Advertisement

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

Open AccessPublished:March 08, 2011DOI:https://doi.org/10.1074/jbc.M110.200691
      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 (InsP6) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP6 (<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 InsP6 was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP6 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 InsP6-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 InsP6 and subsequent processing of the toxin.

      Introduction

      Various pathogens of the genus Clostridium produce highly potent glucosylating toxins, clostridial glucosylating toxins (CGTs),
      The abbreviations used are: CGT
      clostridial glucosylating toxin
      CPD
      cysteine protease domain
      InsP6
      inositol hexakisphosphate
      GD
      glucosyltransferase domain.
      which act on host target cells by modification and inactivation of Rho and Ras GTPases (
      • Just I.
      • Selzer J.
      • Wilm M.
      • von Eichel-Streiber C.
      • Mann M.
      • Aktories K.
      ,
      • Selzer J.
      • Hofmann F.
      • Rex G.
      • Wilm M.
      • Mann M.
      • Just I.
      • Aktories K.
      ,
      • Just I.
      • Selzer J.
      • Hofmann F.
      • Green G.A.
      • Aktories K.
      ,
      • Just I.
      • Gerhard R.
      ,
      • Voth D.E.
      • Ballard J.D.
      ). This group of toxins, comprising Clostridium difficile toxins A and B, Clostridium novyi α-toxin, and Clostridium sordellii lethal and hemorrhagic toxins, are major virulence factors (
      • Boriello S.P.
      • Aktories K.
      ). Whereas C. difficile toxins A and B cause antibiotic-associated diarrhea and pseudomembranous colitis (
      • Kelly C.P.
      • LaMont J.T.
      ), C. sordellii lethal toxin is implicated in toxic shock syndrome (e.g. after medically induced abortion) (
      • Ho C.S.
      • Bhatnagar J.
      • Cohen A.L.
      • Hacker J.K.
      • Zane S.B.
      • Reagan S.
      • Fischer M.
      • Shieh W.J.
      • Guarner J.
      • Ahmad S.
      • Zaki S.R.
      • McDonald L.C.
      ) and, like C. novyi α-toxin, plays a pathogenic role in gas gangrene syndrome (
      • Cohen A.L.
      • Bhatnagar J.
      • Reagan S.
      • Zane S.B.
      • D'Angeli M.A.
      • Fischer M.
      • Killgore G.
      • Kwan-Gett T.S.
      • Blossom D.B.
      • Shieh W.J.
      • Guarner J.
      • Jernigan J.
      • Duchin J.S.
      • Zaki S.R.
      • McDonald L.C.
      ,
      • Tsokos M.
      • Schalinski S.
      • Paulsen F.
      • Sperhake J.P.
      • Püschel K.
      • Sobottka I.
      ).
      CGTs are single-chain proteins subdivided into at least four functional domains (
      • Jank T.
      • Aktories K.
      ). Binding of the toxins to cell-surface receptors is mediated by a C-terminal region including a series of repetitive oligopeptides (
      • von Eichel-Streiber C.
      • Sauerborn M.
      • Kuramitsu H.K.
      ). After uptake of the toxins by clathrin-dependent endocytosis (
      • Papatheodorou P.
      • Zamboglou C.
      • Genisyuerek S.
      • Guttenberg G.
      • Aktories K.
      ), a central region harboring a pattern of hydrophobic amino acids is suggested to mediate pore formation and translocation from endosomes into the cytosol (
      • von Eichel-Streiber C.
      • Sauerborn M.
      • Kuramitsu H.K.
      ,
      • Genisyuerek S.
      • Papatheodorou P.
      • Guttenberg G.
      • Schubert R.
      • Benz R.
      • Aktories K.
      ). The glucosyltransferase domain is located at the N-terminal end of the toxins (
      • Hofmann F.
      • Busch C.
      • Prepens U.
      • Just I.
      • Aktories K.
      ). As shown for toxins A and B, this domain is released into the cytosol by autocatalytic cleavage induced by an adjacent cysteine protease domain (CPD) (
      • Pfeifer G.
      • Schirmer J.
      • Leemhuis J.
      • Busch C.
      • Meyer D.K.
      • Aktories K.
      • Barth H.
      ,
      • Egerer M.
      • Giesemann T.
      • Jank T.
      • Satchell K.J.
      • Aktories K.
      ,
      • Reineke J.
      • Tenzer S.
      • Rupnik M.
      • Koschinski A.
      • Hasselmayer O.
      • Schrattenholz A.
      • Schild H.
      • von Eichel-Streiber C.
      ,
      • Rupnik M.
      • Pabst S.
      • Rupnik M.
      • von Eichel-Streiber C.
      • Urlaub H.
      • Söling H.D.
      ).
      Previous studies revealed that autocatalytic processing of CGTs depends on inositol hexakisphosphate (InsP6) (
      • Egerer M.
      • Giesemann T.
      • Jank T.
      • Satchell K.J.
      • Aktories K.
      ,
      • Reineke J.
      • Tenzer S.
      • Rupnik M.
      • Koschinski A.
      • Hasselmayer O.
      • Schrattenholz A.
      • Schild H.
      • von Eichel-Streiber C.
      ). It was shown for C. difficile toxins A and B that InsP6 can bind to the intrinsic CPD (
      • Egerer M.
      • Giesemann T.
      • Herrmann C.
      • Aktories K.
      ,
      • Pruitt R.N.
      • Chagot B.
      • Cover M.
      • Chazin W.J.
      • Spiller B.
      • Lacy D.B.
      ). Binding of InsP6 causes conformational changes in the catalytic center of the CPD, resulting in activation of the protease, cleavage of the toxin molecule between the glucosyltransferase domain and the CPD, and release of the glucosyltransferase into the cytosol (
      • Pruitt R.N.
      • Chagot B.
      • Cover M.
      • Chazin W.J.
      • Spiller B.
      • Lacy D.B.
      ,
      • Egerer M.
      • Satchell K.J.
      ,
      • Prochazkova K.
      • Satchell K.J.
      ).
      This study deals with the biochemical characterization of the InsP6-induced autocatalytic cleavage of C. sordellii lethal toxin and C. novyi α-toxin. For this purpose, exclusively recombinant toxins were used. Although no major differences could be observed in the binding affinity of InsP6 for the CPDs of all CGTs, significant differences between CGTs were observed in the InsP6-induced autocatalytic processing of the holotoxins. In particular, we report on the activation of the InsP6-induced autoproteolytic cleavage of C. sordellii lethal toxin, which suggests differences in the modular organization and function of the toxin domains of the various members of the CGT family.

      DISCUSSION

      Here, we studied the InsP6-dependent autocatalytic processing of three CGTs. Initial studies with short fragments of the toxins covering the glucosyltransferase (GlcNAc-transferase of α-toxin) domain and the CPD revealed that all toxins needed InsP6 for cleavage. This is not surprising because the CPDs of lethal toxin and α-toxin are 78 and 34% identical to the CPD of toxin B, and the proposed catalytic triad (toxin B Asp-587/His-653/Cys-698) and the InsP6-binding sites are highly conserved among the toxins. Accordingly, we determined very similar Kd values for the binding of InsP6 to the toxin protease domains. Moreover, the concentration range of InsP6 needed for cleavage was similar, indicating that the mechanism of InsP6-induced activation of the protease activity is similar among all these toxins. In addition, we predicted and confirmed the cleavage sites of all three toxins by mutagenesis studies, indicating that the toxins are split after a conserved leucine residue.
      Whereas the short fragments covering the glucosyltransferase (GlcNAc-transferase of α-toxin) domains and CPDs of the toxins exhibited similar properties with respect to InsP6 binding and activation, full-length toxins differed significantly. Although full-length toxin B and α-toxin exhibited InsP6 sensitivity at 1 and 100 μm, respectively, lethal toxin was much more resistant against autocatalytic cleavage even at high concentrations of InsP6 (≥1 mm). Moreover, we were not able to detect significant binding of InsP6 to lethal toxin in filter binding assays, whereas toxin B and α-toxin exhibited InsP6 binding under these conditions. This discrepancy was not caused by misfolding of recombinant B. megaterium-expressed proteins because glucosyltransferase activity and cytotoxicity were confirmed, indicating proper folding of the proteins.
      To study the impact of toxin processing on cytotoxicity of full-length toxins, we changed the catalytic cysteine residues of the CPD to alanine. This resulted in inhibition of cytotoxicity of all three toxins, including lethal toxin, which was largely resistant to in vitro processing even in the presence of a high concentration of InsP6. These findings indicate that also processing of lethal toxin is essential for full cytotoxicity.
      As CGTs translocate from acidic endosomal compartments into the cytosol (
      • Jank T.
      • Aktories K.
      ,
      • Barth H.
      • Pfeifer G.
      • Hofmann F.
      • Maier E.
      • Benz R.
      • Aktories K.
      ,
      • Qa'Dan M.
      • Spyres L.M.
      • Ballard J.D.
      ), we wondered whether incubation at low pH might affect the susceptibility of the toxins to InsP6. We observed that lowering the pH strongly increased the autocatalytic cleavage of full-length lethal toxin in the presence of InsP6. In line with this finding, we observed that the binding of InsP6 to full-length lethal toxin was largely increased at low pH. This effect was most likely caused by conformational changes in the overall structure of lethal toxin and not only by charge effects at the binding site. A decrease in the pH should rather reduce the binding of the negatively charged InsP6 to the toxin. This was confirmed in a direct comparison of the InsP6 binding of full-length lethal toxin with that of full-length toxin B at different pH values. Whereas the binding of InsP6 to lethal toxin increased at low pH, the reverse was true for toxin B.
      The notion that, at low pH, the binding of InsP6 to its binding pocket at the cysteine protease is reduced was confirmed by isothermal titration calorimetry with the isolated protease domain of lethal toxin, showing an ∼10-fold reduced binding affinity for InsP6. Thus, these findings indicate that the pH shift causes major conformational changes in the overall structure of lethal toxin, resulting in exposure and/or structural changes in the CPD, allowing InsP6 binding.
      Major pH-dependent structural changes in lethal toxin have been reported before. Ballard and co-workers (
      • Qa'Dan M.
      • Spyres L.M.
      • Ballard J.D.
      ) showed that the cytotoxicity of C. sordellii lethal toxin is increased in a pH-dependent manner. A short pH pulse strongly increased the velocity of cytotoxic effects. They showed major conformational changes in lethal toxin by fluorescence methods at low pH, which were reversible (
      • Qa'Dan M.
      • Spyres L.M.
      • Ballard J.D.
      ). These data are in agreement with our studies, indicating that, at low pH, a major conformational change in the toxin exposes the InsP6-binding site and allows binding of InsP6 and subsequent autocatalytic processing. As processing is important for activity, low pH exposure of the toxin results in an increase in cytotoxicity.
