Function of the usher N-terminus in catalysing pilus assembly

The chaperone/usher (CU) pathway is a conserved bacterial secretion system that assembles adhesive fibres termed pili or fimbriae. Pilus biogenesis by the CU pathway requires a periplasmic chaperone and an outer membrane (OM) assembly platform termed the usher. The usher catalyses formation of subunit-subunit interactions to promote polymerization of the pilus fibre and provides the channel for fibre secretion. The mechanism by which the usher catalyses pilus assembly is not known. Using the P and type 1 pilus systems of uropathogenic Escherichia coli, we show that a conserved N-terminal disulphide region of the PapC and FimD ushers, as well as residue F4 of FimD, are required for the catalytic activity of the ushers. PapC disulphide loop mutants were able to bind PapDG chaperone-subunit complexes, but did not assemble PapG into pilus fibres. FimD disulphide loop and F4 mutants were able to bind chaperone-subunit complexes and initiate assembly of pilus fibres, but were defective for extending the pilus fibres, as measured using in vivo co-purification and in vitro pilus polymerization assays. These results suggest that the catalytic activity of PapC is required to initiate pilus biogenesis, whereas the catalytic activity of FimD is required for extension of the pilus fibre.     

Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis

Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface. We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli. Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains. Several periplasmic regions were localized, including two domains containing conserved cysteine pairs. Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding. The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo. Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo. These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.     

Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminus

Uropathogenic strains of Escherichia coli assemble type 1 and P pili to colonize the bladder and kidney respectively. These pili are prototype structures assembled by the chaperone/usher secretion pathway. In this pathway, a periplasmic chaperone works together with an outer membrane (OM) usher to control the folding of pilus subunits, their assembly into a pilus fibre and secretion of the fibre to the cell surface. The usher serves as the assembly and secretion platform in the OM. The usher has distinct functional domains, with the N-terminus providing the initial targeting site for chaperone-subunit complexes and the C-terminus required for subsequent stages of pilus biogenesis. In this study, we investigated the molecular interactions occurring at the usher during pilus biogenesis and the function of the usher C-terminus. We provide genetic and biochemical evidence that the usher functions as a complex in the OM and that interaction of the pilus adhesin with the usher is critical to prime the usher for pilus biogenesis. Analysis of C-terminal truncation and substitution mutants of the P pilus usher PapC demonstrated that the C-terminus is required for proper binding of chaperone-subunit complexes to the usher and plays an important role in assembly of complete pili.     

The outer membrane usher forms a twin-pore secretion complex

The PapC usher is an outer membrane protein required for assembly and secretion of P pili in uropathogenic Escherichia coli. P pilus biogenesis occurs by the chaperone/usher pathway, a terminal branch of the general secretory pathway. Periplasmic chaperone-subunit complexes target to the PapC usher for fiber assembly and secretion through the usher to the cell surface. The molecular details of pilus biogenesis at the usher, and protein secretion across the outer membrane in general, are unclear. We studied the structure and oligomeric state of PapC by gel filtration, dynamic light scattering, and electron microscopy and image analysis. Two-dimensional crystals of wild-type PapC and a C-terminal deletion mutant of PapC were produced by reconstituting detergent purified usher into E.coli lipids. PapC formed a dimer both in detergent solution and in the phospholipid bilayer. Cryo-electron microscopy revealed that the usher forms a twin-pore complex. Removal of the C-terminal domain did not change the basic shape of the PapC molecule, but altered the dimeric association of the usher, suggesting that the C terminus forms part of the dimerization interface. The overall molecular size (11 nm), pore size (2 nm), and twin-pore configuration of PapC resemble that of the Tom40 complex, a mitochondrial outer membrane protein translocase.     

Fiber formation across the bacterial outer membrane by the chaperone/usher pathway

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.     

Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD

Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface. Here we report nuclear magnetic resonance and X-ray protein structures of the N-terminal substrate recognition domain of FimD (FimD(N)) before and after binding of a chaperone-subunit complex. FimD(N) consists of a flexible N-terminal segment of 24 residues, a structured core with a novel fold, and a C-terminal hinge segment. In the ternary complex, residues 1-24 of FimD(N) specifically interact with both FimC and the subunit, acting as a sensor for loaded FimC molecules. Together with in vivo complementation studies, we show how this mechanism enables recognition and discrimination of different chaperone-subunit complexes by bacterial pilus assembly platforms.     

The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS

The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.     

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.     

Twin ushers guide pili across the bacterial outer membrane

The chaperone/usher pathway is responsible for the assembly of adhesive pili on the surface of gram-negative pathogenic bacteria. In this issue, Remaut et al. (2008) present the crystal structure of the PapC usher translocation domain and images of the FimD usher bound to a pilus translocation intermediate. These new structures provide the first detailed view of a translocase in action.     

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.     

Modulating Effects of the Plug, Helix, and N- and C-terminal Domains on Channel Properties of the PapC Usher

The chaperone/usher system is one of the best characterized pathways for protein secretion and assembly of cell surface appendages in Gram-negative bacteria. In particular, this pathway is used for biogenesis of the P pilus, a key virulence factor used by uropathogenic Escherichia coli to adhere to the host urinary tract. The P pilus individual subunits bound to the periplasmic chaperone PapD are delivered to the outer membrane PapC usher, which serves as an assembly platform for subunit incorporation into the pilus and secretion of the pilus fiber to the cell surface. PapC forms a dimeric, twin pore complex, with each monomer composed of a 24-stranded transmembrane β-barrel channel, an internal plug domain that occludes the channel, and globular N- and C-terminal domains that are located in the periplasm. Here we have used planar lipid bilayer electrophysiology to characterize the pore properties of wild type PapC and domain deletion mutants for the first time. The wild type pore is closed most of the time but displays frequent short-lived transitions to various open states. In comparison, PapC mutants containing deletions of the plug domain, an α-helix that caps the plug domain, or the N- and C-terminal domains form channels with higher open probability but still exhibiting dynamic behavior. Removal of the plug domain results in a channel with extremely large conductance. These observations suggest that the plug gates the usher channel closed and that the periplasmic domains and α-helix function to modulate the gating activity of the PapC twin pore.     

The Outer Membrane Usher Guarantees the Formation of Functional Pili by Selectively Catalyzing Donor-Strand Exchange between Subunits That Are Adjacent in the Mature Pilus

Type 1 pili from uropathogenic Escherichia coli are a prototype of adhesive surface organelles assembled and secreted by the conserved chaperone/usher pathway. They are composed of four different homologous protein subunits that need to be assembled in a defined order. In the periplasm, the pilus chaperone FimC donates a β-strand segment to the subunits to complete their imperfect immunoglobulin-like fold. During subunit assembly, this segment of the chaperone is displaced by an amino-terminal extension of an incoming subunit in a reaction termed donor-strand exchange. To date, the molecular mechanisms underlying the coordinated subunit assembly, in particular the role of the outer membrane usher FimD, are still poorly understood. Here we show that the binding of complexes between FimC and the different pilus subunits to the amino-terminal substrate recognition domain of FimD is an extremely fast process, with association rate constants in the range of 10⁷-10⁸ M^− 1 s^− 1 at 20 °C. Furthermore, we demonstrate that the ordered assembly of pilus subunits is a consequence of the usher's ability to selectively catalyze the assembly of defined subunit-subunit pairs that are adjacent in the mature pilus. The usher therefore coordinates the assembly of pilus subunits at the stage of donor-strand exchange between pairs of subunits and not at the level of the initial binding of chaperone-subunit complexes.     

Ramifications of kinetic partitioning on usher-mediated pilus biogenesis.

The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone-adhesin complex with the usher. A study of association and dissociation events of chaperone-subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone-adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone-adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone-adhesin-usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone-adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly.     

