N-terminal Glucosyltransferase Research Articles

Increasing Domain Coverage Improves Neutralizing Potency of C. difficile Toxin-specific Antibodies

Abstract Clostridiumdifficile (C.difficile)is a significant human pathogen. C.difficile infection (CDI) causesclinical symptoms ranging from diarrhea to life-threatening fulminant pseudo membranous colitis. The pathogenesis of C. difficile is mediated by two large exotoxins, toxins A and B. These two toxins are highly homologous, single chain proteins consisting of four functional domains: N-terminal glucosyl transferase domain (GTD), cysteine protease domain (CPD), translocation domain (TLD) and a C-terminal receptor-binding domain (RBD). The important role played by anti-toxin sera and antibodies in the prevention of primary and recurrent CDI has been described and demonstrated in both clinical and pre-clinical studies; however, work focused on the impact of the toxin domain specificity of these antibodies is limited. To address this deficit, sera from a C. difficile vaccine immunized hamsters and toxin-specific human monoclonal antibodies (mAbs) were used to assess the impact of toxin domain specificity on antibody mediated inhibition of cytotoxicity in a Vero cell-based functional assay. Results from toxin domain immunoabsorption assays using hamster anti-toxinsera indicated that no single domain fragment from either toxin A or toxin B could inhibit neutralizing activities. Anti-toxin A activity was prevented with a combination of GTD and CTD fragments while anti-toxin B activity required the GTD, CTD and CPD fragments to block activity. Assays with human mAbs demonstrated that combining mAbs that target multiple toxin domains greatly improves neutralizing potency when compared to equivalent concentrations of either a single mAb or a combination of mAbs against a single domain.

A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells

Clostridium difficile infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against C. difficile infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of C. difficile strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of C. difficile toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

Clostridium difficile Toxin Biology.

Clostridium difficile is the cause of antibiotics-associated diarrhea and pseudomembranous colitis. The pathogen produces three protein toxins: C. difficile toxins A (TcdA) and B (TcdB), and C. difficile transferase toxin (CDT). The single-chain toxins TcdA and TcdB are the main virulence factors. They bind to cell membrane receptors and are internalized. The N-terminal glucosyltransferase and autoprotease domains of the toxins translocate from low-pH endosomes into the cytosol. After activation by inositol hexakisphosphate (InsP6), the autoprotease cleaves and releases the glucosyltransferase domain into the cytosol, where GTP-binding proteins of the Rho/Ras family are mono-O-glucosylated and, thereby, inactivated. Inactivation of Rho proteins disturbs the organization of the cytoskeleton and affects multiple Rho-dependent cellular processes, including loss of epithelial barrier functions, induction of apoptosis, and inflammation. CDT, the third C. difficile toxin, is a binary actin-ADP-ribosylating toxin that causes depolymerization of actin, thereby inducing formation of the microtubule-based protrusions. Recent progress in understanding of the toxins' actions include insights into the toxin structures, their interaction with host cells, and functional consequences of their actions.

Crystal structure of Clostridium difficile toxin A.

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor.

Clostridium difficile may induce antibiotic-associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB-induced rise in intracellular-free Ca(2+) and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR-1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR-1. Domain analyses revealed that the N-terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro-inflammatory ligand effect can be triggered even by cleaved and, thus, non-cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved 'non-cytotoxic' fragments and (iii) an interaction of the N-terminal glucosyltransferase domain instead of the C-terminal receptor binding domain of TcdB with target cells.

Domain organization of Legionella effector SetA

Legionella pneumophila is a human pathogen causing severe pneumonia called Legionnaires' disease. Multiple Legionella effectors are type IV-secreted into the host cell to establish a specific vesicular compartment for pathogen replication. Recently, it has been reported that the Legionella effector SetA shares sequence similarity with glycosyltransferases and interferes with vesicular trafficking of host cells. Here we show that SetA possesses glycohydrolase and mono-O-glucosyltransferase activity by using UDP-glucose as a donor substrate. Whereas the catalytic activity is located at the N terminus of SetA, the C terminus (amino acids 401-644) is essential for guidance of SetA to vesicular compartments of host cells. EGFP-SetA expressed in HeLa cells localizes to early endosomes by interacting with phosphatidylinositol 3-phosphate. EGFP-SetA, transiently expressed in RAW 264.7 macrophages, associates with early phagosomes after infection with Escherichia coli and L. pneumophila. Only the combined expression of the C- and N-terminal domains induces growth defects in yeast similar to full-length SetA. The data indicate that SetA is a multidomain protein with an N-terminal glucosyltransferase domain and a C-terminal phosphatidylinositol 3-phosphate-binding domain, which guides the Legionella effector to the surface of the Legionella-containing vacuole. Both, the localization and the glucosyltransferase domains of SetA are crucial for cellular functions.