      More recently, it was proposed that lethal toxin forms high molecular complexes, which dissociate at low pH and reassociate after an increase in pH (
      • Voth D.E.
      • Qa'Dan M.
      • Hamm E.E.
      • Pelfrey J.M.
      • Ballard J.D.
      ). We suggest that these studies performed with native lethal toxin purified from C. sordellii might be biased by pH-dependent impurities, which could affect complex formation. We used recombinant toxins expressed in B. megaterium. By gel filtration of recombinant full-length lethal toxin, we could confirm a monodisperse preparation of monomers. Therefore, formation of multimeric toxin complexes most likely did not play a major role in our studies. However, we also observed reversibility of the pH-dependent effect. After readjustment of the low pH to pH 7.4, the binding of InsP6 and the processing of the toxin were again inhibited. This finding indicates that the proposed pH-dependent conformational change, which allows InsP6 binding, is largely reversible.
      Recent studies solved the crystal structures of the GDs of C. difficile toxin B (
      • Reinert D.J.
      • Jank T.
      • Aktories K.
      • Schulz G.E.
      ) and C. novyi α-toxin and C. sordellii lethal toxin (
      • Ziegler M.O.
      • Jank T.
      • Aktories K.
      • Schulz G.E.
      ); the CPD of toxin A (
      • Pruitt R.N.
      • Chagot B.
      • Cover M.
      • Chazin W.J.
      • Spiller B.
      • Lacy D.B.
      ); and parts of the C-terminal repetitive oligopeptide domain (
      • Ho J.G.
      • Greco A.
      • Rupnik M.
      • Ng K.K.
      ,
      • Greco A.
      • Ho J.G.
      • Lin S.J.
      • Palcic M.M.
      • Rupnik M.
      • Ng K.K.
      ). However, the structure of the holotoxin is still enigmatic. Related CPD structures are available not only for toxin A but also for Vibrio cholerae MARTX toxin. Notably, both CPDs share conserved lysine residues, which are involved in InsP6 binding. However, the conformation of the bound allosteric activator is largely different in the CPDs of toxin A and MARTX (
      • Pruitt R.N.
      • Chagot B.
      • Cover M.
      • Chazin W.J.
      • Spiller B.
      • Lacy D.B.
      ,
      • Egerer M.
      • Satchell K.J.
      ,
      • Prochazkova K.
      • Shuvalova L.A.
      • Minasov G.
      • Voburka Z.
      • Anderson W.F.
      • Satchell K.J.
      ). Because the InsP6-binding properties of the isolated CPDs of toxin B, α-toxin, and lethal toxin are very similar, we do not suggest that major structural differences exist in the CPDs of these toxins. Therefore, structural features located distantly to the CPD may affect the conformation and binding of InsP6 to the CPD in full-length lethal toxin. A recent study with negative stain electron microscopy revealed a model of holotoxins A and B and proposed conformational changes occurring at the low pH of endosomes (
      • Pruitt R.N.
      • Chambers M.G.
      • Ng K.K.
      • Ohi M.D.
      • Lacy D.B.
      ). In this model, the large extension of the C-terminal domain is remarkable, not even excluding interaction with N-terminal structures of the toxins. Especially the C-terminal polypeptide repeat domain, which has an extended solenoid-like structure, differs between the various CGTs. Therefore, it remains to be studied whether the C-terminal domain is involved in the different susceptibility of holotoxins to InsP6.
      What is the physiological reason for the unique, low pH-dependent InsP6-binding properties of lethal toxin? One might speculate that C. sordellii has adopted to environments that frequently exhibit high concentrations of extracellular InsP6. The disintegration of cells in wounds and injured tissues (frequent sites of C. sordellii infection) would allow cytosolic InsP6 to reach extracellular compartments. Unique structural characteristics of lethal toxin under neutral pH conditions thereby prevent activation of the CPD by InsP6 binding prior to cell entry. Such prerequisites might not be necessary for C. difficile toxin B, for example, because phytase enzymes that hydrolyze extracellular luminal InsP6 efficiently are present in the small intestine (
      • Iqbal T.H.
      • Lewis K.O.
      • Cooper B.T.
      ). After endocytosis, however, all CGTs are exposed to endosomal acidification, resulting in partial unfolding and/or activation of domains involved in membrane insertion and delivery of the enzyme portions across the endosomal membrane. Eventually, the CPD is accessible for InsP6-driven activation, leading to the release of the glucosyltransferase into the cytosol. However, one has to keep in mind that activation of the CPD and release of the GD should not occur before entering the cytosol. Otherwise, the delivery of the biologically active part is altered. Therefore, it makes sense that the affinity of toxin B for InsP6 is reduced at low pH (e.g. which is observed in endosomes).

