Corynebacterium diphtheriae employs specific minor pilins to target human pharyngeal epithelial cells

Adherence to host tissues mediated by pili is pivotal in the establishment of infection by many bacterial pathogens. Corynebacterium diphtheriae assembles on its surface three distinct pilus structures. The function and the mechanism of how various pili mediate adherence, however, have remained poorly understood. Here we show that the SpaA-type pilus is sufficient for the specific adherence of corynebacteria to human pharyngeal epithelial cells. The deletion of the spaA gene, which encodes the major pilin forming the pilus shaft, abolishes pilus assembly but not adherence to pharyngeal cells. In contrast, adherence is greatly diminished when either minor pilin SpaB or SpaC is absent. Antibodies directed against either SpaB or SpaC block bacterial adherence. Consistent with a direct role of the minor pilins, latex beads coated with SpaB or SpaC protein bind specifically to pharyngeal cells. Therefore, tissue tropism of corynebacteria for pharyngeal cells is governed by specific minor pilins. Importantly, immunoelectron microscopy and immunofluorescence studies reveal clusters of minor pilins that are anchored to cell surface in the absence of a pilus shaft. Thus, the minor pilins may also be cell wall anchored in addition to their incorporation into pilus structures that could facilitate tight binding to host cells during bacterial infection.     

Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae

Many surface proteins in Gram-positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres--SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. We demonstrate here the function of SrtF, the so-called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall-linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface-linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild-type levels of the SpaA-type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two-stage model of pilus assembly: pilins are first polymerized by a pilus-specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase.     

A thiol‐disulfide oxidoreductase of the Gram‐positive pathogen Corynebacterium diphtheriae is essential for viability,pilus assembly,toxin production and virulence

The Gram‐positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane‐bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein‐folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol‐disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin‐like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature‐sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.     

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.     

The basal and major pilins in the Corynebacterium diphtheriae SpaA pilus adopt similar structures that competitively react with the pilin polymerase

Many species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus from Corynebacterium diphtheriae is built by the ^CdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show that ^CdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (^NSpaA) that is also crosslinked by ^CdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “latch” mechanism of isopeptide bond formation. Competition experiments using an inactive SpaB variant and additional NMR studies suggest that SpaB terminates SpaA polymerization by outcompeting ^NSpaA for access to a shared thioester enzyme–substrate reaction intermediate.     

Cell surface display of minor pilin adhesins in the form of a simple heterodimeric assembly in Corynebacterium diphtheriae

Pilus assembly in Gram-positive bacteria occurs by a two-step mechanism, whereby pilins are polymerized and then covalently anchored to the cell wall. In Corynebacterium diphtheriae, the pilin-specific sortase SrtA catalyses polymerization of the SpaA-type pilus, consisting of the shaft pilin SpaA, tip pilin SpaC and minor pilin SpaB. Cell wall anchoring of the SpaA polymers is triggered when SrtA incorporates SpaB into the pilus base via lysine-mediated transpeptidation; anchoring to the cell wall peptidoglycan is subsequently catalysed by the housekeeping sortase SrtF. Here we show that SpaB and SpaC formed a heterodimer independent of SpaA polymerization. SrtA was absolutely required for the formation of the SpaBC heterodimer, while SrtF facilitated the optimal cell wall anchoring of this heterodimer. Alanine substitution of the SpaB lysine residue K139 or truncation of the SpaB cell wall-sorting signal (CWSS) abolished assembly of the SpaBC heterodimer, hence underscoring SpaB function in transpeptidation and cell wall linkage. Importantly, sortase specificity for the cell wall-anchoring step was found to be dependent on the LAFTG motif within the SpaB CWSS. Thus, C. diphtheriae employs a common sortase-catalysed mechanism involving lysine-mediated transpeptidation to generate both adhesive pilus and simple heterodimeric structures on the bacterial the cell wall.     

Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.     

Characterization of the SpaCBA pilus fibers in the probiotic Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG is a human intestinal isolate that has been studied intensively because of its probiotic properties. We have previously shown that L. rhamnosus GG produces proteinaceous pili that earlier had been observed only in Gram-positive pathogens (M. Kankainen et al., Proc. Natl. Acad. Sci. U. S. A. 106:17193-17198, 2009). These pili were found to be encoded by the spaCBA gene cluster, and the pilus-associated SpaC pilin was shown to confer on the cells a mucus-binding ability. In addition to the spaCBA cluster, another putative pilus cluster, spaFED, was predicted from the L. rhamnosus GG genome sequence. Herein, we show that only SpaCBA pili are produced by L. rhamnosus, and we describe a detailed analysis of cell wall-associated and affinity-purified SpaCBA pili by Western blotting and immunogold electron microscopy. Our results indicate that SpaCBA pili are heterotrimeric protrusions with a SpaA subunit as the shaft-forming major pilin. Only a few SpaB subunits could be observed in pilus fibers. Instead, SpaB pilins were found at pilus bases, as assessed by immunogold double labeling of thin sections of cells, suggesting that SpaB is involved in the termination of pilus assembly. The SpaC adhesin was present along the whole pilus length at numbers nearly equaling those of SpaA. The relative amount and uniform distribution of SpaC within pili not only makes it possible to exert both long-distance and intimate contact with host tissue but also provides mucus-binding strength, which explains the prolonged intestinal residency times observed for L. rhamnosus GG compared to that of nonpiliated lactobacilli.     

Acyl Enzyme Intermediates in Sortase-Catalyzed Pilus Morphogenesis in Gram-Positive Bacteria

