Structure of Clostridium difficile PilJ exhibits unprecedented divergence from known type IV pilins.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface.

Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure.

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated "ileal loop." This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1ß. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.

Mutagenic analysis of the Clostridium difficile flagellar proteins, FliC and FliD, and their contribution to virulence in hamsters.

Although toxins A and B are known to be important contributors to the acute phase of Clostridium difficile infection, the role of colonization and adherence to host tissues in the overall pathogenesis of these organisms remains unclear. Consequently, we used the recently introduced intron-based ClosTron gene interruption system to eliminate the expression of two reported C. difficile colonization factors, the major flagellar structural subunit (FliC) and the flagellar cap protein (FliD), to gain greater insight into how flagella and motility contribute to C. difficile's pathogenic strategy. The results demonstrate that interrupting either the fliC or the fliD gene results in a complete loss of flagella, as well as motility, in C. difficile. However, both the fliC and fliD mutant strains adhered better than the wild-type 630Δerm strain to human intestine-derived Caco-2 cells, suggesting that flagella and motility do not contribute to, or may even interfere with, C. difficile adherence to epithelial cell surfaces in vitro. Moreover, we found that the mutant strains were more virulent in hamsters, indicating either that flagella are unnecessary for virulence or that repression of motility may be a pathogenic strategy employed by C. difficile in hamsters.

Binding of Clostridium difficile toxins to human milk oligosaccharides.

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(ß1-4)Glc, Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc, Fuc(α1-2)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Fuc(α1-4)]GlcNAc(ß1-3)Gal(ß1-4)Glc and Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-3)Gal(ß1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3 )M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.

N-acetyllactosamine-induced retraction of bundle-forming pili regulates virulence-associated gene expression in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant morbidity and mortality due to diarrhoea in developing countries. The pathogenesis of EPEC is dependent on a coordinated multi-step process culminating in the intimate adherence of the organisms to the host's intestinal mucosa. During the initial stages of the EPEC colonization process, the fimbrial adhesin, bundle-forming pili (BFP), plays an integral role. We previously reported that the major BFP structural subunit, bundlin, displays lectin-like properties, which enables BFP to initially tether EPEC to N-acetyllactosamine (LacNAc) glycan receptors on host cell surfaces. We also reported that incubating EPEC with synthetic LacNAc-bearing neoglycoconjugates not only inhibits their adherence to host cells, but also induces BFP retraction and subsequent degradation of the bundlin subunits. Herein, we demonstrate that the periplasmic serine protease, DegP, is required for degrading bundlin during this process. We also show that DegP appears to act as a bundlin chaperone during BFP assembly and that LacNAc-BSA-induced BFP retraction is followed by transcriptional upregulation of the BFP operon and downregulation of the locus of enterocyte effacement operons in EPEC.

In vivo supramolecular templating enhances the activity of multivalent ligands: a potential therapeutic against the Escherichia coli O157 AB5 toxins.

We demonstrate that interactions between multimeric receptors and multivalent ligands are dramatically enhanced by recruiting a complementary templating receptor such as an endogenous multimeric protein but only when individual ligands are attached to a polymer as preorganized, covalent, heterobifunctional pairs. This effect cannot be replicated by a multivalent ligand if the same recognition elements are independently arrayed on the scaffold. Application of this principle offers an approach to create high-avidity inhibitors for multimeric receptors. Judicious selection of the ligand that engages the templating protein allows appropriate effector function to be incorporated in the polymeric construct, thereby providing an opportunity for therapeutic applications. The power of this approach is exemplified by the design of exceptionally potent Escherichia coli Shiga toxin antagonists that protect transgenic mice that constitutively express a human pentraxin, serum amyloid P component.

From alpha to beta: identification of amino acids required for the N-acetyllactosamine-specific lectin-like activity of bundlin.

