Advertisement

Molecular Characteristics of Clostridium perfringens TpeL Toxin and Consequences of Mono-O-GlcNAcylation of Ras in Living Cells*

  • Gregor Guttenberg
    Affiliations
    Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Sven Hornei
    Affiliations
    Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Thomas Jank
    Affiliations
    Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Carsten Schwan
    Affiliations
    Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Wei Lü
    Affiliations
    Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Oliver Einsle
    Affiliations
    Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Panagiotis Papatheodorou
    Affiliations
    Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Klaus Aktories
    Correspondence
    To whom correspondence should be addressed: Albert-Ludwigs-Universität Freiburg, Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albertstr. 25, Otto-Krayer-Haus, D-79104 Freiburg. Tel.: +49-(0)-761-203-5301; Fax: +49-(0)-761-203-5311
    Affiliations
    Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany
    Search for articles by this author
  • Author Footnotes
    * This work was supported by the Deutsche Forschungsgemeinschaft (AK6/16-3) and the Center for Biological Signaling Studies (BIOSS) in Freiburg (Germany).
    This article contains supplemental Figs. S1–S6.
Open AccessPublished:June 04, 2012DOI:https://doi.org/10.1074/jbc.M112.347773
      Background: TpeL is a member of the family of clostridial glucosylating toxins, produced by Clostridium perfringens.
      Results: TpeL enters target cells by self-mediated entry and mono-glycosylates Ras proteins at Thr-35.
      Conclusion: TpeL inhibits Ras signaling and induces apoptosis in target cells.
      Significance: TpeL is a new glucosylating toxin produced by C. perfringens.

      Introduction

      Clostridium perfringens is a widespread pathogen, which is responsible for a number of severe diseases in humans and animals (
      • Borriello S.P.
      • Aktories K.
      ,
      • Hatheway C.L.
      Toxigenic clostridia.
      ). In 70 to 80% of the cases it is the causative agent of the gas gangrene syndrome. The bacteria are equipped with a broad arsenal of toxins (
      • Smedley 3rd, J.G.
      • Fisher D.J.
      • Sayeed S.
      • Chakrabarti G.
      • McClane B.A.
      The enteric toxins of Clostridium perfringens.
      ,
      • Rood J.I.
      • Cole S.T.
      Molecular genetics and pathogenesis of Clostridium perfringens.
      ,
      • Rood J.I.
      Virulence genes of Clostridium perfringens.
      ,
      • Petit L.
      • Gibert M.
      • Popoff M.R.
      Clostridium perfringens: toxinotype and genotype.
      ). C. perfringens is further classified into five serotypes A to E. C. perfringens type C produces the two major toxins α- and β-toxin, but not ϵ- and ι-toxin. Strains of this serotype cause a severe life-threatening necrotic enteritis of the jejunum and ileum mainly due to the production of β-toxin (
      • Stevens D.L.
      ).
      Recently TpeL was identified and isolated from the supernatant of C. perfringens type C strain MC18 (
      • Amimoto K.
      • Noro T.
      • Oishi E.
      • Shimizu M.
      A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.
      ). TpeL is a C-terminally truncated homologue of clostridial glucosylating toxins (CGTs),
      The abbreviations used are: CGTs
      clostridial glucosylating toxins
      UDP-Glc
      UDP-glucose
      CPD
      cysteine protease domain
      InsP6
      inositol hexakisphosphate
      UDP-GlcNAc
      UDP-N-acetylglucosamine
      DABCO
      1,4-diazabicyclo[2.2.2]octane.
      encompassing toxin A and B from C. difficile, lethal and hemorrhagic toxin from C. sordellii and α-toxin from C. novyi (
      • Amimoto K.
      • Noro T.
      • Oishi E.
      • Shimizu M.
      A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.
      ,
      • Jank T.
      • Giesemann T.
      • Aktories K.
      Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function.
      ,
      • Just I.
      • Gerhard R.
      Large clostridial cytotoxins.
      ,
      • Voth D.E.
      • Ballard J.D.
      Clostridium difficile toxins: mechanism of action and role in disease.
      ). CGTs share a similar structure-function relationship. The toxins consist of at least 4 major domains (ABCD model) (
      • Jank T.
