Electrostatic networks control plug stabilization in the PapC usher

Abstract

The PapC usher, a β-barrel pore in the outer membrane of uropathogenic Escherichia coli, is used for assembly of the P pilus, a key virulence factor in bacterial colonization of human kidney cells. Each PapC protein is composed of a 24-stranded β-barrel channel, flanked by N- and C-terminal globular domains protruding into the periplasm, and occluded by a plug domain (PD). The PD is displaced from the channel towards the periplasm during pilus biogenesis, but the molecular mechanism for PD displacement remains unclear. Two structural features within the β-barrel, an α-helix and β5-6 hairpin loop, may play roles in controlling plug stabilization. Here we have tested clusters of residues at the interface of the plug, barrel, α-helix and hairpin, which participate in electrostatic networks. To assess the roles of these residues in plug stabilization, we used patch-clamp electrophysiology to compare the activity of wild-type and mutant PapC channels containing alanine substitutions at these sites. Mutations interrupting each of two salt bridge networks were relatively ineffective in disrupting plug stabilization. However, mutation of two pairs of arginines located at the inner and the outer surfaces of the PD resulted in an enhanced propensity for plug displacement. One arginine pair involved in a repulsive interaction between the linkers that tether the plug to the β-barrel was particularly sensitive to mutation. These results suggest that plug displacement, which is necessary for pilus assembly and translocation, may require a weakening of key electrostatic interactions between the plug linkers, and the plug and the α-helix.     

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.     

The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events

Pilus biogenesis on the surface of uropathogenic Escherichia coli requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.     

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.     

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.     

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.     

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.     

Topology of the outer membrane usher PapC determined by site-directed fluorescence labeling

In contrast to typical membrane proteins that span the lipid bilayer via transmembrane alpha-helices, bacterial outer membrane proteins adopt a beta-barrel architecture composed of antiparallel transmembrane beta-strands. The topology of outer membrane proteins is difficult to predict accurately using computer algorithms, and topology mapping protocols commonly used for alpha-helical membrane proteins do not work for beta-barrel proteins. We present here the topology of the PapC usher, an outer membrane protein required for assembly and secretion of P pili by the chaperone/usher pathway in uropathogenic Escherichia coli. An initial attempt to map PapC topology by insertion of protease cleavage sites was largely unsuccessful due to lack of cleavage at most sites and the requirement to disrupt the outer membrane to identify periplasmic sites. We therefore adapted a site-directed fluorescence labeling technique to permit topology mapping of outer membrane proteins using small molecule probes in intact bacteria. Using this method, we demonstrated that PapC has the potential to encode up to 32 transmembrane beta-strands. Based on experimental evidence, we propose that the usher consists of an N-terminal beta-barrel domain comprised of 26 beta-strands and that a distinct C-terminal domain is not inserted into the membrane but is located instead within the lumen of the N-terminal beta-barrel similar to the plug domains encoded by the outer membrane iron-siderophore uptake proteins.     

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.     

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.     

Characterization of FimC, a periplasmic assembly factor for biogenesis of type 1 pili in Escherichia coli

Assembly of type 1 pili from Escherichia coli is mediated by FimC, a periplasmic chaperone (assembly factor) consisting of two immunoglobulin-like domains. FimC is assumed to recognize the individual pilus subunits in the periplasm mainly via their conserved C-terminal segments and to deliver the subunits to an assembly platform in the outer membrane. Here we present the first biochemical characterization of a periplasmic pilus chaperone and analyze the importance of the two chaperone domains for stability and function. Comparison of the isolated C-terminal domain with wild-type FimC revealed a strongly reduced thermodynamic stability, indicating strong interdomain interactions. The affinity of FimC toward a peptide corresponding to the 11 C-terminal residues of the type 1 pilus adhesin FimH is at least 1000-fold lower compared to binding of intact FimH, confirming that bacterial pilus chaperones, unlike other chaperones, specifically interact with folded pilus subunits.     

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.     

Evidence for a novel domain of bacterial outer membrane ushers

Many pathogenic bacteria possess adhesive surface organelles (called pili), anchored to their outer membrane, which mediate the first step of infection by binding to host tissue. Pilus biogenesis occurs via the "chaperone-usher" pathway: the usher, a large outer membrane protein, binds complexes of a periplasmic chaperone with pilus subunits, unloads the subunits from the chaperone, and assembles them into the pilus, which is extruded into the extracellular space. Ushers comprise an N-terminal periplasmic domain, a large transmembrane beta-barrel central domain, and a C-terminal periplasmic domain. Since structural data are available only for the N-terminal domain, we performed an in-depth bioinformatic analysis of bacterial ushers. Our analysis led us to the conclusion that the transmembrane beta-barrel region of ushers contains a so far unrecognized soluble domain, the "middle domain", which possesses a beta-sandwich fold. Two other bacterial beta-sandwich domains, the TT0351 protein from Thermus thermophilus and the carbohydrate binding module CBM36 from Paenibacillus polymyxa, are possible distant relatives of the usher "middle domain". Several mutations reported to abolish in vivo pilus formation cluster in this region, underlining its functional importance.     

Structure and Assembly of a Trans-Periplasmic Channel for Type IV Pili in Neisseria meningitidis

Type IV pili are polymeric fibers which protrude from the cell surface and play a critical role in adhesion and invasion by pathogenic bacteria. The secretion of pili across the periplasm and outer membrane is mediated by a specialized secretin protein, PilQ, but the way in which this large channel is formed is unknown. Using NMR, we derived the structures of the periplasmic domains from N. meningitidis PilQ: the N-terminus is shown to consist of two β-domains, which are unique to the type IV pilus-dependent secretins. The structure of the second β-domain revealed an eight-stranded β-sandwich structure which is a novel variant of the HSP20-like fold. The central part of PilQ consists of two α/β fold domains: the structure of the first of these is similar to domains from other secretins, but with an additional α-helix which links it to the second α/β domain. We also determined the structure of the entire PilQ dodecamer by cryoelectron microscopy: it forms a cage-like structure, enclosing a cavity which is approximately 55 Å in internal diameter at its largest extent. Specific regions were identified in the density map which corresponded to the individual PilQ domains: this allowed us to dock them into the cryoelectron microscopy density map, and hence reconstruct the entire PilQ assembly which spans the periplasm. We also show that the C-terminal domain from the lipoprotein PilP, which is essential for pilus assembly, binds specifically to the first α/β domain in PilQ and use NMR chemical shift mapping to generate a model for the PilP:PilQ complex. We conclude that passage of the pilus fiber requires disassembly of both the membrane-spanning and the β-domain regions in PilQ, and that PilP plays an important role in stabilising the PilQ assembly during secretion, through its anchorage in the inner membrane.     

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.     

Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus.

The class of proteins collectively known as periplasmic immunoglobulin-like chaperones play an essential role in the assembly of a diverse set of adhesive organelles used by pathogenic strains of Gram-negative bacteria. Herein, we present a combination of genetic and structural data that sheds new light on chaperone-subunit and subunit-subunit interactions in the prototypical P pilus system, and provides new insights into how PapD controls pilus biogenesis. New crystallographic data of PapD with the C-terminal fragment of a subunit suggest a mechanism for how periplasmic chaperones mediate the extraction of pilus subunits from the inner membrane, a prerequisite step for subunit folding. In addition, the conserved N- and C-terminal regions of pilus subunits are shown to participate in the quaternary interactions of the mature pilus following their uncapping by the chaperone. By coupling the folding of subunit proteins to the capping of their nascent assembly surfaces, periplasmic chaperones are thereby able to protect pilus subunits from premature oligomerization until their delivery to the outer membrane assembly site.     

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.     

Periplasmic peptidyl prolyl cis-trans isomerases are not essential for viability, but SurA is required for pilus biogenesis in Escherichia coli

In Escherichia coli, FkpA, PpiA, PpiD, and SurA are the four known periplasmic cis-trans prolyl isomerases. These isomerases facilitate proper protein folding by increasing the rate of transition of proline residues between the cis and trans states. Genetic inactivation of all four periplasmic isomerases resulted in a viable strain that exhibited a decreased growth rate and increased susceptibility to certain antibiotics. Levels of the outer membrane proteins LamB and OmpA in the quadruple mutant were indistinguishable from those in the surA single mutant. In addition, expression of P and type 1 pili (adhesive organelles produced by uropathogenic strains of E. coli and assembled by the chaperone/usher pathway) were severely diminished in the absence of the four periplasmic isomerases. Maturation of the usher was significantly impaired in the outer membranes of strains devoid of all four periplasmic isomerases, resulting in a defect in pilus assembly. Moreover, this defect in pilus assembly and usher stability could be attributed to the absence of SurA. The data presented here suggest that the four periplasmic isomerases are not essential for growth under laboratory conditions but may have significant roles in survival in environmental and pathogenic niches, as indicated by the effect on pilus production.     

Components of SurA required for outer membrane biogenesis in uropathogenic Escherichia coli

Background

SurA is a periplasmic peptidyl-prolyl isomerase (PPIase) and chaperone of Escherichia coli and other Gram-negative bacteria. In contrast to other PPIases, SurA appears to have a distinct role in chaperoning newly synthesized porins destined for insertion into the outer membrane. Previous studies have indicated that the chaperone activity of SurA rests in its “core module” (the N- plus C-terminal domains), based on in vivo envelope phenotypes and in vitro binding and protection of non-native substrates.

Methodology/Principal Findings

In this study, we determined the components of SurA required for chaperone activity using in vivo phenotypes relevant to disease causation by uropathogenic E. coli (UPEC), namely membrane resistance to permeation by antimicrobials and maturation of the type 1 pilus usher FimD. FimD is a SurA-dependent, integral outer membrane protein through which heteropolymeric type 1 pili, which confer bladder epithelial binding and invasion capacity upon uropathogenic E. coli, are assembled and extruded. Consistent with prior results, the in vivo chaperone activity of SurA in UPEC rested primarily in the core module. However, the PPIase domains I and II were not expendable for wild-type resistance to novobiocin in broth culture. Steady-state levels of FimD were substantially restored in the UPEC surA mutant complemented with the SurA N- plus C-terminal domains. The addition of PPIase domain I augmented FimD maturation into the outer membrane, consistent with a model in which domain I enhances stability of and/or substrate binding by the core module.

Conclusions/Significance

Our results confirm the core module of E. coli SurA as a potential target for novel anti-infective development.