Domains within the Vibrio cholerae toxin coregulated pilin subunit that mediate bacterial colonization

Several experimental approaches have provided evidence suggesting that a domain within the C-terminal region of the TcpA pilin, delineated by the single disulfide loop, is directly responsible for the colonization function mediated by the toxin coregulated pilus (TCP) of Vibrio cholerae. This evidence includes the mapping of domains recognized by protective monoclonal antibodies to this region, the ability of peptides from within this region to elicit cholera protective antibody, the construction of tcpA missense mutations that abolish TCP function, and the requirement of a periplasmic disulfide isomerase to produce functional TCP.     

Type IV pilin structures: insights on shared architecture, fiber assembly, receptor binding and type II secretion

Type IV pili are long, flexible filaments that extend from the surface of Gram-negative bacteria and are formed by the polymerization of pilin subunits. This review focuses on the structural information available for each pilin subclass, type IVa and type IVb, highlighting the contributions crystal and nuclear magnetic resonance structures have made in understanding pilus function and assembly. In addition, the type II secretion pseudopilus subunit structure and helical assembly is compared to that of the type IV pilus. The pilin subunits adopt an alphabeta-roll fold formed by the hydrophobic packing of the C-terminal half of a long alpha-helix against an antiparallel beta-sheet. The conserved N-terminal half of the same alpha-helix, as well as two sequence- and structurally-variable regions, protrude from this globular head domain. Filament models have a hydrophobic core formed by the signature long alpha-helices, with variable regions at the filament surface.     

Crystallographic structure reveals phosphorylated pilin from Neisseria: phosphoserine sites modify type IV pilus surface chemistry and fibre morphology

Understanding the structural biology of type IV pili, fibres responsible for the virulent attachment and motility of numerous bacterial pathogens, requires a detailed understanding of the three-dimensional structure and chemistry of the constituent pilin subunit. X-ray crystallographic refinement of Neisseria gonorrhoeae pilin against diffraction data to 2.6 A resolution, coupled with mass spectrometry of peptide fragments, reveals phosphoserine at residue 68. Phosphoserine is exposed on the surface of the modelled type IV pilus at the interface of neighbouring pilin molecules. The site-specific mutation of serine 68 to alanine showed that the loss of the phosphorylation alters the morphology of fibres examined by electron microscopy without a notable effect on adhesion, transformation, piliation or twitching motility. The structural and chemical characterization of protein phosphoserine in type IV pilin subunits is an important indication that this modification, key to numerous regulatory aspects of eukaryotic cell biology, exists in the virulence factor proteins of bacterial pathogens. These O-linked phosphate modifications, unusual in prokaryotes, thus merit study for possible roles in pilus biogenesis and modulation of pilin chemistry for optimal in vivo function.     

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.     

Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus

Type IV pili are multifunctional filaments displayed on many bacterial pathogens. Members of the Type IVa pilus subclass are found on a diverse group of human pathogens, whereas Type IVb pili are found almost exclusively on enteric bacteria. The Type IVa and IVb subclasses are distinguished by differences in the pilin subunits, including the fold of the globular domain. To understand the implications of the distinct pilin folds, we compared the stabilities of pilin subunits and pilus filaments for the Type IVa GC pilus from Neisseria gonorrhoeae and the Type IVb toxin-coregulated pilus (TCP) from Vibrio cholerae. We show that while recombinant TCP pilin is more stable than GC pilin, the GC pili are more resistant to proteolysis, heat and chemical denaturation than TCP, remaining intact in 8?M urea. To understand these differences, we determined the TCP structure by electron microscopy and three-dimensional image reconstruction. TCP have an architecture similar to that of GC pili, with subunits arranged in a right-handed 1-start helix and related by an 8.4-? axial rise and a 96.8° azimuthal rotation. However, the TCP subunits are not as tightly packed as GC pilins, and the distinct Type IVb pilin fold exposes a segment of the α-helical core of TCP. Hydrophobic interactions dominate for both pilus subtypes, but base stacking by aromatic residues conserved among the Type IVa pilins may contribute to GC pilus stability. The extraordinary stability of GC pili may represent an adaptation of the Type IVa pili to harsh environments and the need to retract against external forces.     