      • Aktories K.
      Structure and mode of action of clostridial glucosylating toxins: the ABCD model.
      ). The N terminus harbors the biological active glucosyltransferase activity (domain A) (
      • Hofmann F.
      • Busch C.
      • Prepens U.
      • Just I.
      • Aktories K.
      Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin.
      ). This region is followed by a cysteine protease domain (C domain) (
      • Egerer M.
      • Giesemann T.
      • Jank T.
      • Satchell K.J.
      • Aktories K.
      Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.
      ). The receptor binding domain (B domain), which consists of polypeptide repeats (
      • von Eichel-Streiber C.
      • Sauerborn M.
      Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases.
      ,
      • Greco A.
      • Ho J.G.
      • Lin S.J.
      • Palcic M.M.
      • Rupnik M.
      • Ng K.K.
      Carbohydrate recognition by Clostridium difficile toxin A.
      ), is located C-terminally and was shown to bind certain carbohydrates. Between the C domain and the putative receptor binding domain, a toxin part is localized, which is probably responsible for the delivery (D domain) of the glucosyltransferase into the cytosol of target cells (
      • Genisyuerek S.
      • Papatheodorou P.
      • Guttenberg G.
      • Schubert R.
      • Benz R.
      • Aktories K.
      Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B.
      ). Toxin up-take is suggested to start with receptor binding and clathrin-mediated endocytosis of the toxin-receptor complex into endosomal compartments (
      • Papatheodorou P.
      • Zamboglou C.
      • Genisyuerek S.
      • Guttenberg G.
      • Aktories K.
      Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.
      ). At low pH of endosomes, insertion into membranes is accomplished and toxin translocation initiated. After autoproteolytical processing by the intrinsic cysteine protease domain only the glucosyltransferase domain is released into the cytosol. Once in the cytosol, the toxins catalyze the mono-O-glucosylation of a threonine residue of Rho/Ras proteins, which is crucial for function of the small GTPases (
      • Just I.
      • Selzer J.
      • Wilm M.
      • von Eichel-Streiber C.
      • Mann M.
      • Aktories K.
      Glucosylation of Rho proteins by Clostridium difficile toxin B.
      ,
      • Selzer J.
      • Hofmann F.
      • Rex G.
      • Wilm M.
      • Mann M.
      • Just I.
      • Aktories K.
      Clostridium novyi α-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.
      ). Notably, while C. difficile toxins A, B, and C. sordellii lethal toxin utilize UDP-glucose as a sugar donor (
      • Just I.
      • Selzer J.
      • Wilm M.
      • von Eichel-Streiber C.
      • Mann M.
      • Aktories K.
      Glucosylation of Rho proteins by Clostridium difficile toxin B.
      ,
      • Just I.
      • Wilm M.
      • Selzer J.
      • Rex G.
      • von Eichel-Streiber C.
      • Mann M.
      • Aktories K.
      The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.
      ,
      • Just I.
      • Selzer J.
      • Hofmann F.
      • Green G.A.
      • Aktories K.
      Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation.
      ,
      • Popoff M.R.
      • Chaves-Olarte E.
      • Lemichez E.
      • von Eichel-Streiber C.
      • Thelestam M.
      • Chardin P.
      • Cussac D.
      • Antonny B.
      • Chavrier P.
      • Flatau G.
      • Giry M.
      • de Gunzburg J.
      • Boquet P.
      Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.
      ), C. novyi toxin transfers N-acetylglucosamine from UDP-N-acetylglucosamine onto the target protein (
      • Selzer J.
      • Hofmann F.
      • Rex G.
      • Wilm M.
      • Mann M.
      • Just I.
      • Aktories K.
      Clostridium novyi α-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.
      ). The molecular basis for these differences in the donor substrate specificity were recognized to be due to changes of 2 amino acid residues in the glucosyltransferase domain of the toxins (
      • Jank T.
      • Reinert D.J.
      • Giesemann T.
      • Schulz G.E.
      • Aktories K.
      Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis.
      ,
      • Reinert D.J.
      • Jank T.
      • Aktories K.
      • Schulz G.E.
      Structural basis for the function of Clostridium difficile toxin B.
      ).
