Type IV pilin structure and assembly: X-ray and EM analyses of Vibrio cholerae toxin-coregulated pilus and Pseudomonas aeruginosa PAK pilin

Pilin assembly into type IV pili is required for virulence by bacterial pathogens that cause diseases such as cholera, pneumonia, gonorrhea, and meningitis. Crystal structures of soluble, N-terminally truncated pilin from Vibrio cholera toxin-coregulated pilus (TCP) and full-length PAK pilin from Pseudomonas aeruginosa reveal a novel TCP fold, yet a shared architecture for the type IV pilins. In each pilin subunit a conserved, extended, N-terminal alpha helix wrapped by beta strands anchors the structurally variable globular head. Inside the assembled pilus, characterized by cryo-electron microscopy and crystallography, the extended hydrophobic alpha helices make multisubunit contacts to provide mechanical strength and flexibility. Outside, distinct interactions of adaptable heads contribute surface variation for specificity of pilus function in antigenicity, motility, adhesion, and colony formation.     

NMR structure of the Escherichia coli type 1 pilus subunit FimF and its interactions with other pilus subunits

Type 1 pili from uropathogenic Escherichia coli strains mediate bacterial attachment to target receptors on the host tissue. They are composed of up to 3000 copies of the subunit FimA, which form the stiff, helical pilus rod, and the subunits FimF, FimG, and FimH, which form the linear tip fibrillum. All subunits in the pilus interact via donor strand complementation, in which the incomplete immunoglobulin-like fold of each subunit is complemented by insertion of an N-terminal extension from the following subunit. We determined the NMR structure of a monomeric, self-complemented variant of FimF, FimF_F, which has a second FimF donor strand segment fused to its C-terminus that enables intramolecular complementation of the FimF fold. NMR studies on bimolecular complexes between FimF_F and donor strand-depleted variants of FimF and FimG revealed that the relative orientations of neighboring domains in the tip fibrillum cover a wide range. The data provide strong support for the intrinsic flexibility of the tip fibrillum. They lend further support to the hypothesis that this flexibility would significantly increase the probability that the adhesin at the distal end of the fibrillum successfully targets host cell receptors.     

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.     

Crystallization of a type I 3-dehydroquinase from Salmonella typhi.

Crystals have been grown of a type I 3-dehydroquinase from both Escherichia coli and Salmonella typhi. However, only those from S. typhi diffract to a resolution of 2.3 A on a conventional X-ray source and are suitable for structure determination. The space group has been determined as P2(1)2(1)2 with unit cell dimensions a = 48.01 A, b = 114.29 A, c = 42.87 A. There is one subunit in the asymmetric unit.     

High resolution x-ray structure of tyvelose epimerase from Salmonella typhi

Tyvelose epimerase catalyzes the last step in the biosynthesis of tyvelose by converting CDP-d-paratose to CDP-d-tyvelose. This unusual 3,6-dideoxyhexose occurs in the O-antigens of some types of Gram-negative bacteria. Here we describe the cloning, protein purification, and high-resolution x-ray crystallographic analysis of tyvelose epimerase from Salmonella typhi complexed with CDP. The enzyme from S. typhi is a homotetramer with each subunit containing 339 amino acid residues and a tightly bound NAD+ cofactor. The quaternary structure of the enzyme displays 222 symmetry and can be aptly described as a dimer of dimers. Each subunit folds into two distinct lobes: the N-terminal motif responsible for NAD+ binding and the C-terminal region that harbors the binding site for CDP. The analysis described here demonstrates that tyvelose epimerase belongs to the short-chain dehydrogenase/reductase superfamily of enzymes. Indeed, its active site is reminiscent to that observed for UDP-galactose 4-epimerase, an enzyme that plays a key role in galactose metabolism. Unlike UDP-galactose 4-epimerase where the conversion of configuration occurs about C-4 of the UDP-glucose or UDP-galactose substrates, in the reaction catalyzed by tyvelose epimerase, the inversion of stereochemistry occurs at C-2. On the basis of the observed binding mode for CDP, it is possible to predict the manner in which the substrate, CDP-paratose, and the product, CDP-tyvelose, might be accommodated within the active site of tyvelose epimerase.     