Bundle-forming pili (BFP) promote the adherence of typical enteropathogenic Escherichia coli (EPEC) to human intestinal epithelial cells. BFP are polymers of bundlin and nine bundlin alleles have been identified in EPEC isolated from diverse sources. These alleles are divided into two main groups, alpha and beta, based on their amino acid sequences. Alpha bundlins are also N-acetyllactosamine- (LacNAc) specific lectins and bind to HEp-2 cells, whereas beta bundlins do not display these characteristics. The four surface-exposed regions of amino acid sequence heterogeneity between alpha and beta bundlin were therefore investigated as potential LacNAc-specific carbohydrate-binding domains in a bundlin. Mutation of one of these domains, 137-GENNI-141, in alpha(1) bundlin to that of beta bundlin (136-SPDST-140) resulted in BFP that no longer bound to LacNAc or HEp-2 cells. Conversely, mutating the beta3 bundlin gene to encode the alpha bundlin sequence at this domain resulted in the gain of HEp-2 cell adherence. The importance of this domain in carbohydrate binding is supported by the finding that introducing the mutation GENNI-->GENNT altered the alpha1 bundlin carbohydrate-binding specificity from LacNAc to the Lewis X glycan sequence.

Assembly and stability of the shiga toxins investigated by electrospray ionization mass spectrometry.

A systematic investigation into the assembly and stability of native and modified subunits of the Shiga toxins (Stx) in vitro is described. Analysis of the assembly of native and modified B subunits of Stx1 and Stx2 in solution, carried out using electrospray ionization mass spectrometry (ES-MS), suggests that the lower thermodynamic stability of the B subunit homopentamer of Stx2, compared to that of Stx1, is due to the presence of a repulsive interaction involving Asp70 of the Stx2 B subunit. In Stx1 B, the corresponding (spatially) residue is Arg. Using temperature-controlled ES-MS, it is shown that the Stx1 and Stx2 holotoxins exhibit differences in their resistance to temperature- and acid-induced dissociation. However, both Stx1 and Stx2 are fully assembled at pH >3.5 and 37 degrees C. This finding has several important biological implications. First, it argues against the likelihood that the difference in Stx1 and Stx2 toxicity arises from differential dissociation of the toxins during the intracellular trafficking steps of the cellular intoxication process. Second, it implies that the activation of the A subunits of Stx1 and Stx2 by enzymatic cleavage must occur while the A subunit is assembled with the B subunit homopentamer. It is, therefore, proposed that the differential toxicities of Stx1 and Stx2 reflect the relative efficiencies of intracellular activation of the A subunits.

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin.

Synthetic N-acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants (K(assoc)), to synthetic LacNAc-Benzene and LacNAc-O(CH(2))(8)CONH(2) glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express alpha bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the alpha bundlin monomer. Collectively, these results suggest that alpha bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of alpha bundlin-expressing EPEC strains to host intestinal epithelial cells.

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile.

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked alpha Gal(1,3)betaGal(1,4)beta Glc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-alpha Gal(1,3)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

Differential binding of Shiga toxin 2 to human and murine neutrophils.

Shiga toxins (Stx1 and Stx2) are responsible for initiating haemolytic uraemic syndrome, a serious extraintestinal complication caused by enterohaemorrhagic Escherichia coli O157 : H7 infection in humans. Shiga toxins are classical AB(5)-type exotoxins, consisting of a globotriaosylceramide (Gb(3))-binding B subunit pentamer and an enzymic A subunit. It is demonstrated in this study that Stx2 binds to human neutrophils by a non-classical mechanism that is independent of Gb(3). In contrast, the investigation revealed that Stx2 binds to murine neutrophils by the classical Gb(3)-dependent mechanism. Moreover, whereas the human serum amyloid P (HuSAP) component inhibited Stx2 binding to murine neutrophils, HuSAP increased Stx2 binding to human neutrophils by 84.2 % (P< or =0.002, Student's t-test). These observations may explain why HuSAP protects mice from the lethal effects of Stx2, whereas there is no indication that HuSAP plays a similar protective role in humans infected by E. coli O157 : H7.

Basis for N-acetyllactosamine-mediated inhibition of enteropathogenic Escherichia coli localized adherence.