RetargetingClostridium difficileToxin B to Neuronal Cells as a Potential Vehicle for Cytosolic Delivery of Therapeutic Biomolecules to Treat Botulism

Botulinum neurotoxins (BoNTs) deliver a protease to neurons which can cause a flaccid paralysis called botulism. Development of botulism antidotes will require neuronal delivery of agents that inhibit or destroy the BoNT protease. Here, we investigated the potential of engineering Clostridium difficile toxin B (TcdB) as a neuronal delivery vehicle by testing two recombinant TcdB chimeras. For AGT-TcdB chimera, an alkyltransferase (AGT) was appended to the N-terminal glucosyltransferase (GT) of TcdB. Recombinant AGT-TcdB had alkyltransferase activity, and the chimera was nearly as toxic to Vero cells as wild-type TcdB, suggesting efficient cytosolic delivery of the AGT/GT fusion. For AGT-TcdB-BoNT/A-Hc, the receptor-binding domain (RBD) of TcdB was replaced by the equivalent RBD from BoNT/A (BoNT/A-Hc). AGT-TcdB-BoNT/A-Hc was >25-fold more toxic to neuronal cells and >25-fold less toxic to Vero cells than AGT-TcdB. Thus, TcdB can be engineered for cytosolic delivery of biomolecules and improved targeting of neuronal cells.

Inositol Hexakisphosphate-dependent Processing of Clostridium sordellii Lethal Toxin and Clostridium novyi α-Toxin

Clostridium sordellii lethal toxin and Clostridium novyi α-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (α-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi α-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin.

Structural determinants for membrane insertion, pore formation and translocation ofClostridium difficiletoxin B

Clostridium difficile toxins A and B bind to eukaryotic target cells, are endocytosed and then deliver their N-terminal glucosyltransferase domain after processing into the cytosol. Whereas glucosyltransferase, autoprocessing and cell-binding domains are well defined, structural features involved in toxin delivery are unknown. Here, we studied structural determinants that define membrane insertion, pore formation and translocation of toxin B. Deletion analyses revealed that a large region, covering amino acids 1501-1753 of toxin B, is dispensable for cytotoxicity in Vero cells. Accordingly, a chimeric toxin, consisting of amino acids 1-1550 and the receptor-binding domain of diphtheria toxin, caused cytotoxic effects. A large N-terminal part of toxin B (amino acids 1-829) was not essential for pore formation (measured by (86) Rb(+) release in mammalian cells). Studies using C-terminal truncation fragments of toxin B showed that amino acid residues 1-990 were still capable of inducing fluorescence dye release from large lipid vesicles and led to increased electrical conductance in black lipid membranes. Thereby, we define the minimal pore-forming region of toxin B within amino acid residues 830 and 990. Moreover, we identify within this region a crucial role of the amino acid pair glutamate-970 and glutamate-976 in pore formation of toxin B.

Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166's participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.

Four Distinct Structural Domains in Clostridium difficile Toxin B Visualized Using SAXS

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of ∼275 Å. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.

Autocatalytic Processing of Clostridium difficile Toxin B: BINDING OF INOSITOL HEXAKISPHOSPHATE

Clostridium difficile toxins A and B are major virulence factors responsible for induction of pseudomembranous colitis and antibiotic-associated diarrhea in men. The toxins possess a multidomain structure and only the N-terminal glucosyltransferase domain, which inactivates Rho GTPases by glucosylation, is translocated into the cytosol of target cells. Processing of the toxin occurs by autocatalytic cleavage and is activated by inositol hexakisphosphate (InsP6). Here we studied the inherent protease activity in fragments of toxin B and determined the site of toxin B that interacts with InsP6. We report that a fragment of toxin B, comprised of residues 1-955, is cleaved in the presence of InsP6. In contrast, mutants of the catalytic triad of the putative cysteine protease domain did not cleave this fragment. [3H]InsP6 bound to holotoxin B and to the fragment 1-955, but not to a fragment comprising residues 900-2366 or the glucosyltransferase domain (residues 1-544). Binding to the putative cysteine protease domain (residues 544-955) was also observed. InsP6-binding was specific and saturable. Isothermal titration calorimetry revealed a Kd value of 2.4 microm for binding of InsP6 to toxin fragment 544-955 with a stoichiometry factor of 0.86. Lysine 600 of toxin B was identified as essential amino acid for InsP6 binding and for InsP6-dependent activation of the protease activity.

Auto-catalytic Cleavage of Clostridium difficile Toxins A and B Depends on Cysteine Protease Activity

The action of Clostridium difficile toxins A and B depends on processing and translocation of the catalytic glucosyltransferase domain into the cytosol of target cells where Rho GTPases are modified. Here we studied the processing of the toxins. Dithiothreitol and beta-mercaptoethanol induced auto-cleavage of purified native toxin A and toxin B into approximately 250/210- and approximately 63-kDa fragments. The 63-kDa fragment was identified by mass spectrometric analysis as the N-terminal glucosyltransferase domain. This cleavage was blocked by N-ethylmaleimide or iodoacetamide. Exchange of cysteine 698, histidine 653, or aspartate 587 of toxin B prevented cleavage of full-length recombinant toxin B and of an N-terminal fragment covering residues 1-955 and inhibited cytotoxicity of full-length toxin B. Dithiothreitol synergistically increased the effect of myo-inositol hexakisphosphate, which has been reported to facilitate auto-cleavage of toxin B (Reineke, J., Tenzer, S., Rupnik, M., Koschinski, A., Hasselmayer, O., Schrattenholz, A., Schild, H., and Von Eichel-Streiber, C. (2007) Nature 446, 415-419). N-Ethylmaleimide blocked auto-cleavage induced by the addition of myo-inositol hexakisphosphate, suggesting that cysteine residues are essential for the processing of clostridial glucosylating toxins. Our data indicate that clostridial glucosylating cytotoxins possess an inherent cysteine protease activity related to the cysteine protease of Vibrio cholerae RTX toxin, which is responsible for auto-cleavage of glucosylating toxins.