      Acknowledgments

      We greatly appreciate the excellent technical assistance of Otilia Wunderlich and Sven Hornei.

      REFERENCES

        • Just I.
        • Selzer J.
        • Wilm M.
        • von Eichel-Streiber C.
        • Mann M.
        • Aktories K.
        Nature. 1995; 375: 500-503
        • Selzer J.
        • Hofmann F.
        • Rex G.
        • Wilm M.
        • Mann M.
        • Just I.
        • Aktories K.
        J. Biol. Chem. 1996; 271: 25173-25177
        • Just I.
        • Selzer J.
        • Hofmann F.
        • Green G.A.
        • Aktories K.
        J. Biol. Chem. 1996; 271: 10149-10153
        • Just I.
        • Gerhard R.
        Rev. Physiol. Biochem. Pharmacol. 2004; 152: 23-47
        • Voth D.E.
        • Ballard J.D.
        Clin. Microbiol. Rev. 2005; 18: 247-263
        • Boriello S.P.
        • Aktories K.
        Boriello S.P. Murray P.R. Funke G. Topley & Wilson's Microbiology & Microbial Infections. Hodder Arnold, Ltd., London2005: 1089-1136
        • Kelly C.P.
        • LaMont J.T.
        N. Engl. J. Med. 2008; 359: 1932-1940
        • Ho C.S.
        • Bhatnagar J.
        • Cohen A.L.
        • Hacker J.K.
        • Zane S.B.
        • Reagan S.
        • Fischer M.
        • Shieh W.J.
        • Guarner J.
        • Ahmad S.
        • Zaki S.R.
        • McDonald L.C.
        Am. J. Obstet. Gynecol. 2009; 201: 459-467
        • Cohen A.L.
        • Bhatnagar J.
        • Reagan S.
        • Zane S.B.
        • D'Angeli M.A.
        • Fischer M.
        • Killgore G.
        • Kwan-Gett T.S.
        • Blossom D.B.
        • Shieh W.J.
        • Guarner J.
        • Jernigan J.
        • Duchin J.S.
        • Zaki S.R.
        • McDonald L.C.
        Obstet. Gynecol. 2007; 110: 1027-1033
        • Tsokos M.
        • Schalinski S.
        • Paulsen F.
        • Sperhake J.P.
        • Püschel K.
        • Sobottka I.
        Int. J. Legal Med. 2008; 122: 35-41
        • Jank T.
        • Aktories K.
        Trends Microbiol. 2008; 16: 222-229
        • von Eichel-Streiber C.
        • Sauerborn M.
        • Kuramitsu H.K.
        J. Bacteriol. 1992; 174: 6707-6710
        • Papatheodorou P.
        • Zamboglou C.
        • Genisyuerek S.
        • Guttenberg G.
        • Aktories K.
        PLoS ONE. 2010; 5: e10673
        • Genisyuerek S.
        • Papatheodorou P.
        • Guttenberg G.
        • Schubert R.
        • Benz R.
        • Aktories K.
        Mol. Microbiol. 2011; 79: 1643-1654
        • Hofmann F.
        • Busch C.
        • Prepens U.
        • Just I.
        • Aktories K.
        J. Biol. Chem. 1997; 272: 11074-11078
        • Pfeifer G.
        • Schirmer J.
        • Leemhuis J.
        • Busch C.
        • Meyer D.K.
        • Aktories K.
        • Barth H.
        J. Biol. Chem. 2003; 278: 44535-44541
        • Egerer M.
        • Giesemann T.
        • Jank T.
        • Satchell K.J.
        • Aktories K.
        J. Biol. Chem. 2007; 282: 25314-25321
        • Reineke J.
        • Tenzer S.
        • Rupnik M.
