the host cells.