Recognition of the N-terminal lectin domain of FimH adhesin by the usher FimD is required for type 1 pilus biogenesis

In this work we discover that a specific recognition of the N-terminal lectin domain of FimH adhesin by the usher FimD is essential for the biogenesis of type 1 pili in Escherichia coli. These filamentous organelles are assembled by the chaperone-usher pathway, in which binary complexes between fimbrial subunits and the periplasmic chaperone FimC are recognized by the outer membrane protein FimD (the usher). FimH adhesin initiates fimbriae polymerization and is the first subunit incorporated in the filament. Accordingly, FimD shows higher affinity for the FimC/FimH complex although the structural basis of this specificity is unknown. We have analysed the assembly into fimbria, and the interaction with FimD in vivo, of FimH variants in which the N-terminal lectin domain of FimH was deleted or substituted by different immunoglobulin (Ig) domains, or in which these Ig domains were fused to the N-terminus of full-length FimH. From these data, along with the analysis of a FimH mutant with a single amino acid change (G16D) in the N-terminal lectin domain, we conclude that the lectin domain of FimH is recognized by FimD usher as an essential step for type 1 pilus biogenesis.     

Crystal structure of the ternary FimC-FimF(t)-FimD(N) complex indicates conserved pilus chaperone-subunit complex recognition by the usher FimD

Type 1 pili, anchored to the outer membrane protein FimD, enable uropathogenic Escherichia coli to attach to host cells. During pilus biogenesis, the N-terminal periplasmic domain of FimD (FimD(N)) binds complexes between the chaperone FimC and pilus subunits via its partly disordered N-terminal segment, as recently shown for the FimC-FimH(P)-FimD(N) ternary complex. We report the structure of a new ternary complex (FimC-FimF(t)-FimD(N)) with the subunit FimF(t) instead of FimH(p). FimD(N) recognizes FimC-FimF(t) and FimC-FimH(P) very similarly, predominantly through hydrophobic interactions. The conserved binding mode at a "hot spot" on the chaperone surface could guide the design of pilus assembly inhibitors.     

Chaperone-subunit-usher interactions required for donor strand exchange during bacterial pilus assembly

The assembly of type 1 pili on the surface of uropathogenic Escherichia coli proceeds via the chaperone-usher pathway. Chaperone-subunit complexes interact with one another via a process termed donor strand complementation whereby the G1beta strand of the chaperone completes the immunoglobulin (Ig) fold of the pilus subunit. Chaperone-subunit complexes are targeted to the usher, which forms a channel across the outer membrane through which pilus subunits are translocated and assembled into pili via a mechanism known as donor strand exchange. This is a mechanism whereby chaperone uncapping from a subunit is coupled with the simultaneous assembly of the subunit into the pilus fiber. Thus, in the pilus fiber, the N-terminal extension of every subunit completes the Ig fold of its neighboring subunit by occupying the same site previously occupied by the chaperone. Here, we investigated details of the donor strand exchange assembly mechanism. We discovered that the information necessary for targeting the FimC-FimH complex to the usher resides mainly in the FimH protein. This interaction is an initiating event in pilus biogenesis. We discovered that the ability of an incoming subunit (in a chaperone-subunit complex) to participate in donor strand exchange with the growing pilus depended on a previously unrecognized function of the chaperone. Furthermore, the donor strand exchange assembly mechanism between subunits was found to be necessary for subunit translocation across the outer membrane usher.     