        • Koschinski A.
        • Hasselmayer O.
        • Schrattenholz A.
        • Schild H.
        • von Eichel-Streiber C.
        Nature. 2007; 446: 415-419
        • Rupnik M.
        • Pabst S.
        • Rupnik M.
        • von Eichel-Streiber C.
        • Urlaub H.
        • Söling H.D.
        Microbiology. 2005; 151: 199-208
        • Egerer M.
        • Giesemann T.
        • Herrmann C.
        • Aktories K.
        J. Biol. Chem. 2009; 284: 3389-3395
        • Pruitt R.N.
        • Chagot B.
        • Cover M.
        • Chazin W.J.
        • Spiller B.
        • Lacy D.B.
        J. Biol. Chem. 2009; 284: 21934-21940
        • Egerer M.
        • Satchell K.J.
        PLoS Pathog. 2010; 6: e1000942
        • Prochazkova K.
        • Satchell K.J.
        J. Biol. Chem. 2008; 283: 23656-23664
        • Yang G.
        • Zhou B.
        • Wang J.
        • He X.
        • Sun X.
        • Nie W.
        • Tzipori S.
        • Feng H.
        BMC Microbiol. 2008; 8: 192
        • Just I.
        • Selzer J.
        • von Eichel-Streiber C.
        • Aktories K.
        J. Clin. Invest. 1995; 95: 1026-1031
        • 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.
        J. Biol. Chem. 1996; 271: 10217-10224
        • Kreimeyer I.
        • Euler F.
        • Marckscheffel A.
        • Tatge H.
        • Pich A.
        • Olling A.
        • Schwarz J.
        • Just I.
        • Gerhard R.
        Naunyn-Schmiedeberg's Arch. Pharmacol. 2011; 383: 253-262
        • Barth H.
        • Pfeifer G.
        • Hofmann F.
        • Maier E.
        • Benz R.
        • Aktories K.
        J. Biol. Chem. 2001; 276: 10670-10676
        • Qa'Dan M.
        • Spyres L.M.
        • Ballard J.D.
        Infect. Immun. 2000; 68: 2470-2474
        • Qa'Dan M.
        • Spyres L.M.
        • Ballard J.D.
        Infect. Immun. 2001; 69: 5487-5493
        • Voth D.E.
        • Qa'Dan M.
        • Hamm E.E.
        • Pelfrey J.M.
        • Ballard J.D.
        Infect. Immun. 2004; 72: 3366-3372
        • Reinert D.J.
        • Jank T.
        • Aktories K.
        • Schulz G.E.
        J. Mol. Biol. 2005; 351: 973-981
        • Ziegler M.O.
        • Jank T.
        • Aktories K.
        • Schulz G.E.
        J. Mol. Biol. 2008; 377: 1346-1356
        • Ho J.G.
        • Greco A.
        • Rupnik M.
        • Ng K.K.
        Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 18373-18378
        • Greco A.
        • Ho J.G.
        • Lin S.J.
        • Palcic M.M.
        • Rupnik M.
        • Ng K.K.
        Nat. Struct. Mol. Biol. 2006; 13: 460-461
        • Prochazkova K.
        • Shuvalova L.A.
        • Minasov G.
        • Voburka Z.
        • Anderson W.F.
        • Satchell K.J.
        J. Biol. Chem. 2009; 284: 26557-26568
        • Pruitt R.N.
        • Chambers M.G.
        • Ng K.K.
        • Ohi M.D.
        • Lacy D.B.
        Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 13467-13472
        • Iqbal T.H.
        • Lewis K.O.
        • Cooper B.T.
        Gut. 1994; 35: 1233-1236