Delineation of pilin domains required for bacterial association into microcolonies and intestinal colonization by Vibrio cholerae

The toxin-co-regulated pilus (TCP), a type 4 pilus that is expressed by epidemic strains of Vibrio cholerae O1 and O139, is required for colonization of the human intestine. The TCP structure is assembled as a polymer of repeating subunits of TcpA pilin that form long fibres, which laterally associate into bundles. Previous passive immunization studies have suggested that the C-terminal region of TcpA is exposed on the surface of the pilus fibre and has a critical role in mediating the colonization functions of TCP. In the present study, we have used site-directed mutagenesis to delineate two domains within the C-terminal region that contribute to TCP structure and function. Alterations in the first domain, termed the structural domain, result in altered pilus stability or morphology. Alterations in the second domain, termed the interaction domain, affect colonization and/or infection by CTX-bacteriophage without affecting pilus morphology. In vitro and in vivo analyses of the tcpA mutants revealed that a major function of TCP is to mediate bacterial interaction through direct pilus-pilus contact required for microcolony formation and productive intestinal colonization. The importance of this function is supported by the finding that intragenic suppressor mutations that restore colonization ability to colonization-deficient mutants simultaneously restore pilus-mediated bacterial interactions. The alterations resulting from the suppressor mutations also provide insight into the molecular interactions between pilin subunits within and between pilus fibres.     

Outer membrane translocation arrest of the TcpA pilin subunit in rfb mutants of Vibrio cholerae O1 strain 569B.

The toxin-coregulated pilus (TCP) of Vibrio cholerae is a type 4-related fimbrial adhesin and a useful model for the study of type 4 pilus biogenesis and related bacterial macromolecular transport pathways. Transposon mutagenesis of the putative perosamine biosynthesis genes in the rfb operon of V. cholerae 569B eliminates lipopolysaccharide (LPS) O-antigen biosynthesis but also leads to a specific defect in TCP export. Localization of TcpA is made difficult by the hydrophobic nature of this bundle-forming pilin, which floats anomalously in sucrose density gradients, but the processed form of TcpA can be found in membrane and periplasmic fractions prepared from these strains. While TcpA cannot be detected by surface immunogold labelling in transmission electron microscope preparations, EDTA pretreatment facilitates immunofluorescent antibody labelling of whole cells, and ultrathin cryosectioning techniques confirm membrane and periplasmic accumulation of TcpA. Salt and detergent extraction, protease accessibility, and chemical cross-linking experiments suggest that although TcpA has not been assembled on the cell surface, subunit interactions are otherwise identical to those within TCP. In addition, TcpA-mediated fucose-resistant hemagglutination of murine erythrocytes is preserved in whole-cell lysates, suggesting that TcpA has obtained its mature conformation. These data localize a stage of type 4 pilin translocation to the outer membrane, at which stage export failure leads to the accumulation of pilin subunits in a configuration similar to that within the mature fiber. Possible candidates for the outer membrane defect are discussed.     

Pseudomonas aeruginosa pilin activates the inflammasome

IL-1β is produced from inactive pro-IL-1β by activation of caspase-1 brought about by a multi-subunit protein platform called the inflammasome. Many bacteria can trigger inflammasome activity through flagellin activation of the host protein NLRC4. However, strains of the common human pathogen Pseudomonas aeruginosa lacking flagellin can still activate the inflammasome. We set out to identify what non-flagellin components could produce this activation. Using mass spectroscopy, we identified an inflammasome-activating factor from P. aeruginosa as pilin, the major component of the type IV bacterial pilus. Purified pilin introduced into mouse macrophages by liposomal delivery activated caspase-1 and led to secretion of mature IL-1β, as did recombinant pilin purified from Escherichia coli. This was dependent on caspase-1 but not on the host inflammasome proteins NLRC4, NLRP3 or ASC. Mutants of P. aeruginosa strain PA103 lacking pilin did not activate the inflammasome following infection of macrophages with live bacteria. Type III secretion remained intact in the absence of pili, showing this was not due to a lack of effector delivery. Our observations show pilin is a novel activator of the inflammasome in addition to flagellin and the recently described PrgJ protein family, the basal body rod component of the type III apparatus.     

Minor pilin subunits are conserved in Vibrio cholerae type IV pili

The nucleotide sequences of five open reading frames within the Vibrio cholerae NAGV14 type IV pilus gene cluster were determined. The genes showed high homology to the mannose-sensitive hemagglutinin (MSHA) pilus genes mshB, mshC, mshD, mshO and mshP. PCR analysis showed that a MSHA-like gene cluster is highly conserved among different V. cholerae strains, with the exception of the previously reported major pilin subunit. Recombinant MshB and MshO proteins were purified and specific antiserum was raised to each of them. Western blotting analyses showed that these antisera reacted with purified NAGV14 and MSHA pili. The results suggested that MshB and MshO are minor components of the pilus fiber. Although there was no cross-reaction between the major pilin subunits of NAGV14 and MSHA pili, minor components seemed to be highly homologous and immunologically cross-reactive.     

