Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins

Large clostridial glucosylating toxins (LCGTs) are produced by toxigenic strains of Clostridium difficile, Clostridium perfringens, Clostridium novyi and Clostridium sordellii. While most C. sordellii strains solely produce lethal toxin (TcsL), C. sordellii strain VPI9048 co‐produces both hemorrhagic toxin (TcsH) and TcsL. Here, the sequences of TcsH‐9048 and TcsL‐9048 are provided, showing that both toxins retain conserved LCGT features and that TcsL and TcsH are highly related to Toxin A (TcdA) and Toxin B (TcdB) from C. difficile strain VPI10463. The substrate profile of the toxins was investigated with recombinant LCGT transferase domains (rN) and a wide panel of small GTPases. rN‐TcsH‐9048 and rN‐TcdA‐10463 glucosylated preferably Rho‐GTPases but also Ras‐GTPases to some extent. In this respect, rN‐TcsH‐9048 and rN‐TcdA‐10463 differ from the respective full‐length TcsH‐9048 and TcdA‐10463, which exclusively glucosylate Rho‐GTPases. rN‐TcsL‐9048 and full length TcsL‐9048 glucosylate both Rho‐ and Ras‐GTPases, whereas rN‐TcdB‐10463 and full length TcdB‐10463 exclusively glucosylate Rho‐GTPases. Vero cells treated with full length TcsH‐9048 or TcdA‐10463 also showed glucosylation of Ras, albeit to a lower extent than of Rho‐GTPases. Thus, in vitro analysis of substrate spectra using recombinant transferase domains corresponding to the auto‐proteolytically cleaved domains, predicts more precisely the in vivo substrates than the full length toxins. Except for TcdB‐1470, all LCGTs evoked increased expression of the small GTPase RhoB, which exhibited cytoprotective activity in cells treated with TcsL isoforms, but pro‐apoptotic activity in cells treated with TcdA, TcdB, and TcsH. All LCGTs induced a rapid dephosphorylation of pY118‐paxillin and of pS144/141‐PAK1/2 prior to actin filament depolymerization indicating that disassembly of focal adhesions is an early event leading to the disorganization of the actin cytoskeleton.     

Functional implications of lethal toxin-catalysed glucosylation of (H/K/N)Ras and Rac1 in Clostridium sordellii-associated disease

Clostridium sordellii-based diseases in humans and livestock rely on the activity of the major virulence factors, the single-chain protein toxins TcsL and TcsH, both belonging to the large clostridial glucosylating toxins. TcsL exclusively glucosylates Rho and Ras low molecular weight GTP-binding proteins. TcsL-induced loss of barrier function in epithelial (diarrhoea) and endothelial cells (extravasation of blood fluid) is based on Rac glucosylation whereas induction of apoptosis results from glucosylation of Ras. Intracellular glucosylation of Rac and Ras can be tracked by immunoblot applying the glucosylation-sensitive antibodies Rac1(Mab 102) and Ras(Mab 27H5). Induction of apoptosis especially of phagocytotic cells is crucial for the severity of C. sordellii-associated disease. The inhibition of TcsL-induced apoptosis by tauroursodeoxycholic acid (TUDCA) may be a promising therapeutic option.     

Bacterial intracellularly active toxins: Membrane localisation of the active domain

Numerous bacterial toxins exert their activity by inactivating or modulating a specific intracellular host target. For this purpose, these toxins have developed efficient strategies to overcome the different host cell defences including specific binding to cell surface, internalisation, passage through the endosome or plasma membrane, exploiting intracellular trafficking and addressing to intracellular targets. Several intracellularly active toxins deliver an active domain into the cytosol that interacts with a target localised to the inner face of the plasma membrane. Thus, the large clostridial glucosylating toxins (LCGTs) target Rho/Ras‐GTPases, certain virulence factors of Gram negative bacteria, Rho‐GTPases, while Pasteurella multocida toxin (PMT) targets trimeric G‐proteins. Others such as botulinum neurotoxins and tetanus neurotoxin have their substrate on synaptic vesicle membrane. LCGTs, PMT, and certain virulence factors from Vibrio sp. show a particular structure constituted of a four‐helix bundle membrane (4HBM) protruding from the catalytic site that specifically binds to the membrane phospholipids and then trap the catalytic domain at the proximity of the membrane anchored substrate. Structural and functional analysis indicate that the 4HBM tip of the Clostridium sordellii lethal toxin (TcsL) from the LCGT family contain two loops forming a cavity that mediates the binding to phospholipids and more specifically to phosphatidylserine.     

Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function

Clostridium difficile causes pseudomembranous colitis and is responsible for many cases of nosocomial antibiotic-associated diarrhea. Major virulence factors of C. difficile are the glucosylating exotoxins A and B. Both toxins enter target cells in a pH- dependent manner from endosomes by forming pores. They translocate the N-terminal catalytic domains into the cytosol of host cells and inactivate Rho guanosine triphosphatases by glucosylation. The crystal structure of the catalytic domain of toxin B was solved in a complex with uridine diphosphate, glucose, and manganese ion, exhibiting a folding of type A family glycosyltransferases. Crystallization of fragments of the C-terminus of toxin A, which is characterized by polypeptide repeats, revealed a solenoid-like structure often found in bacterial cell surface proteins. These studies, which provide new insights into structure, uptake, and function of the family of clostridial glucosylating toxins, are reviewed.     

The membrane localization domains of two distinct bacterial toxins form a 4‐helix‐bundle in solution

Membrane localization domain (MLD) was first proposed for a 4‐helix‐bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1‐specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats‐in‐toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1‐C2 domains of PMT, including a putative MLD. We have determined the solution structure for the MLDs in PMT and in RRSP using solution state NMR. We conclude that the MLDs in these two toxins assume a 4‐helix‐bundle structure in solution.     

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.     

Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis

Large clostridial toxins mono‐O‐glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [¹⁴C]glucosylation, sequential [³²P]ADP‐ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS‐based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo‐2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R‐Ras2.     

Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain

Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.     

Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway

(3R,5R)‐Clavulanic acid (CA) is a clinically important inhibitor of Class A β‐lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5‐related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2₁ space groups) of CBG were obtained; in both forms one molecule of acetyl‐CoA (AcCoA) was bound to the N‐terminal GNAT domain, with the C‐terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl‐CoA molecule can bind to CBG. Succinyl‐CoA and myristoyl‐CoA displayed the strongest binding to the “second” CoA binding site, which is likely in the C‐terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N‐terminal GNAT domain plays a structural role whereas the C‐terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl‐CoA occurs preferentially to monomeric rather than dimeric CBG. The N‐terminal AcCoA binding site and the proposed C‐terminal acyl‐CoA binding site of CBG are compared with acyl‐CoA binding sites of other tandem and single domain GNAT proteins. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Non-toxigenic Clostridium sordellii: clinical and microbiological features of a case of cholangitis-associated bacteremia

Toxigenic Clostridium sordellii strains are increasingly recognized to cause highly lethal infections in humans that are typified by a toxic shock syndrome (TSS). Two glucosylating toxins, lethal toxin (TcsL) and hemorrhagic toxin (TcsH) are believed to be important in the pathogenesis of TSS. While non-toxigenic strains of C. sordellii demonstrate reduced cytotoxicity in vitro and lower virulence in animal models of infection, there are few data regarding their behavior in humans. Here we report a non-TSS C. sordellii infection in the context of a polymicrobial bacterial cholangitis. The C. sordellii strain associated with this infection did not carry either the TcsL-encoding tcsL gene or the tcsH gene for TcsH. In addition, the strain was neither cytotoxic in vitro nor lethal in a murine sepsis model. These results provide additional correlative evidence that TcsL and TcsH increase the risk of mortality during C. sordellii infections.     

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.     

Distinct kinetics of (H/K/N)Ras glucosylation and Rac1 glucosylation catalysed by Clostridium sordellii lethal toxin

Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptotic cell death. One yet unsolved problem in studies on TcsL is the lack of a method allowing the specific detection of (H/K/N)Ras glucosylation. In this study, we identify the Ras(Mab 27H5) antibody as a glucosylation-sensitive antibody capable for the immunoblot detection of (H/K/N)Ras glucosylation in TcsL-treated cells. Alternative Ras antibodies including the K-Ras(Mab F234) antibody or the v-H-Ras(Mab Y13-159) antibody recognize Ras proteins regardless of glucosylation. (H/K)Ras are further shown to be more efficaciously glucosylated by TcsL than Rac1 in rat basophilic leukemia cells as well as in a cell-free system.

Structured summary

MINT-7261742: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) H-RAS (uniprotkb:P01112) by enzymatic studies (MI:0415)MINT-7261729: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) Rac1 (uniprotkb:P63000) by enzymatic studies (MI:0415)MINT-7261772: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) K-RAS (uniprotkb:P01116) by enzymatic studies (MI:0415)MINT-7261784: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) N-RAS (uniprotkb:P01111) by enzymatic studies (MI:0415)     

Rac1 inactivation by lethal toxin from Clostridium sordellii modifies focal adhesions upstream of actin depolymerization

Inactivation of different small GTPases upon their glucosylation by lethal toxin from Clostridium sordellii strain IP82 (LT‐82) is already known to lead to cell rounding, adherens junction (AJ) disorganization and actin depolymerization. In the present work, we observed that LT‐82 induces a rapid dephosphorylation of paxillin, a protein regulating focal adhesion (FA), independently of inactivation of paxillin kinases such as Src, Fak and Pyk2. Among the small GTPases inactivated by this toxin, including Rac, Ras, Rap and Ral, we identified Rac1, as responsible for paxillin dephosphorylation using cells overexpressing Rac1^V12. Rac1 inactivation by LT‐82 modifies interactions between proteins from AJ and FA complexes as shown by pull‐down assays. We showed that in Triton X‐100‐insoluble membrane proteins from these complexes, namely E‐cadherin, β‐catenin, p120‐catenin and talin, are decreased upon LT‐82 intoxication, a treatment that also induces a rapid decrease in cell phosphoinositide content. Therefore, we proposed that Rac inactivation by LT‐82 alters phosphoinositide metabolism leading to FA and AJ complex disorganization and actin depolymerization.     

Backbone and side-chain assignments of an effector membrane localization domain from Vibrio vulnificus MARTX toxin

¹H, ¹³C, and ¹⁵N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain from the domain of unknown function 5 (DUF5) effector (MLD_VvDUF5) of the MARTX toxin from Vibrio vulnificus in its solution state. We have assigned 97 % of all backbone and side-chain carbon atoms, including 96 % of all backbone residues. Secondary chemical shift analysis using TALOS+ demonstrates four helices that align with those predicted by structure homology modeling using the MLDs of Pasteurella multocida toxin (PMT) and the clostridial TcdB and TcsL toxins as templates. Future studies will be towards solving the structure and determining the dynamics in the solution state.     

Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata

C‐glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C‐glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C‐glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C‐glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C‐glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C‐glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over‐expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.     

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-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.     

Purification,molecular cloning and functional characterization of flavonoid C‐glucosyltransferases from Fagopyrum esculentum M. (buckwheat) cotyledon

C‐Glycosides are characterized by their C–C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C‐Glycosides are remarkably stable, as their C–C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C‐glycosylflavonoids; however, the genes encoding for enzymes that catalyze C‐glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C‐glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C‐glucosylation of 2‐hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C‐glucosylation activity towards 2‐hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2′,4′,6′‐trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C‐glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.     

Low pH-induced formation of ion channels by clostridium difficile toxin B in target cells

Clostridium difficile toxin B (269 kDa), which is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis, inactivates Rho GTPases by glucosylation. Here we studied the uptake and membrane interaction of the toxin with eukaryotic target cells. Bafilomycin A1, which prevents acidification of endosomal compartments, blocked the cellular uptake of toxin B in Chinese hamster ovary cells cells. Extracellular acidification (pH 相似文献   

Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre‐loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site‐directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine‐rich transmembrane helix 1 with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild‐type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol.