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.     

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.     

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.     

The fimbrial usher FimD follows the SurA-BamB pathway for its assembly in the outer membrane of Escherichia coli

Fimbrial ushers are the largest β-barrel outer membrane proteins (OMPs) known to date, which function in the polymerization of fimbriae and their translocation to the bacterial surface. Folding and assembly of these complex OMPs are not characterized. Here, we investigate the role of periplasmic chaperones (SurA, Skp, DegP, and FkpA) and individual components of the β-barrel assembly machinery (BAM) complex (BamA, BamB, BamC, and BamE) in the folding of the Escherichia coli FimD usher. The FimD level is dramatically reduced (～30-fold) in a surA null mutant, but a strong cell envelope stress is constitutively activated with upregulation of DegP (～10-fold). To demonstrate a direct role of SurA, FimD folding was analyzed in a conditional surA mutant in which SurA expression was controlled. In this strain, FimD is depleted from bacteria in parallel to SurA without significant upregulation of DegP. Interestingly, the dependency on SurA is higher for FimD than for other OMPs. We also demonstrate that a functional BAM complex is needed for folding of FimD. In addition, FimD levels were strongly reduced (～5-fold) in a mutant lacking the accessory lipoprotein BamB. The critical role of BamB for FimD folding was confirmed by complementation and BamB depletion experiments. Similar to SurA dependency, FimD showed a stronger dependency on BamB than OMPs. On the other hand, folding of FimD was only marginally affected in bamC and bamE mutants. Collectively, our results indicate that FimD usher follows the SurA-BamB pathway for its assembly. The preferential use of this pathway for the folding of OMPs with large β-barrels is discussed.     

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.     

Assembly of complex organelles: pilus biogenesis in gram-negative bacteria as a model system

Pathogenic bacteria assemble a variety of adhesive structures on their surface for attachment to host cells. Some of these structures are quite complex. For example, the hair-like organelles known as pili or fimbriae are generally composed of several components and often exhibit composite morphologies. In gram-negative bacteria assembly of pili requires that the subunits cross the cytoplasmic membrane, fold correctly in the periplasm, target to the outer membrane, assemble into an ordered structure, and cross the outer membrane to the cell surface. Thus, pilus biogenesis provides a model for a number of basic biological problems including protein folding, trafficking, secretion, and the ordered assembly of proteins into complex structures. P pilus biogenesis represents one of the best-understood pilus systems. P pili are produced by 80-90% of all pyelonephritic Escherichia coli and are a major virulence determinant for urinary tract infections. Two specialized assembly factors known as the periplasmic chaperone and outer membrane usher are required for P pilus assembly. A chaperone/usher pathway is now known to be required for the biogenesis of more than 30 different adhesive structures in diverse gram-negative pathogenic bacteria. Elucidation of the chaperone/usher pathway was brought about through a powerful combination of molecular, biochemical, and biophysical techniques. This review discusses these approaches as they relate to pilus assembly, with an emphasis on newer techniques.     

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.     

Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

β-Barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the nonviable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli.     

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 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.     

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.     

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.     

Substitutions in the BamA β-barrel domain overcome the conditional lethal phenotype of a ΔbamB ΔbamE strain of Escherichia coli

BamA interacts with the BamBCDE lipoproteins, and together they constitute the essential β-barrel assembly machine (BAM) of Escherichia coli. The simultaneous absence of BamB and BamE confers a conditional lethal phenotype and a severe β-barrel outer membrane protein (OMP) biogenesis defect. Without BamB and BamE, wild-type BamA levels are significantly reduced, and the folding of the BamA β-barrel, as assessed by the heat-modifiability assay, is drastically compromised. Single-amino-acid substitutions in the β-barrel domain of BamA improve both bacterial growth and OMP biogenesis in a bamB bamE mutant and restore BamA levels close to the BamB(+) BamE(+) level. The substitutions alter BamA β-barrel folding, and folding in the mutants becomes independent of BamB and BamE. Remarkably, BamA β-barrel alterations also improve OMP biogenesis in cells lacking the major periplasmic chaperone, SurA, which, together with BamB, is thought to facilitate the transfer of partially folded OMPs to the soluble POTRA (polypeptide-transport-associated) domain of BamA. Unlike the bamB bamE mutant background, the absence of BamB or SurA does not affect BamA β-barrel folding. Thus, substitutions in the outer membrane-embedded BamA β-barrel domain overcome OMP biogenesis defects that occur at the POTRA domain of BamA in the periplasm. Based on the structure of FhaC, the altered BamA residues are predicted to lie on a highly conserved loop that folds inside the β-barrel and in regions pointing outside the β-barrel, suggesting that they influence BamA function by both direct and indirect mechanisms.     

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.     

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.     

Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli

Some strains of enterotoxigenic Escherichia coli associated with human diarrhoeal disease produce a class of pili represented by those called CS1. For the assembly of the major-pilin subunit, CooA, into pili, each of four linked genes, cooB,A,C, and D, is required. In this study, we have determined the subcellular localization of CooB, C and D, and investigated the molecular interactions of these proteins using specific antisera. CooD appears to be an integral pilus protein because it co-purifies with, and is strongly associated with, CS1 pili. In keeping with its role as an assembly protein, the CooD minor pilin (when overexpressed in CS1-piliated strains) was detected in periplasmic inter-molecular complexes with the major-pilin subunit CooA. CooB is an assembly protein found exclusively in the periplasm of CS1-piliated strains. CooB also forms periplasmic intermolecular complexes with CooA, but does not constitute part of the final pilus structure. Immunoblot analysis of cell fractions showed that CooC is an outer membrane protein of CS1-piliated E. coli. Based on this information, we have proposed a model for CS1 -pilus assembly which is very similar to the model for polymerization of the PapA pilin of uropathogenic E. coli. As the assembly proteins of Pap and CS1 pili are structurally unrelated, this may represent a case of convergent evolution.     

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.     

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.     

Role for Skp in LptD Assembly in Escherichia coli

The periplasmic chaperone Skp has long been implicated in the assembly of outer membrane proteins (OMPs) in Escherichia coli. It has been shown to interact with unfolded OMPs, and the simultaneous loss of Skp and the main periplasmic chaperone in E. coli, SurA, results in synthetic lethality. However, a Δskp mutant displays only minor OMP assembly defects, and no OMPs have been shown to require Skp for their assembly. Here, we report a role for Skp in the assembly of the essential OMP LptD. This role may be compensated for by other OMP assembly proteins; in the absence of both Skp and FkpA or Skp and BamB, LptD assembly is impaired. Overexpression of SurA does not restore LptD levels in a Δskp ΔfkpA double mutant, nor does the overexpression of Skp or FkpA restore LptD levels in the ΔsurA mutant, suggesting that Skp acts in concert with SurA to efficiently assemble LptD in E. coli. Other OMPs, including LamB, are less affected in the Δskp ΔfkpA and Δskp bamB::kan double mutants, suggesting that Skp is specifically necessary for the assembly of certain OMPs. Analysis of an OMP with a domain structure similar to that of LptD, FhuA, suggests that common structural features may determine which OMPs require Skp for their assembly.     

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.