Structural Homology between the C-Terminal Domain of the PapC Usher and Its Plug

P pili are extracellular appendages responsible for the targeting of uropathogenic Escherichia coli to the kidney. They are assembled by the chaperone-usher (CU) pathway of pilus biogenesis involving two proteins, the periplasmic chaperone PapD and the outer membrane assembly platform, PapC. Many aspects of the structural biology of the Pap CU pathway have been elucidated, except for the C-terminal domain of the PapC usher, the structure of which is unknown. In this report, we identify a stable and folded fragment of the C-terminal region of the PapC usher and determine its structure using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These structures reveal a β-sandwich fold very similar to that of the plug domain, a domain of PapC obstructing its translocation domain. This structural similarity suggests similar functions in usher-mediated pilus biogenesis, playing out at different stages of the process. This structure paves the way for further functional analysis targeting surfaces common to both the plug and the C-terminal domain of PapC.Adhesive surface organelles termed pili mediate the adhesion of bacteria to host cells. Pili assembled by the chaperone-usher (CU) pathway form one of five major classes of nonflagellar surface appendages in Gram-negative bacteria, with the P pilus system from uropathogenic Escherichia coli being one of the two best-characterized CU systems. These pili are multisubunit structures consisting of two distinct subassemblies, a rigid rod with a diameter of 6.8 nm and a distal flexible tip fibrillum with a diameter of 2 nm (18, 21). In P pili the helical rod is comprised of more than 1,000 copies of the PapA subunits arranged in a right-handed helical cylinder with 3.3 subunits per turn (3, 8, 14), and the tip fibrillum is comprised of 5 to 10 copies of the PapE subunits (21). Two “adaptor” subunits, PapK and PapF, connect the PapE tip fibrillum to the PapA rod and the PapE tip fibrillum to the distal PapG adhesin (16, 21). The proximal end of the pilus is terminated by the PapH subunit (2, 50). The PapG adhesin mediates the bacterial colonization of the kidney (25, 40) by binding to the globoseries of glycolipids present in the human kidney (25, 40) (Fig. ​(Fig.1A),1A), an event that is critical in pyelonephritis.Open in a separate windowFIG. 1.(A) Schematic diagram of a P pilus assembled in the usher translocation platform. Subunits are represented by oval shapes, and N-terminal extensions are represented by short rectangular shapes. The usher homodimer is represented in the outer membrane (OM). In the usher protomer through which the nascent pilus passes, two positions of the plug are indicated by P where the plug is positioned to the side of the transmembrane barrel''s lumen and P′ where the plug has swung into the periplasmic space. (B) Domain organization of the PapC usher based on amino acid sequence. The C-terminal domain sequences are indicated in marine blue. The constructs used in this study are schematically represented underneath; all converge to a fragment containing residues 722 to 809, termed the “PapC CTD.” Ntd, N-terminal domain. (C) Identification of a discrete folding unit at the C terminus of PapC. Shown is an SDS-PAGE gel stained with Coomassie blue of the eluted PapC C-terminal fragments obtained with a construct comprising residues 641 to 809 after the first purification step. PS, prestained protein standards; Inj, loaded sample; FT, flowthrough.The assembly of pili is a coordinated process requiring two proteins: a chaperone and an outer membrane assembly platform, the usher. Pilus subunits are translocated into the periplasm via the general secretory machinery (38, 47). The binding of the PapD chaperone to the nascently translocated subunits facilitates their folding on the chaperone template. The chaperone remains bound to the folded subunits capping their interactive surfaces, thus preventing nonproductive interactions in the periplasm (7). Chaperone-subunit complexes are then targeted to the usher (PapC), where subunits polymerize in an ordered fashion and translocate across the outer membrane through the usher pore (47, 52). Subunit folding and stabilization occur when the chaperone and subunit form a complex through a mechanism termed donor strand complementation (DSC) (9, 41). In this mechanism the C-terminally truncated Ig-like fold of the pilus subunits, which contains only six of the seven β-strands that constitute the canonical Ig fold, is complemented by the donation of a β-strand from the chaperone (9, 41). Chaperone-subunit complexes are then targeted to the outer membrane usher, where the chaperone is released and subunits are noncovalently joined to preceding subunits in the nascent pilus fiber. This polymerization process is made possible by the presence of a disordered N-terminal extension sequence (NTES) in each subunit (except the adhesin) (41), which during pilus assembly displaces the strand donated by the chaperone, thereby substituting for the missing secondary structure in the previously assembled subunit. This mechanism is called donor-strand exchange (DSE) (9, 41, 42, 55). It is believed that this structural reorganization provides the driving force for pilus biogenesis, since no ATP hydrolysis or other type of external energy source is required (17, 56).DSE occurs at the outer membrane usher, which acts as a catalyst for polymerization (34). Biophysical and cryo-electron microscopy (EM) studies of the FimD usher (a close homolog of PapC) have shown that the usher is a twinned pore in both detergent and lipid bilayers (23, 46). Only one pore is used for secretion, but two pores are required for subunit recruitment (39). For PapC, both monomers and dimers have been described (15, 39). The usher has four functional domains (Fig. ​(Fig.1B):1B): a translocation domain forming a β-barrel with 24 transmembrane β-strands (15, 39), a plug domain in the middle of the translocation domain, and two periplasmic domains, one at each of the N- and C-terminal ends of the usher polypeptide (35, 48). The plug domain has a β-sandwich fold and completely occludes the pore in the inactive usher. Its function, besides gating the channel, seems to be further associated with pilus biogenesis since the deletion of the plug domain abolishes pilus formation in vitro and in vivo (15, 26, 54). The N-terminal domain selectively binds chaperone-subunit complexes (12, 33). The structure of the N-terminal domain of FimD bound to chaperone-subunit complexes indicated that the first 24 residues of FimD are involved in the recognition of chaperone-subunit complexes; the deletion of this region was shown previously to abolish pilus biogenesis (12, 32, 33).The role of the usher C-terminal domain (CTD) is not well understood. The binding of the chaperone-adhesin complex to the usher C terminus was previously demonstrated in vitro (46), while protease susceptibility in FimD shows that, following targeting to the usher N terminus, the chaperone-adhesin complex forms stable interactions with the FimD C terminus, inducing a conformational change in FimD that may be fundamental in the activation step of pilus biogenesis (29, 30, 43). The structure of the C-terminal domain is unknown and is the only part of the CU pilus biogenesis pathway not yet represented in structural terms. Here we provide evidence for the presence of a discrete folding unit in the PapC CTD and report its structure determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography.     

Specific residues in the N-terminal domain of FimH stimulate type 1 fimbriae assembly in Escherichia coli following the initial binding of the adhesin to FimD usher

Type 1 fimbriae are assembled by the chaperone–usher pathway where periplasmic protein complexes formed between fimbrial subunits and the FimC chaperone are recruited by the outer membrane protein FimD (the usher) for their ordered polymerization and export. FimH adhesin initiates and stimulates type 1 fimbriae polymerization by interacting with FimD. Previously we showed that the N-terminal lectin domain of FimH (N-FimH) is necessary for binding of the adhesin to FimD. In this work, we have selected mutants in N-FimH that reduce the levels of adhesin and type 1 fimbriae displayed in Escherichia coli without altering the levels of FimH in the periplasm. The selected mutations are mostly concentrated in residues G15, N46 and D47. In contrast to other mutations isolated that simply affect binding of FimH to FimD (e.g. C3Y), these variants associate to FimD and alter its susceptibility to trypsin digestion similarly to wild-type FimH. Importantly, their mutant phenotype is rescued when FimD is activated in vivo by the coexpression of wild-type FimH. Altogether, these data indicate that residues G15, N46 and D47 play an important role following initial binding of FimH to FimD for efficient type 1 fimbriae polymerization by this outer membrane usher.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

The chaperone/usher pathway: a major terminal branch of the general secretory pathway

Gram-negative bacteria assemble a variety of adhesive organelles on their surface, including the thread-like structures known as pili. Recent studies on pilus assembly by the chaperone/usher pathway have revealed new insights into the mechanisms of pilus subunit export into the periplasm and targeting to the outer membrane. Signaling events controlling pilus biogenesis have begun to emerge and investigations of the usher have yielded insights into pilus translocation across the outer membrane.