      Recently, Nagahama et al. proposed that in contrast to other members of the CGTs, TpeL could utilize both UDP-Glc and UDP-GlcNAc as a donor substrate to modify certain Rho- and Ras-GTPases (
      • Nagahama M.
      • Ohkubo A.
      • Oda M.
      • Kobayashi K.
      • Amimoto K.
      • Miyamoto K.
      • Sakurai J.
      Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
      ). Here we characterized the enzymatic activities of TpeL in detail and show that Ras is the preferred acceptor and N-acetylglucosamine the preferred donor substrate. The donor substrate specificity of TpeL is defined by amino acid residue alanine-383. A cysteine protease domain, located next to the glucosyltransferase domain, mediates InsP6-dependent autocatalytic cleavage of the TpeL toxin. Furthermore, we report that a TpeL full-length variant, which is ∼15 kDa larger than previously described, causes cytotoxic effects in HeLa cells, induces apoptosis and inhibits Ras signaling pathways in rat pheochromocytoma PC12 cells. Intoxication of HeLa cells with TpeL followed by mass spectrometric analysis of immunoprecipitated Ras indicates that N-acetylglucosamine is the preferred modification of Ras in living cells.

      DISCUSSION

      TpeL is the most recently discovered member of the family of CGTs (
      • Amimoto K.
      • Noro T.
      • Oishi E.
      • Shimizu M.
      A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.
      ,
      • Nagahama M.
      • Ohkubo A.
      • Oda M.
      • Kobayashi K.
      • Amimoto K.
      • Miyamoto K.
      • Sakurai J.
      Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
      ). Accordingly, important amino acids in the glucosyltransferase domain (e.g. DXD motif, donor substrate binding motif) and the cysteine protease domain (catalytic DHC triad) are conserved among the different TpeL variants described in this study and C. difficile toxin B, a typical member of CGTs (supplemental Fig. S6). Here we characterized the toxin in respect to its substrate specificity, autocatalytic activation and cytotoxic effects in intact cells. Depending on the toxin type, the various CGTs specifically modify Rho/Ras proteins by glucosylation or GlcNAcylation. Mainly based on studies with toxin fragments, a recent report suggests that TpeL possesses both glucosylation and GlcNAcylation activity to modify Rac and Ras (
      • Nagahama M.
      • Ohkubo A.
      • Oda M.
      • Kobayashi K.
      • Amimoto K.
      • Miyamoto K.
      • Sakurai J.
      Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
      ). Using the recombinant glucosyltransferase domain of TpeL (TpeL1–542) we observed the modification of Ha-, K-, N-, and R-Ras in the presence of UDP-Glc and UDP-GlcNAc with a slight preference for Ha-Ras and N-Ras. By using the indirect method of differential glucosylation, Nagahama et al. suggested that Ras is modified in threonine 35 (
      • Nagahama M.
      • Ohkubo A.
      • Oda M.
      • Kobayashi K.
      • Amimoto K.
      • Miyamoto K.
      • Sakurai J.
      Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
      ). We confirmed this suggestion by direct mass spectrometry analysis. Moreover, we observed that Rap1B and Rap2A as well as the Rho family GTPases Rac1, Rac2, and Rac3 were modified in the presence of UDP-GlcNAc but not with UDP-Glc. However, RalA that was described as a substrate of TpeL by the recent study of Nagahama et al. (
      • Nagahama M.
      • Ohkubo A.
      • Oda M.
      • Kobayashi K.
      • Amimoto K.
      • Miyamoto K.
      • Sakurai J.
      Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
      ), was not modified by the TpeL glucosyltransferase domain in our study.
      The activity of TpeL1–542 to hydrolyze UDP-GlcNAc was almost 20-fold higher than the hydrolysis of UDP-Glc and the KD value of the toxin fragment for binding of UDP-GlcNAc was ∼3-fold lower than for UDP-Glc. Therefore, TpeL is an enzyme which prefers UDP-GlcNAc. The structural determinants for donor sugar specificity of CGTs have been identified as a short motif in the catalytic site, covering residues 383INQ385 of toxin A or lethal toxin (utilizing UDP-Glc) and 385SNA387 of α-toxin (utilizing UDP-GlcNAc) (
      • Jank T.