Structural characterization of CFA/III and Longus type IVb pili from enterotoxigenic Escherichia coli

The type IV pili are helical filaments found on many Gram-negative pathogenic bacteria, with multiple diverse roles in pathogenesis, including microcolony formation, adhesion, and twitching motility. Many pathogenic enterotoxigenic Escherichia coli (ETEC) isolates express one of two type IV pili belonging to the type IVb subclass: CFA/III or Longus. Here we show a direct correlation between CFA/III expression and ETEC aggregation, suggesting that these pili, like the Vibrio cholerae toxin-coregulated pili (TCP), mediate microcolony formation. We report a 1.26-Å resolution crystal structure of CofA, the major pilin subunit from CFA/III. CofA is very similar in structure to V. cholerae TcpA but possesses a 10-amino-acid insertion that replaces part of the α2-helix with an irregular loop containing a 3₁₀-helix. Homology modeling suggests a very similar structure for the Longus LngA pilin. A model for the CFA/III pilus filament was generated using the TCP electron microscopy reconstruction as a template. The unique 3₁₀-helix insert fits perfectly within the gap between CofA globular domains. This insert, together with differences in surface-exposed residues, produces a filament that is smoother and more negatively charged than TCP. To explore the specificity of the type IV pilus assembly apparatus, CofA was expressed heterologously in V. cholerae by replacing the tcpA gene with that of cofA within the tcp operon. Although CofA was synthesized and processed by V. cholerae, no CFA/III filaments were detected, suggesting that the components of the type IVb pilus assembly system are highly specific to their pilin substrates.     

Characterization and sequence analysis of pilin from F-like plasmids.

Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.     

Ultrahigh resolution and full-length pilin structures with insights for filament assembly, pathogenic functions, and vaccine potential

Pilin proteins assemble into Type IV pili (T4P), surface-displayed bacterial filaments with virulence functions including motility, attachment, transformation, immune escape, and colony formation. However, challenges in crystallizing full-length fiber-forming and membrane protein pilins leave unanswered questions regarding pilin structures, assembly, functions, and vaccine potential. Here we report pilin structures of full-length DnFimA from the sheep pathogen Dichelobacter nodosus and FtPilE from the human pathogen Francisella tularensis at 2.3 and 1 ? resolution, respectively. The DnFimA structure reveals an extended kinked N-terminal α-helix, an unusual centrally located disulfide, conserved subdomains, and assembled epitopes informing serogroup vaccines. An interaction between the conserved Glu-5 carboxyl oxygen and the N-terminal amine of an adjacent subunit in the crystallographic dimer is consistent with the hypothesis of a salt bridge between these groups driving T4P assembly. The FtPilE structure identifies an authentic Type IV pilin and provides a framework for understanding the role of T4P in F. tularensis virulence. Combined results define a unified pilin architecture, specialized subdomain roles in pilus assembly and function, and potential therapeutic targets.     

An Aeromonas salmonicida type IV pilin is required for virulence in rainbow trout Oncorhynchus mykiss

Aeromonas salmonicida expresses a large number of proven and suspected virulence factors including bacterial surface proteins, extracellular degradative enzymes, and toxins. We report the isolation and characterization of a 4-gene cluster, tapABCD, from virulent A. salmonicida A450 that encodes proteins homologous to components required for type IV pilus biogenesis. One gene, tapA, encodes a protein with high homology to type IV pilus subunit proteins from many gram-negative bacterial pathogens, including Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio vulnificus. A survey of A. salmonicida isolates from a variety of sources shows that the tapA gene is as ubiquitous in this species as it is in other members of the Aeromonads. Immunoblotting experiments demonstrate that it is expressed in vitro and is antigenically conserved among the A. salmonicida strains tested. A mutant A. salmonicida strain defective in expression of TapA was constructed by allelic exchange and found to be slightly less pathogenic for juvenile Oncorhynchus mykiss (rainbow trout) than wild type when delivered by intraperitoneal injection. In addition, fish initially challenged with a high dose of wild type were slightly more resistant to rechallenge with wild type than those initially challenged with the tapA mutant strain, suggesting that presence of TapA contributes to immunity. Two of the other three genes identified, tapB and tapC, encode proteins with homology to factors known to be required for type IV pilus assembly in P. aeruginosa, but in an as yet unidentified manner. TapB is a member of the ABC-transporter family of proteins that contain characteristic nucleotide-binding regions, and which may provide energy for type IV pilus assembly through the hydrolysis of ATP. TapC homologs are integral cytoplasmic membrane proteins that may play a role in pilus anchoring or initiation of assembly. The fourth gene, tapD, encodes a product that shares homology with a family of proteins with a known biochemical function, namely, the type IV prepilin leader peptidases. These bifunctional enzymes proteolytically cleave the leader peptide from the pilin precursor (prepilin) and then N-methylate the newly exposed N-terminal amino acid prior to assembly of the subunits into the pilus structure. We demonstrate that A. salmonicida TapD is able to restore type IV pilus assembly and type II secretion in a P. aeruginosa strain carrying a mutation in its type IV peptidase gene, suggesting that it plays the same role in A. salmonicida.     

Sortases and pilin elements involved in pilus assembly of Corynebacterium diphtheriae

Corynebacterium diphtheriae SpaA pili are composed of three pilin subunits, SpaA, SpaB and SpaC. SpaA, the major pilin protein, is distributed uniformly along the pilus shaft, whereas SpaB is observed at regular intervals, and SpaC seems to be positioned at the pilus tip. Pilus assembly in C. diphtheriae requires the pilin motif and the C-terminal sorting signal of SpaA, and is proposed to occur by a mechanism of ordered cross-linking, whereby pilin-specific sortase enzymes cleave precursor proteins at sorting signals and involve the side-chain amino groups of pilin motif sequences to generate covalent linkages between pilin subunits. We show here that two elements of SpaA pilin precursor, the pilin motif and the sorting signal, are together sufficient to promote the polymerization of an otherwise secreted protein by a process requiring the function of the sortase A gene (srtA). Five other sortase genes are dispensable for SpaA pilus assembly. Further, the incorporation of SpaB into SpaA pili requires a glutamic acid residue within the E box motif of SpaA, a feature that is found to be conserved in other Gram-positive pathogens that encode sortase and pilin subunit genes with sorting signals and pilin motifs. When the main fimbrial subunit of Actinomyces naeslundii type I fimbriae, FimA, is expressed in corynebacteria, C. diphtheriae strain NCTC13129 polymerized FimA to form short fibres. Although C. diphtheriae does not depend on other actinomycetal genes for FimA polymerization, this process involves the pilin motif and the sorting signal of FimA as well as corynebacterial sortase D (SrtD). Thus, pilus assembly in Gram-positive bacteria seems to occur by a universal mechanism of ordered cross-linking of precursor proteins, the multiple conserved features of which are recognized by designated sortase enzymes.     

Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili

Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.     

NMR structure of a type IVb pilin from Salmonella typhi and its assembly into pilus

The structure of the N-terminal-truncated Type IVb structural pilin (t-PilS) from Salmonella typhi was determined by NMR. Although topologically similar to the recently determined x-ray structure of pilin from Vibrio cholerae toxin-coregulated pilus, the only Type IVb pilin with known structure, t-PilS contains many distinct structural features. The protein contains an extra pair of beta-strands in the N-terminal alphabeta loop that align with the major beta-strands to form a continuous 7-stranded antiparallel beta-sheet. The C-terminal disulfide-bonded region of t-PilS is only half the length of that of toxin-coregulated pilus pilin. A model of S. typhi pilus has been proposed and mutagenesis studies suggested that residues on both the alphabeta loop and the C-terminal disulfide-bonded region of PilS might be involved in binding specificity of the pilus. This model structure reveals an exposed surface between adjacent subunits of PilS that could be a potential binding site for the cystic fibrosis transmembrane conductance regulator.     

Gene conversion involving the pilin structural gene correlates with pilus+ in equilibrium with pilus- changes in Neisseria gonorrhoeae

Gonococci (Gc) exhibit pilus+----pilus- "phase transitions" at high frequency, but only some of the pilus- Gc can revert to pilus+ phenotype. We examined reversible phase transitions between pilus+ Gc and a particular pilus- variant (P-rp+ phenotype) whose pilin mRNA carries a unique block of nucleotides encoding an "assembly missense" pilin polypeptide. The results show that Gc pilus+ in equilibrium with P-rp+ transitions can result from intragenic recombination in which there is nonreciprocal exchange of partially homologous DNA sequences from a partial pilin gene (in silent, storage form) into the expression locus' complete pilin gene. Hence Gc pilus phase variation, like pilus antigenic variation, can occur by gene conversion of the pilin structural gene expression locus.     

TcpA pilin sequences and colonization requirements for O1 and O139 Vibrio cholerae

The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non-O1 serotypes.