In a previous article, the authors reported that exposing wild-type enteropathogenic Escherichia coli (EPEC) to chemically synthesized N-acetyllactosamine glycosides covalently coupled to BSA (LacNAc-BSA) inhibited localized adherence (LA) by these organisms and also caused them to lose their bundle-forming pili (BFP), the filamentous surface appendages responsible for their LA phenotype. This effect has now been further investigated by screening a panel of LacNAc-BSA-related glycosides for their ability to inhibit EPEC LA, which revealed that LacNAc-BSA retained its status as the most effective inhibitor of EPEC LA. It was also shown that LacNAc-BSA did not cause the loss of BFP in an EPEC strain containing a non-polar mutation in the bfpF gene and, as a consequence, unable to retract its BFP. LacNAc-BSA also effectively inhibited LA of the bfpF mutant EPEC. Taken together, these observations suggest that, as well as triggering BfpF-mediated BFP retraction, LacNAc-BSA likely functions as a competitive inhibitor of EPEC binding to LacNAc-related receptors on host cells. Moreover, transmission electron microscopy revealed that LacNAc conjugated to gold nanoparticles bound specifically to BFP. This observation indicated that either the major BFP structural subunit (BfpA) itself or, possibly, an accessory protein co-assembled with BfpA into the BFP filaments, contains a LacNAc-specific EPEC adhesin. The results suggest a mechanism whereby the initial binding of EPEC to LacNAc-like receptors on host cells triggers BfpF-mediated BFP retraction. This could then expedite the intimate adherence phase of the multi-step EPEC colonization process by drawing the organisms closer to the host-cell plasma membrane.

Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7.

Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active as an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to approximately 1.8 angstroms and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.

Stability of the homopentameric B subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry.

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B(5) homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60 degrees C and subunit concentrations ranging from 5 to 85 microM, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 microM, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B(5) ions of Stx2 at the +12 and +13 charge states proceeds, at reaction temperatures of 120 to 180 degrees C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B(5) ions at the +12 and +13 charge states were compared with values reported for the corresponding B(5) ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E(a)) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E(a) values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins.

Structural and evolutionary analyses show unique stabilization strategies in the type IV pili of Clostridium difficile.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion, and horizontal gene transfer. However, many Gram-positive species, including Clostridium difficile, also produce type IV pili. Here, we identify the major subunit of the type IV pili of C. difficile, PilA1, and describe multiple 3D structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the type IVb pilins from Gram-negative bacteria that suggest that the type IV pili of C. difficile are involved in microcolony formation.

Virulence potential and adherence properties of Escherichia coli that produce CTX-M and NDM ß-lactamases.

The success of certain sequence types such as ST131 that produce CTX-M or NDM ß-lactamases, and ST405 that produce CTX-M ß-lactamases, among extraintestinal Escherichia coli (ExPEC) had previously been linked to a combination of antimicrobial resistance and certain virulence factors. The adherence properties of these sequence types to gastro-intestinal epithelial cells had not been investigated. A study was therefore designed to investigate the phylogenetic groups, virulence factors and adherence properties of E. coli sequence types ST101, ST131 and ST405 that produce CTX-M-15 and NDM-1. Our results show that ST131 was positive for phylogenetic group B2, ST101 for B1 and ST404 for D. ST131 had more virulence factors than ST101 or ST405. Interestingly, ST101 adhered more avidly to HEp-2 and Caco-2 cells than did ST131 and ST405. Our study showed that adherence to gastro-intestinal cells did not seem to play an important role in the worldwide epidemiological success of ST131 and ST405. The exact role of ExPEC-associated virulence genes is unknown and it is unlikely that one set of factors determines the virulence properties and epidemiological success of certain sequence types. Future investigations should be undertaken to study the microbiological and ecological factors that make certain sequence types among ExPEC such successful pathogens.

Lipopolysaccharide renders transgenic mice expressing human serum amyloid P component sensitive to Shiga toxin 2.

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2. The enhanced susceptibility to Stx2 correlated with an increased expression of genes encoding the pro-inflammatory cytokine TNFα and chemokines of the CXC and CC families in the kidneys of LPS-treated mice, 48 hours after the Stx2/LPS challenge. Co-administering the glucocorticoid dexamethasone, but not the LPS neutralizing cationic peptide LL-37, protected LPS-sensitized HuSAP-transgenic mice from lethal doses of Stx2. Dexamethasone protection was specifically associated with decreased expression of the same inflammatory mediators (CXC and CC-type chemokines and TNFα) linked to enhanced susceptibility caused by LPS. The studies reveal further details about the complex cascade of host-related events that are initiated by Stx2 as well as establish a new animal model system in which to investigate strategies for diminishing serious Stx2-mediated complications in humans infected with enterohemorrhagic E. coli strains.

Impact of the nature and size of the polymeric backbone on the ability of heterobifunctional ligands to mediate shiga toxin and serum amyloid p component ternary complex formation.

Inhibition of AB(5)-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin's pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB(5) complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor's activity.

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells.

Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection.