Type IV-like pili formed by the type II secreton: specificity,composition, bundling,polar localization,and surface presentation of peptides

The secreton or type II secretion machinery of gram-negative bacteria includes several type IV pilin-like proteins (the pseudopilins) that are absolutely required for secretion. We previously reported the presence of a bundled pilus composed of the pseudopilin PulG on the surface of agar-grown Escherichia coli K-12 cells expressing the Klebsiella oxytoca pullulanase (Pul) secreton genes at high levels (N. Sauvonnet, G. Vignon, A. P. Pugsley, and P. Gounon, EMBO J. 19:2221-2228, 2000). We show here that PulG is the only pseudopilin in purified pili and that the phenomenon is not restricted to the Pul secreton reconstituted in E. coli or to PulG. For example, high-level expression of the endogenous E. coli gsp secreton genes caused production of bundled pili composed of the pseudopilin GspG, and the Pul secreton was able to form pili composed of PulG-like proteins from secreton systems of other bacteria. PulG derivatives in which the C terminus was extended by the addition of eight different peptides were also assembled into pili and functioned in secretion. Three of the C-terminal peptides were shown to be exposed along the entire length of the assembled pili. Hence, the C terminus of PulG may represent a permissive site for the insertion of immunogenic epitopes or other peptide sequences. One of these PulG variants, with a six-histidine tag at its C terminus, formed nonpolar, nonbundled pili, suggesting that bundle formation and polar localization are not correlated with the ability of PulG to function in secretion. We propose that the PulG pilus is an artifactual manifestation of a periplasmic "pseudopilus" and that cycles of pseudopilus extension and retraction within the periplasm propel pullulanase through secretin channels in the outer membrane. Abnormally long pili that extend beyond the outer membrane are produced only when pilus length control and retraction are deregulated by overproduction of the major pseudopilus subunit (PulG).     

Structure and assembly of the pseudopilin PulG

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.     

Disulfide bond formation in secreton component PulK provides a possible explanation for the role of DsbA in pullulanase secretion

When expressed in Escherichia coli, the 15 Klebsiella oxytoca pul genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these pul genes, efficient pullulanase secretion also requires the host dsbA gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a dsbA mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a dsbA mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.     

Type II protein secretion in Pseudomonas aeruginosa: the pseudopilus is a multifibrillar and adhesive structure

The type II secretion pathway of Pseudomonas aeruginosa is involved in the extracellular release of various toxins and hydrolytic enzymes such as exotoxin A and elastase. This pathway requires the function of a macromolecular complex called the Xcp secreton. The Xcp secreton shares many features with the machinery involved in type IV pilus assembly. More specifically, it involves the function of five pilin-like proteins, the XcpT-X pseudopilins. We show that, upon overexpression, the XcpT pseudopilin can be assembled in a pilus, which we call a type II pseudopilus. Image analysis and filtering of electron micrographs indicated that these appendages are composed of individual fibrils assembled together in a bundle structure. Our observations thus revealed that XcpT has properties similar to those of type IV pilin subunits. Interestingly, the assembly of the type II pseudopilus is not exclusively dependent on the Xcp machinery but can be supported by other similar machineries, such as the Pil (type IV pilus) and Hxc (type II secretion) systems of P. aeruginosa. In addition, heterologous pseudopilins can be assembled by P. aeruginosa into a type II pseudopilus. Finally, we showed that assembly of the type II pseudopilus confers increased bacterial adhesive capabilities. These observations confirmed the ability of pseudopilins to form a pilus structure and raise questions with respect to their function in terms of secretion and adhesion, two crucial biological processes in the course of bacterial infections.     

Pseudopilin residue E5 is essential for recruitment by the type 2 secretion system assembly platform

Type II secretion systems (T2SSs) promote secretion of folded proteins playing important roles in nutrient acquisition, adaptation and virulence of Gram‐negative bacteria. Protein secretion is associated with the assembly of type 4 pilus (T4P)‐like fibres called pseudopili. Initially membrane embedded, pseudopilin and T4 pilin subunits share conserved transmembrane segments containing an invariant Glu residue at the fifth position, E5. Mutations of E5 in major T4 pilins and in PulG, the major pseudopilin of the Klebsiella T2SS abolish fibre assembly and function. Among the four minor pseudopilins, only PulH required E5 for secretion of pullulanase, the substrate of the Pul T2SS. Mass‐spectrometry analysis of pili resulting from the co‐assembly of PulG^E5A variant and PulG^WT ruled out an E5 role in pilin processing and N‐methylation. A bacterial two‐hybrid analysis revealed interactions of the full‐length pseudopilins PulG and PulH with the PulJ‐PulI‐PulK priming complex and with the assembly factors PulM and PulF. Remarkably, PulG^E5A and PulH^E5A variants were defective in interaction with PulM but not with PulF, and co‐purification experiments confirmed the E5‐dependent interaction between native PulM and PulG. These results reveal the role of E5 in a recruitment step critical for assembly of the functional T2SS, likely relevant to T4P assembly systems.     