      • Reinert D.J.
      • Giesemann T.
      • Schulz G.E.
      • Aktories K.
      Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis.
      ). This motif is 383ANQ385 in TpeL. In line with an essential role of this sequence, the mutation of this motif to 383INQ385 in TpeL resulted in the change of the UDP-sugar preference. TpeL1–542 (A383I) exhibited higher glucohydrolase and glucotransferase activities as well as higher binding affinity with UDP-Glc than with UDP-GlcNAc. This mutant modified Rac1 in vitro only in the presence of UDP-Glc but not with UDP-GlcNAc. The data indicate that the 383ANQ385 motif of TpeL is a major determinant of its donor substrate specificity.
      Downstream of the glucosyltransferase domain of all CGTs is a cysteine protease domain located, which is involved in the autoproteolytical activation of the toxins (
      • Egerer M.
      • Giesemann T.
      • Jank T.
      • Satchell K.J.
      • Aktories K.
      Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.
      ). This domain is activated by InsP6 (
      • Reineke J.
      • Tenzer S.
      • Rupnik M.
      • Koschinski A.
      • Hasselmayer O.
      • Schrattenholz A.
      • Schild H.
      • von Eichel-Streiber C.
      Autocatalytic cleavage of Clostridium difficile toxin B.
      ) and causes the release of the glucosyltransferase domain into the cytosol (
      • Pfeifer G.
      • Schirmer J.
      • Leemhuis J.
      • Busch C.
      • Meyer D.K.
      • Aktories K.
      • Barth H.
      Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells.
      ). Our study highlights that TpeL also undergoes InsP6-dependent autocatalytic processing and that a cysteine protease domain-like region (TpeL543–805), located next to the glucosyltransferase domain, is sufficient for cleavage. Accordingly, the KD value for the interaction of InsP6 with TpeL543–805, determined by isothermal titration calorimetry, was ∼2.5 μm. This sensitivity characteristics are very similar to those of C. difficile toxin B, C. sordellii lethal toxin, and C. novyi α-toxin (
      • Guttenberg G.
      • Papatheodorou P.
      • Genisyuerek S.
      • Lü W.
      • Jank T.
      • Einsle O.
      • Aktories K.
      Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi α-toxin.
      ).
      Finally, we studied the biological activity of TpeL in intact cells. Amimoto et al. described a full-length variant of TpeL that is produced by C. perfringens strain MC18 (
      • Amimoto K.
      • Noro T.
      • Oishi E.
      • Shimizu M.
      A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.
      ). The same group reported on the poor expression of this TpeL variant in E. coli (
      • Nagahama M.
      • Ohkubo A.
      • Oda M.
      • Kobayashi K.
      • Amimoto K.
      • Miyamoto K.
      • Sakurai J.
      Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
      ). We were able to express reasonable amounts of this TpeL full-length variant in B. megaterium. However, we noticed that although the recombinant protein was active in vitro, no signs of intoxication where observed when added to a variety of cultured cells. Genomic analysis of C. perfringens type C strain JGS1495 uncovered a TpeL full-length variant, which is C-terminally extended and ∼15 kDa larger than the TpeL from the MC18 strain. This recombinant toxin was much more stable, was well expressed in B. megaterium and caused cytotoxic effects in different cell lines and apoptosis in HeLa cells. We conclude that the TpeL full-length variant from strain MC18 may lack C-terminal elements that participate in receptor-binding. Therefore, we believe that TpeL from strain JGS1495 represents the complete ORF for this toxin.
      Treatment of HeLa cells with the toxin caused cytotoxic effects and induced apoptosis of target cells. Toxin-induced apoptosis was characterized by blebbing and cleavage of PARP1. What is the reason for induction of apoptosis? By using glucosylation specific antibodies, we observed that Ras is the preferred substrate of TpeL in intact cells. Moreover, spectrometric analysis definitely show that Ras is modified by attachment of GlcNAc in intact cells. Interestingly, also C. sordellii lethal toxin, which modifies Ras and Rac by glucosylation, prefers Ras in vitro (
      • Busch C.
      • Hofmann F.
      • Selzer J.
      • Munro S.
      • Jeckel D.
      • Aktories K.