Molecular structure of alpha-D-glucose-1-phosphate cytidylyltransferase from Salmonella typhi

Dideoxysugars, which display biological activities ranging from mediating cell-cell interactions to serving as components in some antibiotics, are synthesized in various organisms via complex biochemical pathways that begin with the attachment of alpha-D-glucose 1-phosphate to either CTP or dTTP. Here we describe the three-dimensional structure of the alpha-D-glucose-1-phosphate cytidylyltransferase from Salmonella typhi, which catalyzes the first step in the production of CDP-tyvelose. For this investigation, the enzyme was crystallized in the presence of its product, CDP-glucose. In contrast to previous reports, the enzyme exists as a fully integrated hexamer with 32-point group symmetry. Each subunit displays a "bird-like" appearance with the "body" composed predominantly of a seven-stranded mixed beta-sheet and the two "wings" formed by beta-hairpin motifs. These two wings mediate subunit-subunit interactions along the 3-fold and 2-fold rotational axes, respectively. The six active sites of the hexamer are situated between the subunits related by the 2-fold rotational axes. CDP-glucose is anchored to the protein primarily by hydrogen bonds with backbone carbonyl oxygens and peptidic NH groups. The side chains of Arg111 and Asn188 from one subunit and Glu178 and Lys179 from the second subunit are also involved in hydrogen bonding with the ligand. The topology of the main core domain bears striking similarity to that observed for glucose-1-phosphate thymidylyltransferase and 4-diphosphocytidyl-2-C-methylerythritol synthetase.     

Pseudomonas aeruginosa D-arabinofuranose biosynthetic pathway and its role in type IV pilus assembly

Pseudomonas aeruginosa strains PA7 and Pa5196 glycosylate their type IVa pilins with α1,5-linked D-arabinofuranose (d-Araf), a rare sugar configuration identical to that found in cell wall polymers of the Corynebacterineae. Despite this chemical identity, the pathway for biosynthesis of α1,5-D-Araf in Gram-negative bacteria is unknown. Bioinformatics analyses pointed to a cluster of seven P. aeruginosa genes, including homologues of the Mycobacterium tuberculosis genes Rv3806c, Rv3790, and Rv3791, required for synthesis of a polyprenyl-linked d-ribose precursor and its epimerization to D-Araf. Pa5196 mutants lacking the orthologues of those genes had non-arabinosylated pilins, poor twitching motility, and significantly fewer surface pili than the wild type even in a retraction-deficient (pilT) background. The Pa5196 pilus system assembled heterologous non-glycosylated pilins efficiently, demonstrating that it does not require post-translationally modified subunits. Together the data suggest that pilins of group IV strains need to be glycosylated for productive subunit-subunit interactions. A recombinant P. aeruginosa PAO1 strain co-expressing the genes for d-Araf biosynthesis, the pilin modification enzyme TfpW, and the acceptor PilA(IV) produced arabinosylated pili, confirming that the Pa5196 genes identified are both necessary and sufficient. A P. aeruginosa epimerase knock-out could be complemented with the corresponding Mycobacterium smegmatis gene, demonstrating conservation between the systems of the Corynebacterineae and Pseudomonas. This work describes a novel Gram-negative pathway for biosynthesis of d-Araf, a key therapeutic target in Corynebacterineae.     

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 ? resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.     

Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions

Type IV pili (T4P) are long, thin, flexible filaments on bacteria that undergo assembly-disassembly from inner membrane pilin subunits and exhibit astonishing multifunctionality. Neisseria gonorrhoeae (gonococcal or GC) T4P are prototypic virulence factors and immune targets for increasingly antibiotic-resistant human pathogens, yet detailed structures are unavailable for any T4P. Here, we determined a detailed experimental GC-T4P structure by quantitative fitting of a 2.3 A full-length pilin crystal structure into a 12.5 A resolution native GC-T4P reconstruction solved by cryo-electron microscopy (cryo-EM) and iterative helical real space reconstruction. Spiraling three-helix bundles form the filament core, anchor the globular heads, and provide strength and flexibility. Protruding hypervariable loops and posttranslational modifications in the globular head shield conserved functional residues in pronounced grooves, creating a surprisingly corrugated pilus surface. These results clarify T4P multifunctionality and assembly-disassembly while suggesting unified assembly mechanisms for T4P, archaeal flagella, and type II secretion system filaments.     

FppA, a novel Pseudomonas aeruginosa prepilin peptidase involved in assembly of type IVb pili

Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.     

Homology model of surface antigen OmpC from Salmonella typhi and its functional implications

Homology based 3D structural model of the immunodominant major surface antigen OmpC from Salmonella typhi, an obligatory human pathogen, was built to understand the possible unique conformational features of its antigenic loops with respect to other immunologically cross reacting porins. The homology model was built based on the known crystal structures of the E. coli porins OmpF and PhoE. Structure based sequence alignment helped to define the structurally conserved regions (SCRs). The SCR regions of OmpC were modelled using the coordinates of corresponding regions from reference proteins. Surface exposed variable regions were modelled based on the sequence similarity and loop search in PDB. Structural refinement based on symmetry restrained energy minimization resulted in an agreeable model for the trimer of OmpC. The resulting model was compared with other porin structures, having b-barrel fold with 16 transmembrane beta-strands, and found that the variable regions are unique in terms of sequence and structure. A ranking of the loops taking into account the antigenic index, the sequence variability, the surface accessibility in the context of the trimer, and the structural variability suggests that loop 4 (151-172), loop 5 (194-218) and loop 6 (237-264) are the best ranked B-cell epitopes. The model provides possible explanations for the functional and unique immunological properties associated with the surface exposed regions and outlines the implications for structure based experimental design.     

A model for group B Streptococcus pilus type 1: the structure of a 35-kDa C-terminal fragment of the major pilin GBS80

The Gram-positive pathogen Streptococcus agalactiae, known as group B Streptococcus (GBS), is the leading cause of bacterial septicemia, pneumonia, and meningitis among neonates. GBS assembles two types of pili—pilus islands (PIs) 1 and 2—on its surface to adhere to host cells and to initiate colonization for pathogenesis. The GBS PI-1 pilus is made of one major pilin, GBS80, which forms the pilus shaft, and two secondary pilins, GBS104 and GBS52, which are incorporated into the pilus at various places. We report here the crystal structure of the 35-kDa C-terminal fragment from GBS80, which is composed of two IgG-like domains (N2-N3). The structure was solved by single-wavelength anomalous dispersion using sodium-iodide-soaked crystals and diffraction data collected at the home source. The N2 domain exhibits a cnaA/DEv-IgG fold with two calcium-binding sites, while the N3 domain displays a cnaB/IgG-rev fold. We have built a model for full-length GBS80 (N1, N2, and N3) with the help of available homologous major pilin structures, and we propose a model for the GBS PI-1 pilus shaft. The N2 and N3 domains are arranged in tandem along the pilus shaft, whereas the respective N1 domain is tilted by approximately 20° away from the pilus axis. We have also identified a pilin-like motif in the minor pilin GBS52, which might aid its incorporation at the pilus base.     

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.     

Structure of an essential type IV pilus biogenesis protein provides insights into pilus and type II secretion systems

Type IV pili (T4Ps) are long cell surface filaments, essential for microcolony formation, tissue adherence, motility, transformation, and virulence by human pathogens. The enteropathogenic Escherichia coli bundle-forming pilus is a prototypic T4P assembled and powered by BfpD, a conserved GspE secretion superfamily ATPase held by inner-membrane proteins BfpC and BfpE, a GspF-family membrane protein. Although the T4P assembly machinery shares similarity with type II secretion (T2S) systems, the structural biochemistry of the T4P machine has been obscure. Here, we report the crystal structure of the two-domain BfpC cytoplasmic region (N-BfpC), responsible for binding to ATPase BfpD and membrane protein BfpE. The N-BfpC structure reveals a prominent central cleft between two α/β-domains. Despite negligible sequence similarity, N-BfpC resembles PilM, a cytoplasmic T4P biogenesis protein. Yet surprisingly, N-BfpC has far greater structural similarity to T2S component EpsL, with which it also shares virtually no sequence identity. The C-terminus of the cytoplasmic domain, which leads to the transmembrane segment not present in the crystal structure, exits N-BfpC at a positively charged surface that most likely interacts with the inner membrane, positioning its central cleft for interactions with other Bfp components. Point mutations in surface-exposed N-BfpC residues predicted to be critical for interactions among BfpC, BfpE, and BfpD disrupt pilus biogenesis without precluding interactions with BfpE and BfpD and without affecting BfpD ATPase activity. These results illuminate the relationships between T4P biogenesis and T2S systems, imply that subtle changes in component residue interactions can have profound effects on function and pathogenesis, and suggest that T4P systems may be disrupted by inhibitors that do not preclude component assembly.     

Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD

The outer membrane protein FimD represents the assembly platform of adhesive type 1 pili from Escherichia coli. FimD forms ring-shaped oligomers of 91.4 kDa subunits that recognize complexes between the pilus chaperone FimC and individual pilus subunits in the periplasm and mediate subunit translocation through the outer membrane. Here, we have identified a periplasmic domain of FimD (FimD(N)) comprising the N-terminal 139 residues of FimD. Purified FimD(N) is a monomeric, soluble protein that specifically recognizes complexes between FimC and individual type 1 pilus subunits, but does not bind the isolated chaperone, or isolated subunits. In addition, FimD(N) retains the ability of FimD to recognize different chaperone-subunit complexes with different affinities, and has the highest affinity towards the FimC-FimH complex. Overexpression of FimD(N) in the periplasm of wild-type E.coli cells diminished incorporation of FimH at the tip of type 1 pili, while pilus assembly itself was not affected. The identification of FimD(N) and its ternary complexes with FimC and individual pilus subunits opens the avenue to structural characterization of critical type 1 pilus assembly intermediates.     

Comparative Virulotyping of Salmonella typhi and Salmonella enteritidis

Members of Salmonella enterica are important foodborne pathogens of significant public health concern worldwide. This study aimed to determine a range of virulence genes among typhoidal (S. typhi) and non-typhoidal (S. enteritidis) strains isolated from different geographical regions and different years. A total of 87 S. typhi and 94 S. enteritidis strains were tested for presence of 22 virulence genes by employing multiplex PCR and the genetic relatedness of these strains was further characterized by REP-PCR. In S. typhi, invA, prgH, sifA, spiC, sopB, iroN, sitC, misL, pipD, cdtB, and orfL were present in all the strains, while sopE, agfC, agfA, sefC, mgtC, and sefD were present in 98.8, 97.7, 90.8, 87.4, 87.4 and 17.2 %, of the strains, respectively. No lpfA, lpfC, pefA, spvB, or spvC was detected. Meanwhile, in S. enteritidis, 15 genes, agfA, agfC, invA, lpfA, lpfC, sefD, prgH, spiC, sopB, sopE, iroN, sitC, misL, pipD, and orfL were found in all S. enteritidis strains 100 %, followed by sifA and spvC 98.9 %, pefA, spvB and mgtC 97.8 %, and sefC 90.4 %. cdtB was absent from all S. enteritidis strains tested. REP-PCR subtyped S. typhi strains into 18 REP-types and concurred with the virulotyping results in grouping the strains, while in S. enteritidis, REP-PCR subtyped the strains into eight profiles and they were poorly distinguishable between human and animal origins. The study showed that S. typhi and S. enteritidis contain a range of virulence factors associated with pathogenesis. Virulotyping is a rapid screening method to identify and profile virulence genes in Salmonella strains, and improve an understanding of potential risk for human and animal infections.     

The type IV pilus assembly complex: biogenic interactions among the bundle-forming pilus proteins of enteropathogenic Escherichia coli

Production of type IV bundle-forming pili (BFP) by enteropathogenic Escherichia coli (EPEC) requires the protein products of 12 genes of the 14-gene bfp operon. Antisera against each of these proteins were used to demonstrate that in-frame deletion of individual genes within the operon reduces the abundance of other bfp operon-encoded proteins. This result was demonstrated not to be due to downstream polar effects of the mutations but rather was taken as evidence for protein-protein interactions and their role in the stabilization of the BFP assembly complex. These data, combined with the results of cell compartment localization studies, suggest that pilus formation requires the presence of a topographically discrete assembly complex that is composed of BFP proteins in stoichiometric amounts. The assembly complex appears to consist of an inner membrane component containing three processed, pilin-like proteins, BfpI, -J, and -K, that localize with BfpE, -L, and -A (the major pilin subunit); an outer membrane, secretin-like component, BfpB and -G; and a periplasmic component composed of BfpU. Of these, only BfpL consistently localizes with both the inner and outer membranes and thus, together with BfpU, may articulate between the Bfp proteins in the inner membrane and outer membrane compartments.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.