Crystal structure of the P pilus rod subunit PapA

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G1 beta-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it.     

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.     

The differential affinity of the usher for chaperone–subunit complexes is required for assembly of complete pili

Attachment to host cells via adhesive surface structures is a prerequisite for the pathogenesis of many bacteria. Uropathogenic Escherichia coli assemble P and type 1 pili for attachment to the host urothelium. Assembly of these pili requires the conserved chaperone/usher pathway, in which a periplasmic chaperone controls the folding of pilus subunits and an outer membrane usher provides a platform for pilus assembly and secretion. The usher has differential affinity for pilus subunits, with highest affinity for the tip‐localized adhesin. Here, we identify residues F21 and R652 of the P pilus usher PapC as functioning in the differential affinity of the usher. R652 is important for high‐affinity binding to the adhesin whereas F21 is important for limiting affinity for the PapA major rod subunit. PapC mutants in these residues are specifically defective for pilus assembly in the presence of PapA, demonstrating that differential affinity of the usher is required for assembly of complete pili. Analysis of PapG deletion mutants demonstrated that the adhesin is not required to initiate P pilus biogenesis. Thus, the differential affinity of the usher may be critical to ensure assembly of functional pilus fibres.     

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.     

Integrity of Escherichia coli P pili during biogenesis: properties and role of PapJ

The papJ gene of uropathogenic Escherichia coli is required to maintain the integrity of Gal alpha (1-4)Gal-binding P pili. Electron microscopy and ELISA have established that strains carrying the papJ1 mutant allele have a large amount of pilus antigen free of the cells. In contrast to the whole pili released by strains unable to produce the PapH pilus anchor, the free papJ1 pili consist of variably sized segments that appear to result from internal breakages to the pilus. The DNA sequence of papJ is presented and its gene product identified as an 18kD periplasmic protein that possesses homology with nucleotide-binding proteins. PapJ may function as a 'molecular chaperone' directly or indirectly establishing the correct assembly of PapA subunits in the P pilus.     

The unfolding of the P pili quaternary structure by stretching is reversible, not plastic

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 approximately 10(3) 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.     

Helical structure of P pili from Escherichia coli. Evidence from X-ray fiber diffraction and scanning transmission electron microscopy.

The structure of the P pili from Escherichia coli has been studied using X-ray fiber diffraction and scanning transmission electron microscopy (STEM). Analysis of the fiber diffraction data indicates that the pili are constituted largely of structural subunits arranged helically with approximately 33 subunits in 10 turns in an axial repeat of 244.5 +/- 1.8 A. Radial electron density distributions calculated from equatorial diffraction data and STEM data indicate that the pili are about 65 A in diameter with a small central cavity roughly 15 A across. The principal protein component of the pili is PapA, which has a molecular weight of 16.5 kDa. Assuming that each subunit consists of a single PapA molecule, the mass-per-unit-length of the pili predicted from the X-ray data is 2.23 kDa/A. Measurements of mass-per-unit-length were also made through the analysis of STEM images. These measurements indicate a value of 2.13 +/- 0.14 kDa/A. STEM images demonstrated the presence of thin, thread-like structures emerging from the ends of pili and spanning breaks in the pili structure. These structures, which have been observed under other conditions, have been termed fibrillae. In the STEM images the fibrillae appear about 20 A in diameter. The mass-per-unit-length of the fibrillae was estimated using the STEM data to be 0.4 kDa/A. These data are consistent with the fibrillae representing an unwound or unraveled form of the pili proteins overstretched to about five times the length they would have in the intact pili.     

Localization of a critical interface for helical rod formation of bacterial adhesion P-pili

Pyelonephritic Escherichia coli cause urinary tract infections that involve the kidneys. Initiation of infection is dependent on P-pili expressed on the bacterial surface. In this work, an essential interface for assembly of the helical rod structure of P-pili has been located on the major pilin subunit, PapA. Based on primary sequence alignment, secondary structure analysis, and quaternary structure modeling of the PapA subunit, we predicted the location of a site that is critical for in vivo assembly of the native macromolecular structure of P-pili. A rigid helical rod of PapA subunits comprising most of the pilus length is stabilized by n to n+3 subunit-subunit interactions, and is important for normal function of these pili. Using site-directed mutagenesis, ultrastructural analysis by electron cryomicroscopy, immunocytochemistry, and molecular modeling we show that residues 106-109 (Asn, Gly, Ala, Gly) are essential for assembly of native P-pilus filaments. Mutation of these residues disrupts assembly of the native P-pilus helix. Extended fibrillar structures do still assemble, verifying that n to n+1 subunit-subunit interactions are maintained in the mutant fiber morphology. Observation of this fibrillar morphology in the mutant fiber was predicted by our modeling studies. These mutant P-pili data validate the predictive value of our model for understanding subunit-subunit interactions between PapA monomers. Alteration of the pilus structure from a 7-8 nm helical rod to a 2 nm fibrillar structure may compromise the ability of these bacteria to adhere and remain bound to the host cell, thus providing a possible therapeutic target for antimicrobial drugs.     

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.     

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.     

Biogenesis of E. coli Pap pili: papH, a minor pilin subunit involved in cell anchoring and length modulation

The biogenesis of Escherichia coli Pap pili, encoded by the pap gene cluster, was studied. A novel gene, papH, was identified and found to encode a weakly expressed pilin-like protein. PapH was dispensable for digalactoside-specific binding and for formation of Pap pili. However, in papH deletion mutants 50%-70% of total pilus antigen was found free of the cells. We present evidence showing coregulation of papH and the adjacent gene, papA, which encodes the major pilin subunit. A decrease in the PapA to PapH ratio resulted in a large fraction of cells producing shortened pili, whereas overproduction of PapA relative to PapH resulted in cells with lengthened pili. The data show that PapH has roles in anchoring the pilus to the cell and in modulating pilus length.     

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.     

Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod

The infectious ability of uropathogenic Escherichia coli relies on adhesive fibers, termed pili or fimbriae, that are expressed on the bacterial surface. Pili are multi-protein structures that are formed via a highly preserved assembly and secretion system called the chaperone-usher pathway. We have earlier reported that small synthetic compounds, referred to as pilicides, disrupt both type 1 and P pilus biogenesis in E. coli. In this study, we show that the pilicides do not affect the structure, dynamics or function of the pilus rod. This was demonstrated by first suppressing the expression of P pili in E. coli by pilicide treatment and, next, measuring the biophysical properties of the pilus rod. The reduced abundance of pili was assessed with hemagglutination, atomic force microscopy and Western immunoblot analysis. The biodynamic properties of the pili fibers were determined by optical tweezers force measurements on individual pili and were found to be intact. The presented results establish a potential use of pilicides as chemical tools to study important biological processes e.g. adhesion, pilus biogenesis and the role of pili in infections and biofilm formation.     

PapD, a periplasmic transport protein in P-pilus biogenesis.

The product of the papD gene of uropathogenic Escherichia coli is required for the biogenesis of digalactoside-binding P pili. Mutations within papD result in complete degradation of the major pilus subunit, PapA, and of the pilinlike proteins PapE and PapF and also cause partial breakdown of the PapG adhesin. The papD gene was sequenced, and the gene product was purified from the periplasm. The deduced amino acid sequence and the N-terminal sequence obtained from the purified protein revealed that PapD is a basic and hydrophilic peripheral protein. A periplasmic complex between PapD and PapE was purified from cells that overproduced and accumulated these proteins in the periplasm. Antibodies raised against this complex reacted with purified wild-type P pili but not with pili purified from a papE mutant. In contrast, anti-PapD serum did not react with purified pili or with the culture fluid of piliated cells. However, this serum was able to specifically precipitate the PapE protein from periplasmic extracts, confirming that PapD and PapE were associated as a complex. It is suggested that PapD functions in P-pilus biogenesis as a periplasmic transport protein. Probably PapD forms complexes with pilus subunits at the outer surface of the inner membrane and transports them in a stable configuration across the periplasmic space before delivering them to the site(s) of pilus polymerization.     

Assessing adhesion forces of type I and type IV pili of Xylella fastidiosa bacteria by use of a microfluidic flow chamber

Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 +/- 11 pN. Mutant cells possessing only type I pili required a force of 204 +/- 22 pN for removal, whereas cells possessing only type IV pili required 119 +/- 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.     

Biogenesis and adhesion of type 1 and P pili

BackgroundUropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis.Scope of reviewThe review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly.Major conclusionsThe usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus.Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner.The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described.General significanceThe combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotic resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.     

Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.     

A structural basis for sustained bacterial adhesion: biomechanical properties of CFA/I pili

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized the intrinsic biomechanical properties and kinetics of individual CFA/I pili at the single-organelle level, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding, can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.