      A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins.
      ,
      • Huelsenbeck S.C.
      • Klose I.
      • Reichenbach M.
      • Huelsenbeck J.
      • Genth H.
      Distinct kinetics of (H/K/N)Ras glucosylation and Rac1 glucosylation catalyzed by Clostridium sordellii lethal toxin.
      ) and in intact cells (
      • Huelsenbeck S.C.
      • Klose I.
      • Reichenbach M.
      • Huelsenbeck J.
      • Genth H.
      Distinct kinetics of (H/K/N)Ras glucosylation and Rac1 glucosylation catalyzed by Clostridium sordellii lethal toxin.
      ,
      • Genth H.
      • Just I.
      Functional implications of lethal toxin-catalysed glucosylation of (H/K/N)Ras and Rac1 in Clostridium sordellii-associated disease.
      ). Importantly, our studies on the apoptosis of HeLa cells were performed at rather high concentrations of TpeL (10 nm), where Ras as well as Rac1 is probably modified by the toxin. However, PARP1 cleavage could also be observed with concentrations of TpeL that were not sufficient for Rac1 modification. We propose that the modification of Ras is most important for induction of apoptosis. This notion is supported by recent findings with C. sordellii lethal toxin and the related toxin B variant TcdBF, which shares all protein targets with lethal toxin with the exception of Ras and is not able to induce apoptosis (
      • Dreger S.C.
      • Schulz F.
      • Huelsenbeck J.
      • Gerhard R.
      • Hofmann F.
      • Just I.
      • Genth H.
      Killing of rat basophilic leukemia cells by lethal toxin from Clostridium sordellii: critical role of phosphatidylinositide 3′-OH kinase/Akt signaling.
      ). Therefore, inhibition of the Ras signaling pathway is most likely essential for induction of apoptosis. Our present studies show that modification of Ras by TpeL blocks interaction with the Ras effector Raf and prevents activation of ERK. Another well-studied Ras-dependent signaling pathway is the formation of neurite-like processes in PC12 cells stimulated by nerve growth factor NGF (
      • Vaudry D.
      • Stork P.J.
      • Lazarovici P.
      • Eiden L.E.
      Signaling pathways for PC12 cell differentiation: making the right connections.
      ). In line with the inhibition of the Ras pathway, TpeL blocked formation of neurites in PC12 cells.
      Taken together, our data indicate that TpeL modifies Ras preferentially by GlcNAcylation at threonine 35. Rac is a poor substrate of TpeL and is modified in vitro and in vivo at 1–2 magnitudes higher toxin concentrations. An intrinsic cysteine protease domain is responsible for the autocatalytic cleavage of TpeL in the presence of InsP6. TpeL induces apoptosis in target cells most likely by inhibition of Ras signaling. Effects of the toxin on intact cells depend on TpeL1–1779, a toxin variant, which is extended at the C terminus but lacks the typical C-terminal polypeptide repeat domain that is present in all members of the CGT family.

      Acknowledgments

      We thank Otilia Wunderlich for excellent technical assistance, Andreas Schlosser and Ulrike Lanner (Core Facility Proteomics, ZBSA, Freiburg, Germany) for mass spectrometric analysis, Alfred Wittinghofer (Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany) and Tilman Brummer (Institut für Biologie III und Zentrum für Biosystemanalyse (ZBSA), Freiburg, Germany) for sharing plasmids, Julian Rood (Monash University, Victoria, Australia) for providing C. perfringens strains, Thijn Brummelkamp (Netherlands Cancer Institute) for providing Hap1 cells, and Thomas Wilmes and Björn Schorch for valuable advices.

      REFERENCES

        • Borriello S.P.
        • Aktories K.
        Borriello S.P. Murray P.R. Funke G. Clostridium perfringens, Clostridium difficile, and other Clostridium species, Topley Wilson's Microbiology & Microbial Infections. Hodder Arnold, London2005
        • Hatheway C.L.
        Toxigenic clostridia.
        Clin. Microbiol. Rev. 1990; 3: 66-98
        • Smedley 3rd, J.G.
        • Fisher D.J.
        • Sayeed S.
        • Chakrabarti G.
        • McClane B.A.
        The enteric toxins of Clostridium perfringens.
        Rev. Physiol Biochem. Pharmacol. 2004; 152: 183-204
        • Rood J.I.
        • Cole S.T.
        Molecular genetics and pathogenesis of Clostridium perfringens.
        Microbiol. Rev. 1991; 55: 621-648
        • Rood J.I.
        Virulence genes of Clostridium perfringens.
        Annu. Rev. Microbiol. 1998; 52: 333-360
        • Petit L.
        • Gibert M.
        • Popoff M.R.
        Clostridium perfringens: toxinotype and genotype.
        Trends Microbiol. 1999; 7: 104-110
        • Stevens D.L.
        Rood J. McClane B.A. Songer J.G. Titball R.W. Necrotizing Clostridial Soft Tissue Infections. The Clostridia, Molecular Biology and Pathogenesis. Academic Press, San Diego1997
        • Amimoto K.
        • Noro T.
        • Oishi E.
        • Shimizu M.
        A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.
        Microbiology. 2007; 153: 1198-1206
        • Jank T.
        • Giesemann T.
        • Aktories K.
        Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function.
        Glycobiology. 2007; 17: 15R-22R
        • Just I.
        • Gerhard R.
        Large clostridial cytotoxins.
        Rev. Physiol Biochem. Pharmacol. 2004; 152: 23-47
        • Voth D.E.
        • Ballard J.D.
        Clostridium difficile toxins: mechanism of action and role in disease.
        Clin. Microbiol. Rev. 2005; 18: 247-263
        • Jank T.
        • Aktories K.
        Structure and mode of action of clostridial glucosylating toxins: the ABCD model.
        Trends Microbiol. 2008; 16: 222-229
        • Hofmann F.
        • Busch C.
        • Prepens U.
        • Just I.
        • Aktories K.
        Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin.
        J. Biol. Chem. 1997; 272: 11074-11078
        • Egerer M.
        • Giesemann T.
        • Jank T.
        • Satchell K.J.
        • Aktories K.
        Auto-catalytic cleavage of Clostridium difficile toxins A and B depends on cysteine protease activity.
        J. Biol. Chem. 2007; 282: 25314-25321
        • von Eichel-Streiber C.
        • Sauerborn M.
        Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases.
        Gene. 1990; 96: 107-113
        • Greco A.
        • Ho J.G.
        • Lin S.J.
        • Palcic M.M.
        • Rupnik M.
        • Ng K.K.
        Carbohydrate recognition by Clostridium difficile toxin A.
        Nat. Struct. Mol. Biol. 2006; 13: 460-461
        • Genisyuerek S.
        • Papatheodorou P.
        • Guttenberg G.
        • Schubert R.
        • Benz R.
        • Aktories K.
        Structural determinants for membrane insertion, pore formation and translocation of Clostridium difficile toxin B.
        Mol. Microbiol. 2011; 79: 1643-1654
        • Papatheodorou P.
        • Zamboglou C.
        • Genisyuerek S.
        • Guttenberg G.
        • Aktories K.
        Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis.
        PLoS. One. 2010; 5: e10673
        • Just I.
        • Selzer J.
        • Wilm M.
        • von Eichel-Streiber C.
        • Mann M.
        • Aktories K.
        Glucosylation of Rho proteins by Clostridium difficile toxin B.
        Nature. 1995; 375: 500-503
        • Selzer J.
        • Hofmann F.
        • Rex G.
        • Wilm M.
        • Mann M.
        • Just I.
        • Aktories K.
        Clostridium novyi α-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.
        J. Biol. Chem. 1996; 271: 25173-25177
        • Just I.
        • Wilm M.
        • Selzer J.
        • Rex G.
        • von Eichel-Streiber C.
        • Mann M.
        • Aktories K.
        The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.
        J. Biol. Chem. 1995; 270: 13932-13936
        • Just I.
        • Selzer J.
        • Hofmann F.
        • Green G.A.
        • Aktories K.
        Inactivation of Ras by Clostridium sordellii lethal toxin-catalyzed glucosylation.
        J. Biol. Chem. 1996; 271: 10149-10153
        • Popoff M.R.
        • Chaves-Olarte E.
        • Lemichez E.
        • von Eichel-Streiber C.
        • Thelestam M.
        • Chardin P.
        • Cussac D.
        • Antonny B.
        • Chavrier P.
        • Flatau G.
        • Giry M.
        • de Gunzburg J.
        • Boquet P.
        Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation.
        J. Biol. Chem. 1996; 271: 10217-10224
        • Dreger S.C.
        • Schulz F.
        • Huelsenbeck J.
        • Gerhard R.
        • Hofmann F.
        • Just I.
        • Genth H.
        Killing of rat basophilic leukemia cells by lethal toxin from Clostridium sordellii: critical role of phosphatidylinositide 3′-OH kinase/Akt signaling.
        Biochemistry. 2009; 48: 1785-1792
        • Jank T.
        • Reinert D.J.
        • Giesemann T.
        • Schulz G.E.
        • Aktories K.
        Change of the donor substrate specificity of Clostridium difficile toxin B by site-directed mutagenesis.
        J. Biol. Chem. 2005; 280: 37833-37838
        • Reinert D.J.
        • Jank T.
        • Aktories K.
        • Schulz G.E.
        Structural basis for the function of Clostridium difficile toxin B.
        J. Mol. Biol. 2005; 351: 973-981
        • Nagahama M.
        • Ohkubo A.
        • Oda M.
        • Kobayashi K.
        • Amimoto K.
        • Miyamoto K.
        • Sakurai J.
        Clostridium perfringens TpeL glycosylates the Rac and Ras subfamily proteins.
        Infect. Immun. 2011; 79: 905-910
        • Yang G.
        • Zhou B.
        • Wang J.
        • He X.
        • Sun X.
        • Nie W.
        • Tzipori S.
        • Feng H.
        Expression of recombinant Clostridium difficile toxin A and B in Bacillus megaterium.
        BMC. Microbiol. 2008; 8: 192
        • Egerer M.
        • Satchell K.J.
        Inositol hexakisphosphate-induced autoprocessing of large bacterial protein toxins.
        PLoS. Pathog. 2010; 6: e1000942
        • Guttenberg G.
        • Papatheodorou P.
        • Genisyuerek S.
        • Lü W.
        • Jank T.
        • Einsle O.
        • Aktories K.
        Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi α-toxin.
        J. Biol. Chem. 2011; 286: 14779-14786
        • Karnoub A.E.
        • Weinberg R.A.
        Ras oncogenes: split personalities.
        Nat. Rev. Mol. Cell Biol. 2008; 9: 517-531
        • Vaudry D.
        • Stork P.J.
        • Lazarovici P.
        • Eiden L.E.
        Signaling pathways for PC12 cell differentiation: making the right connections.
        Science. 2002; 296: 1648-1649
        • Reineke J.
        • Tenzer S.
        • Rupnik M.
        • Koschinski A.
        • Hasselmayer O.
        • Schrattenholz A.
        • Schild H.
        • von Eichel-Streiber C.
        Autocatalytic cleavage of Clostridium difficile toxin B.
        Nature. 2007; 446: 415-419
        • Pfeifer G.
        • Schirmer J.
        • Leemhuis J.
        • Busch C.
        • Meyer D.K.
        • Aktories K.
        • Barth H.
        Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells.
        J. Biol. Chem. 2003; 278: 44535-44541
        • Busch C.
        • Hofmann F.
        • Selzer J.
        • Munro S.
        • Jeckel D.
        • Aktories K.
        A common motif of eukaryotic glycosyltransferases is essential for the enzyme activity of large clostridial cytotoxins.
        J. Biol. Chem. 1998; 273: 19566-19572
        • Huelsenbeck S.C.
        • Klose I.
        • Reichenbach M.
        • Huelsenbeck J.
        • Genth H.
        Distinct kinetics of (H/K/N)Ras glucosylation and Rac1 glucosylation catalyzed by Clostridium sordellii lethal toxin.
        FEBS Lett. 2009; 583: 3133-3139
        • Genth H.
        • Just I.
        Functional implications of lethal toxin-catalysed glucosylation of (H/K/N)Ras and Rac1 in Clostridium sordellii-associated disease.
        Eur. J. Cell Biol. 2011; 90: 959-965