Multimers of the precursor of a type IV pilin-like component of the general secretory pathway are unrelated to pili

Both the mature and precursor forms of PulG, a type IV pilin-like component of the general secretory pathway of Klebsiella oxytoca, can be chemically cross-linked into multimers similar to those obtained by cross-linking the components of type IV pili. To explore the possibility that the PulG precursor could form a pilus-like structure, the PulG sequence was altered in a variety of ways, including (i) replacement of the characteristic hydrophobic region, which is required for the assembly of type IV pilins by the MalE signal peptide, or (ii) fusion of β-lactamase (βlaM) to the C-terminus. Neither of these changes affected multimerization. PulG precursor could be post-translationally processed by pre-pilin peptidase (PulO), indicating that the N-terminus of pre PulG remains on the cytoplasmic side of the cytoplasmic membrane where it is accessible to the catalytic site of this enzyme. Finally, precursor and mature forms of PulG could be efficiently cross-linked in a mixed dimer, indicating that at least a subpopu-lation of the two forms of the protein are probably located in clusters in the cytoplasmic membrane. These results provide further evidence that the cross-linked multimers of the precursor form of PulG are unrelated to type IV pilus-like structures. It is still unclear whether a subpopulation of processed PulG can be assembled into a rudimentary pilus-like structure.     

XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus

Pseudomonas aeruginosa is an opportunistic gram-negative pathogen equipped with multiple secretion systems. The type II secretion machinery (Xcp secreton) is involved in the release of toxins and enzymes. The Xcp secreton is a multiprotein complex, and most of its components share homology with proteins involved in type IV pili biogenesis. Among them, the XcpT-X pseudopilins possess characteristics of the major constituent of the type IV pili, the pilin PilA. We have shown previously that XcpT can be assembled in a multifibrillar structure that was called the pseudopilus. By using two different microscopic approaches, we show here that the pseudopili are preferentially isolated fibers rather than tight bundles. Moreover, none of the other four pseudopilins are able to form a pseudopilus, suggesting that the assembly of such a structure is a unique property of XcpT. Moreover, we show that 5 of the 12 Xcp proteins are not required for pseudopilus biogenesis, whereas they are for type II secretion. Most interestingly, we showed that one pseudopilin, XcpX, controls the assembly of XcpT into a pseudopilus. Indeed, when the number of XcpX subunits increases, the length of the pseudopilus decreases. Conversely, in the absence of XcpX, the pseudopilus length is abnormally long. Our results indicate that XcpT and XcpX directly interact with each other. Furthermore, this interaction induces a clear destabilization of XcpT. The interaction between XcpT and XcpX could be part of the molecular mechanism underlying the dynamic control of pseudopilus elongation, which could be crucial for type II-dependent protein secretion.     

The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex

It has been proposed that the four type IV pilin-like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans-periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin-like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X-100 + EDTA or at high pH, but not by Triton X-100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non-sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross-linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N-methylates the PulG precursor, and PulE, a putative ATP-binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.     

Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE

We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.     

An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca

The last gene (pulO) of the pulC-O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC-O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post-translationally. The products of two pulG derivatives with internal inframe deletions were also processed by PulO, but the three PulG-PhoA hybrids, two PulJ-PhoA hybrids and the single PulH-PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction experiments showed that both precursor and mature forms of PulG appear to be associated with low-density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.     

Characterization of type IV pilus genes in plant growth-promoting Pseudomonas putida WCS358.

In a search for factors that could contribute to the ability of the plant growth-stimulating Pseudomonas putida WCS358 to colonize plant roots, the organism was analyzed for the presence of genes required for pilus biosynthesis. The pilD gene of Pseudomonas aeruginosa, which has also been designated xcpA, is involved in protein secretion and in the biogenesis of type IV pili. It encodes a peptidase that processes the precursors of the pilin subunits and of several components of the secretion apparatus. Prepilin processing activity could be demonstrated in P. putida WCS358, suggesting that this nonpathogenic strain may contain type IV pili as well. A DNA fragment containing the pilD (xcpA) gene of P. putida was cloned and found to complement a pilD (xcpA) mutation in P. aeruginosa. Nucleotide sequencing revealed, next to the pilD (xcpA) gene, the presence of two additional genes, pilA and pilC, that are highly homologous to genes involved in the biogenesis of type IV pili. The pilA gene encodes the pilin subunit, and pilC is an accessory gene, required for the assembly of the subunits into pili. In comparison with the pil gene cluster in P. aeruginosa, a gene homologous to pilB is lacking in the P. putida gene cluster. Pili were not detected on the cell surface of P. putida itself, not even when pilA was expressed from the tac promoter on a plasmid, indicating that not all the genes required for pilus biogenesis were expressed under the conditions tested. Expression of pilA of P. putida in P. aeruginosa resulted in the production of pili containing P. putida PilA subunits.     

Roles of the Minor Pseudopilins,XpsH, XpsI and XpsJ,in the Formation of XpsG-Containing Pseudopilus in <Emphasis Type="BoldItalic">Xanthomonas campestris</Emphasis> pv. Campestris

Summary Due to their similarity to type IV pilus (Tfp) subunits, the pseudopilins, XpsG, -H, -I, -J and -K, have been predicted to form a pilus-like structure in the type II secretion (T2S) pathway. While overexpression of GspG can result in the formation of bundle structures, the functions of other pseudopilin are not known yet. In this study, we investigate the mutual interaction among the pseudopilins and characterize the specialized minor pseudopilin, XpsJ. By using gel filtration and Ni-NTA affinity chromatography, a linearly ordered interactive relationship is revealed among the four pseudopilins, XpsG-XpsI-XpsH-XpsJ. Notably, unlike the mutant XpsJ194 staying in the inner membrane, wild type XpsJ stayed in the outer membrane and blocked the extension of overexpressed XpsG to outside of the cell. By analogy with the Type I pilus structures, we hypothesize that the XpsH and XpsI might act as an adaptor to connect XpsJ with the major pseudopilin XpsG, and XpsJ might act as a tip to restrict the out-growth of XpsG in the pilus-like structure of the T2S pathway.     

Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains.

The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. gonorrhoeae prepilin peptidase gene, pilDNg. A single copy of the gene is shown to be present in the chromosome. Its product promotes correct cleavage of the gonococcal prepillin in Escherichia coli cells carrying both the prepilin peptidase gene and the pilin structural gene. PilDNg also cleaves prePulG, a type IV pilin-like protein of Klebsiella oxytoca. Moreover, PilDNg complements a mutation in the gene coding for the prepilin peptidase-like protein of K. oxytoca, pulO, partially restoring PulG-PulO-dependent extracellular secretion of the enzyme pullulanase. Finally, we show that genes homologous to pilDNg are present and expressed in a variety of species in the genus Neisseria, including some commensal strains.     

Insights into type IV pilus biogenesis and dynamics from genetic analysis of a C-terminally tagged pilin: a role for O-linked glycosylation

Type IV pili are surface organelles essential for pathogenicity of many Gram-negative bacteria. In Neisseria gonorrhoeae, the major subunit of type IV pili, PilE, is a target of its general O-linked glycosylation system. This system modifies a diverse set of periplasmic and extracellular gonococcal proteins with a variable set of glycans. Here we show that expression of a particular hexa-histidine-tagged PilE was associated with growth arrest. By studying intra- and extragenic suppressors, we found that this phenotype was dependent on pilus assembly and retraction. Based on these results, we developed a sensitive tool to identify factors with subtle effects on pilus dynamics. Using this approach, we found that glycan chain length has differential effects on the growth arrest that appears to be mediated at the level of pilin subunit-subunit interactions and bidirectional remodelling of pilin between its membrane-associated and assembled states. Gonococcal pilin glycosylation thus plays both an intracellular role in pilus dynamics and potential extracellular roles mediated through type IV pili. In addition to demonstrating the effect of glycosylation on pilus dynamics, the study provides a new way of identifying factors with less dramatic effects on processes involved in type IV pilus biogenesis.     

Type IV Pilin Proteins: Versatile Molecular Modules

Summary: Type IV pili (T4P) are multifunctional protein fibers produced on the surfaces of a wide variety of bacteria and archaea. The major subunit of T4P is the type IV pilin, and structurally related proteins are found as components of the type II secretion (T2S) system, where they are called pseudopilins; of DNA uptake/competence systems in both Gram-negative and Gram-positive species; and of flagella, pili, and sugar-binding systems in the archaea. This broad distribution of a single protein family implies both a common evolutionary origin and a highly adaptable functional plan. The type IV pilin is a remarkably versatile architectural module that has been adopted widely for a variety of functions, including motility, attachment to chemically diverse surfaces, electrical conductance, acquisition of DNA, and secretion of a broad range of structurally distinct protein substrates. In this review, we consider recent advances in this research area, from structural revelations to insights into diversity, posttranslational modifications, regulation, and function.     

New tools in an old trade: CS1 pilus morphogenesis

CS1 pili serve as the prototype for a large class of serologically distinct pili associated with enterotoxigenic Escherichia coli that cause diarrhoea in humans. The four genes essential for CS1 pilus morphogenesis, cooB, A, C and D, are arranged in an operon and encode structural and assembly proteins unlike those of other pilus systems commonly associated with Gram-negative bacteria. CS1 pili are composed primarily of the major pilin subunit, CooA, which determines the serological type of the pilus. The major pilin subunit is assembled into pili by the proteins CooB, CooC and CooD. CooD is both a minor component found at the pilus tip and an essential assembly protein, whereas CooC is an outer membrane protein thought to be involved in pilin transport. CooB is a novel periplasmic chaperone-like protein that forms intermolecular complexes with and stabilizes the major and minor pilins. Unlike other pilin chaperones, CooB also stabilizes the outer membrane component of the assembly system, CooC. The proteins of CS1 pili have no significant homology to those of the well-characterized Pap (pyelonephritis-associated) pili and related systems, although most of the features of pilus morphogenesis are similar. Therefore, these appear to be among the rare cases of convergent evolution. Thus, for CS1 pili, enterotoxigenic E. coli use new protein 'tools' in the old 'trade' of forming functional pili.     

Type III secretion system-associated pilus of Pseudomonas syringae as an epitope display tool

Type III secretion system-associated pili found in several plant pathogenic bacteria are required for injection of virulence proteins from bacteria into the plant cells. The possibility to use the type III secretion pilus of Pseudomonas syringae as an epitope display tool was studied. The advantage of the type III secretion pilus, compared with conventional fimbrial epitope display tools, is that the pilin subunits of the type III secretion pilus can auto-assemble into intact pili in vitro. Various peptides were inserted into the type III secretion pilin subunit, and secretion, assembly and surface properties of the modified pili were monitored. It was concluded that the outwards-projecting N-terminal region of the pilin can bear even 43 amino acids insertion. The three-dimensional structure of the epitope, however, can restrict the use of the pilus as an epitope display tool: a beta-hairpin structure was poorly tolerated.     

Identification of two minor subunits in the pilus of Haemophilus influenzae.

Haemophilus influenzae type b (Hib) organisms produce pili, which mediate attachment to human cells and are multimeric structures composed of a 24-kDa subunit called pilin or HifA. Although pili from other organisms contain additional proteins accessory to pilin, no structural components other than pilin have been identified in Hib pili. Previous analysis of a Hib pilus gene cluster, however, suggested that two genes, hifD and hifE, may encode additional pilus subunits. To determine if hifD and hifE encode pilus components, the genes were overexpressed in Escherichia coli and the resulting proteins were purified and used to raise polyclonal antisera. Antisera raised against C-terminal HifD and HifE fragments reacted with H. influenzae HifD and HifE proteins, respectively, on Western immunoblots. Western immunoblot analysis of immunoprecipitated Hib pili demonstrated that HifD and HifE copurified with pili. In enzyme-linked immunosorbent assays, antisera raised against a recombinant HifE protein that contained most of the mature protein reacted more to piliated Hib than to nonpiliated Hib or to a mutant containing a hifE gene insertion. Immunoelectron microscopy confirmed that the HifE antiserum bound to pili and demonstrated that the antiserum bound predominantly to the pilus tips. These data indicate that HifD and HifE are pilus subunits. Adherence inhibition studies demonstrated that the HifE antiserum completely blocked pilus-mediated hemagglutination, suggesting that HifE mediates pilus adherence.     

Pseudomonas aeruginosa Minor Pilins Prime Type IVa Pilus Assembly and Promote Surface Display of the PilY1 Adhesin

Type IV pili (T4P) contain hundreds of major subunits, but minor subunits are also required for assembly and function. Here we show that Pseudomonas aeruginosa minor pilins prime pilus assembly and traffic the pilus-associated adhesin and anti-retraction protein, PilY1, to the cell surface. PilV, PilW, and PilX require PilY1 for inclusion in surface pili and vice versa, suggestive of complex formation. PilE requires PilVWXY1 for inclusion, suggesting that it binds a novel interface created by two or more components. FimU is incorporated independently of the others and is proposed to couple the putative minor pilin-PilY1 complex to the major subunit. The production of small amounts of T4P by a mutant lacking the minor pilin operon was traced to expression of minor pseudopilins from the P. aeruginosa type II secretion (T2S) system, showing that under retraction-deficient conditions, T2S minor subunits can prime T4P assembly. Deletion of all minor subunits abrogated pilus assembly. In a strain lacking the minor pseudopilins, PilVWXY1 and either FimU or PilE comprised the minimal set of components required for pilus assembly. Supporting functional conservation of T2S and T4P minor components, our 1.4 Å crystal structure of FimU revealed striking architectural similarity to its T2S ortholog GspH, despite minimal sequence identity. We propose that PilVWXY1 form a priming complex for assembly and that PilE and FimU together stably couple the complex to the major subunit. Trafficking of the anti-retraction factor PilY1 to the cell surface allows for production of pili of sufficient length to support adherence and motility.