In gram-positive bacteria, covalently linked pilus polymers are assembled by a specific transpeptidase enzyme called pilus-specific sortase. This sortase is postulated to cleave the LPXTG motif of a pilin precursor between threonine and glycine and to form an acyl enzyme intermediate with the substrate. Pilus polymerization is believed to occur through the resolution of this intermediate upon specific nucleophilic attack by the conserved lysine located within the pilin motif of another pilin monomer, which joins two pilins with an isopeptide bond formed between threonine and lysine. Here, we present evidence for sortase reaction intermediates in Corynebacterium diphtheriae. We show that truncated SrtA mutants that are loosely bound to the cytoplasmic membrane form high-molecular-weight complexes with SpaA polymers secreted into the extracellular milieu. These complexes are not formed with SpaA pilin mutants that have alanine substitutions in place of threonine in the LPXTG motif or lysine in the pilin motif. The same phenotype is observed with alanine substitutions of either the conserved cysteine or histidine residue of SrtA known to be required for catalysis. Remarkably, the assembly of SpaA pili, or the formation of intermediates, is abolished with a SrtA mutant missing the membrane-anchoring domain. We infer that pilus polymerization involves the formation of covalent pilin-sortase intermediates, which occurs within a molecular platform on the exoplasmic face of the cytoplasmic membrane that brings together both sortase and its cognate substrates in close proximity to each other, likely surrounding a secretion apparatus. We present electron microscopic data in support of this picture.Adherence to specific host tissue is a key step in bacterial colonization and the establishment of a successful infection by bacterial pathogens. Bacteria express a variety of adhesive cell surface molecules to bind host cells or other substrates in their natural habitat. The proteinaceous filaments known as pili or fimbriae are a clinically important class of these molecules. Both gram-negative and gram-positive bacteria express pili (6, 8). The gram-positive bacterial pili are unique in three respects (12, 25, 31). First, they represent heterodimeric or heterotrimeric protein polymers in which individual pilin subunits are covalently joined to each other (2, 9, 32). Second, the polymer itself is covalently attached to the cell wall (3, 31). Third, unlike pilus assembly in gram-negative bacteria, many of which require chaperones (26), the polymerization of the gram-positive pili and their cell wall attachment require specific transpeptidase enzymes called sortases (31).Mazmanian and colleagues discovered the sortase SrtA as an enzyme that linked the surface protein A of Staphylococcus aureus to its cell wall (15). Genome sequences revealed that sortases are ubiquitously expressed in gram-positive bacteria, including significant pathogens, such as Actinomyces naeslundii, Bacillus cereus, Corynebacterium diphtheriae, Enterococcus faecalis, Streptococcus agalactiae, and Streptococcus pneumoniae (4, 5, 28). Sortases are classified according to their functions and phylogenic relationships (4, 5). The class that closely matches SrtA of S. aureus in structure and function is now called a housekeeping sortase. Its function is to attach numerous surface proteins to the cell wall (16). Common to each of these cell surface proteins is a cell wall sorting signal with an LPXTG motif that is absolutely necessary for cell wall anchoring (18). Elegant genetic, biochemical, and structural work by the Schneewind laboratory illuminated the universal reaction mechanism of protein sorting in the gram-positive cell wall (14). Cell wall anchoring of surface proteins is catalyzed in two steps. In the first step, SrtA cleaves the TG peptide bond of the LPXTG motif of protein A and forms an acyl enzyme intermediate involving the threonine of protein A and the catalytic cysteine of sortase (22, 27, 29). In the second step, the cleaved protein A is transferred to the cell wall when a nucleophile amine from the lipid II precursor attacks and resolves the acyl enzyme intermediate (20, 21, 30). This seminal work formed the basis of our current model of pilus assembly catalyzed by pilus-specific sortases (12).We have used C. diphtheriae as a model for studies of the mechanism of pilus biogenesis. The corynebacterial genome encodes six different sortases (32). We now know that while five of these sortases (SrtA to -E) are devoted to pilus assembly, even the housekeeping sortase, SrtF, is required for efficient attachment of pili to the cell wall (23). Corynebacteria produce three distinct types of heterotrimeric pili, which are encoded by three pilus islands, each encoding three pilins (namely, SpaABC, SpaDEF, and SpaGHI) plus one or two cognate sortases essential for the assembly of the respective pilus (7, 24, 32). In each case, the prototype pilus represents a shaft structure made of a specific major pilin (namely, SpaA, SpaD, and SpaH) (12). Each type of pilus also contains a minor pilin at the tip (SpaC, SpaF, and SpaG) and another minor pilin dispersed along the shaft, as well as at the base of the pilus (SpaB, SpaE, and SpaI) (12). How are these polymers assembled, and how are they attached to the cell wall? All pilin proteins are predicted to contain in their amino termini a hydrophobic signal sequence necessary for export to the exoplasm by the Sec machinery. In addition, like the cell wall-anchored protein A of S. aureus, a cell wall sorting signal including the LPXTG motif is also present at the carboxy terminus of each of the Spa proteins of corynebacteria and other pilus proteins found in different gram-positive organisms (17). It is thus logical to imagine that the pilus-specific sortase utilizes the LPXTG motif for pilus polymerization, its cell wall anchoring, or both. Substantial genetic, biochemical, and ultrastructural analyses have proved this prediction. Consequently, Ton-That and Schneewind proposed a model of pilus assembly which posited that the basic mechanism of catalysis is conserved between cell wall sorting of surface proteins and the assembly of the pilus (31).According to our current working model (Fig. ​(Fig.1A),1A), the prototype SpaA pilus is assembled as follows. SrtA, which is essential and also specific for SpaA pilus formation, captures and cleaves cognate pilins at the LPXTG motif and forms an acyl enzyme intermediate. To form a dimer of SpaA and SpaC, the proposed tip entity, a conserved lysine in the SpaA pilin motif attacks the Cys-Thr bond of the SpaC-SrtA acyl enzyme intermediate. Shaft formation ensues by the cyclic addition of SpaA to the SpaC-SpaA dimer and the SpaC-SpaA_n oligomer formed in the preceding reaction. When a SpaB is attached to the growing pilus terminus by a similar mechanism involving a critical lysine of SpaB, it acts as a switch, terminating pilus polymerization in favor of cell wall anchoring (11). This happens by the classic resolution reaction mentioned above, which involves the lipid II precursor (28), followed by its linkage to the cell wall (11). Alternatively, the SpaB-containing pilus can elongate further by adding a SpaA subunit to SpaB (11). This model explains all the available genetic and biochemical data we have obtained so far in the corynebacterial system, as well as other systems reported by various investigators.Open in a separate windowFIG. 1.(A) Working model of pilus assembly in C. diphtheriae. Spa pilins are synthesized in the cytoplasm and transported across the cytoplasmic membrane by the Sec machinery. The membrane-bound pilus-specific sortase SrtA cleaves the Spa pilins at the LPXTG motif and forms an acyl enzyme intermediate with the substrates. Pilus polymerization occurs when this intermediate is resolved by a nucleophilic attack by the lysine residue within the pilin motif of an adjacent intermediate. Cell wall anchoring terminates pilus polymerization when SpaB is incorporated into the pilus base by the housekeeping sortase, SrtF (see the text for details). (B) Membrane localization of the pilus-specific sortase SrtA. Corynebacteria grown to mid-log phase were separated from the culture medium (M) by centrifugation. The cell wall (W) was removed from its protoplast by muramidase treatment of the cells. The protoplasts were lysed, and membrane (P*) and cytoplasmic (C) compartments were obtained by ultracentrifugation. Protein samples were separated on 4 to 12% Tris-glycine gradient gels and detected by immunoblotting them with the specific antisera α-SrtA, α-SecA, and α-SpaA. The positions of molecular mass markers (kDa) are indicated. WT, wild type.Significantly, there has been no report demonstrating the proposed intermediates of pilus assembly, to our knowledge. The present study was initiated to explore this key element of our model of pilus assembly, as well as the localization of the sortase in the membrane and its organization in the exoplasmic membrane in relation to the cognate pilins and the general secretion machinery.     

Crystal Structure of the Minor Pilin CofB,the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.     

Assembly of distinct pilus structures on the surface of Corynebacterium diphtheriae

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci. Corynebacterium diphtheriae has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of spaA nor sortase srtA abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of C. diphtheriae.     

Pseudomonas aeruginosa Minor Pilins Are Incorporated into Type IV Pili

Type IV pili are long filamentous appendages required for both adhesion and a unique form of motility known as twitching. Twitching motility involves the extension and retraction of the pilus and requires a number of gene products, including five conserved pilin-like proteins of unknown function (FimU, PilV, PilW, PilX, and PilE in Pseudomonas aeruginosa), termed ‘minor’ pilins. Maintenance of a specific stoichiometric ratio among the minor pilins was important for function, as loss or overexpression of any component impaired motility. Disruption of individual minor pilin genes, or of the AlgR positive regulator of minor pilin operon expression in a strain where pilus retraction was blocked by inactivation of the PilT retraction ATPase, revealed that pili were produced, although levels of piliation were reduced relative to pilT positive control. Differences in the levels of piliation of complemented strains pointed to specific roles for each protein in the assembly process, with FimU and PilX being implicated as key promoters of pilus assembly on the cell surface. Using specific antibodies for each protein, we showed that the minor pilins FimU, PilV, PilW, PilX, and PilE were processed by the pre-pilin peptidase PilD and incorporated throughout the growing pilus filament. This is the first study to demonstrate that the minor pilins, conserved among bacteria expressing type IVa pili, are incorporated into the fiber and support a role for them in the initiation, but not termination, of pilus assembly.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Structural and Functional Studies of the Pseudomonas aeruginosa Minor Pilin,PilE

Many bacterial pathogens, including Pseudomonas aeruginosa, use type IVa pili (T4aP) for attachment and twitching motility. T4aP are composed primarily of major pilin subunits, which are repeatedly assembled and disassembled to mediate function. A group of pilin-like proteins, the minor pilins FimU and PilVWXE, prime pilus assembly and are incorporated into the pilus. We showed previously that minor pilin PilE depends on the putative priming subcomplex PilVWX and the non-pilin protein PilY1 for incorporation into pili, and that with FimU, PilE may couple the priming subcomplex to the major pilin PilA, allowing for efficient pilus assembly. Here we provide further support for this model, showing interaction of PilE with other minor pilins and the major pilin. A 1.25 Å crystal structure of PilE_Δ1–28 shows a typical type IV pilin fold, demonstrating how it may be incorporated into the pilus. Despite limited sequence identity, PilE is structurally similar to Neisseria meningitidis minor pilins PilX_Nm and PilV_Nm, recently suggested via characterization of mCherry fusions to modulate pilus assembly from within the periplasm. A P. aeruginosa PilE-mCherry fusion failed to complement twitching motility or piliation of a pilE mutant. However, in a retraction-deficient strain where surface piliation depends solely on PilE, the fusion construct restored some surface piliation. PilE-mCherry was present in sheared surface fractions, suggesting that it was incorporated into pili. Together, these data provide evidence that PilE, the sole P. aeruginosa equivalent of PilX_Nm and PilV_Nm, likely connects a priming subcomplex to the major pilin, promoting efficient assembly of T4aP.     

The type-4 pilus is the major virulence-associated adhesin of Pseudomonas aeruginosa – a review

Pseudomonas aeruginosa (Pa) produces several surface-associated adherence factors or adhesins which promote attachment to epithelial cells and contribute to the virulence of this pathogen. Among them, the type-4 pilus accounts for about 90% of the adherence capability of Pa to human lung pneumocyte A549 cells. Furthermore, it is responsible for more than 90% of the virulence in AB.Y/SnJ mice. Pa type-4 pili display a tip-base differentiation with the adherence function located at the tip of the pilus. All Pa pili prototypes characterized so far contain an intrachain disulfide loop (DSL) of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin. In Pa, this DSL comprises the epithelial cell-binding domain. Despite little sequence homology, DSL-containing peptides of different pilin prototypes seemingly reveal striking structural similarities. Two β-turns within the loop and the disulfide bridge impose significant structural rigidity on the DSL pilin peptide, suggesting a conformationally conserved binding domain. Insertions of C-terminal pilin peptides with disrupted DSL displayed on the surface of bacterial S-layer mediate the same receptor binding characteristics as pili, indicating that a DSL is not essential in maintaining the functionality of the binding domain. Pa pili bind specifically to the carbohydrate moiety of the glycosphingolipids (GSL) asialo-G_M1 and asialo-G_M2 and, to a much weaker extent, to lactosyl ceramide and ceramide trihexoside. The disaccharide sequence GalNAcβ(1-4)Gal, common in both asialo-G_M1 and asialo-G_M2, likely represents the minimal structural receptor motif recognized by the pili. Pa pili also bind to surface-localized proteins of human epithelial cells and other cell types, suggesting that non-sialylated GSL and (glyco)proteins function as receptors of pili. In addition to the major pilus adhesin, exoenzyme S and, as recent studies indicate, flagella, are further protein adhesins of Pa with GSL receptor binding specificities similar to those of pili.     

Role of type IV pili in virulence of Pseudomonas syringae pv. tabaci 6605: correlation of motility, multidrug resistance, and HR-inducing activity on a nonhost plant

To investigate the role of type IV pili in the virulence of phytopathogenic bacteria, four mutant strains for pilus biogenesis-related genes were generated in Pseudomonas syringae pv. tabaci 6605. PilA encodes the pilin protein as a major subunit of type IV pili, and the pilO product is reported to be required for pilus assembly. The fimU and fimT genes are predicted to produce minor pilins. Western blot analysis revealed that pilA, pilO, and fimU mutants but not the fimT mutant failed to construct type IV pili. Although the swimming motility of all mutant strains was not impaired in liquid medium, they showed remarkably reduced motilities on semisolid agar medium, suggesting that type IV pili are required for surface motilities. Virulence toward host tobacco plants and hypersensitive response-inducing ability in nonhost Arabidopsis leaves of pilA, pilO, and fimU mutant strains were reduced. These results might be a consequence of reduced expression of type III secretion system-related genes in the mutant strains. Further, all mutant strains showed enhanced expression of resistance-nodulation-division family members mexA, mexB, and oprM, and higher tolerance to antimicrobial compounds. These results indicate that type IV pili are an important virulence factor of this pathogen.     

Biogenesis and adhesion of type 1 and P pili

BackgroundUropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis.Scope of reviewThe review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly.Major conclusionsThe usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus.Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner.The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described.General significanceThe combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotic resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.     

The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium’s nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.     

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection.