Clostridium novyi α-Toxin-catalyzed Incorporation of GlcNAc into Rho Subfamily Proteins

The lethal and edema-inducing α-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. α-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the α-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for α-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi α-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins. The lethal and edema-inducing α-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. α-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the α-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for α-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi α-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins. INTRODUCTIONClostridium novyi type A strains have been identified as the causative organisms of gas gangrene infections of humans and animals (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar). Type A strains produce an exotoxin, termed α-toxin, that exhibits in vivo both lethal and edematizing activity (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar). In tissue culture, α-toxin is cytotoxic, causing cell shape changes that are accompanied by disruption of the microfilament cytoskeleton and by minor effects at the vimentin and tubulin system (2Bette P. Oksche A. Mauler F. Eichel-Streiber C. Popoff M.R. Habermann E. Toxicon. 1991; 29: 877-887Crossref PubMed Scopus (51) Google Scholar, 3Müller H. Von Eichel-Streiber C. Habermann E. Infect. Immun. 1992; 60: 3007-3010Crossref PubMed Google Scholar, 4Oksche A. Nakov R. Habermann E. Infect. Immun. 1992; 60: 3002-3006Crossref PubMed Google Scholar). Recently, α-toxin has been cloned and sequenced (5Hofmann F. Herrmann A. Habermann E. Von Eichel-Streiber C. Mol. & Gen. Genet. 1995; 247: 670-679Crossref PubMed Scopus (32) Google Scholar). As deduced from these data, α-toxin has a molecular mass of 250,166 Da and shows 48% homology with Clostridium difficile toxin A (ToxA) 1The abbreviations used are: ToxAC. difficile toxin AToxBC. difficile toxin BGalgalactoseGlcglucoseGlcNAcN-acetyl-galactosaminePAGEpolyacrylamide gel electrophoresisGTPγSguanosine 5ʹ-3-O-(thio)triphosphate. and toxin B (ToxB). ToxA and ToxB are the major virulence factors of pathogenic C. difficile strains and have been identified as the causative agents of the antibiotic-associated diarrhea and the fatal form, the pseudomembranous colitis (6Lyerly D.M. Krivan H.C. Wilkins T.D. Clin. Microbiol. Rev. 1988; 1: 1-18Crossref PubMed Scopus (431) Google Scholar, 7Kelly C.P. Pothoulakis C. LaMont J.T. N. Engl. J. Med. 1994; 330: 257-262Crossref PubMed Scopus (1041) Google Scholar). Furthermore, α-toxin shows 34% homology to the lethal toxin from Clostridium sordellii (5Hofmann F. Herrmann A. Habermann E. Von Eichel-Streiber C. Mol. & Gen. Genet. 1995; 247: 670-679Crossref PubMed Scopus (32) Google Scholar, 8Green G.A. Schué V. Monteil H. Gene (Amst.). 1995; 161: 57-61Crossref PubMed Scopus (26) Google Scholar), which is causally involved in diarrhea and enterotoxaemia in domestic animals and gas gangrene in man (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar, 9McGregor J.A. Soper D.E. Lowell G. Todd J.K. Am. J. Obstet. Gynecol. 1989; 161: 987-995Abstract Full Text PDF PubMed Scopus (90) Google Scholar). These clostridial toxins share common structural features. The C-terminal part of the single-chained toxins covers repetitive peptides that are most likely involved in cell receptor binding, followed by a small hydrophobic intermediate region which probably participates in the translocation of the toxins into the cytoplasm of the target cell (5Hofmann F. Herrmann A. Habermann E. Von Eichel-Streiber C. Mol. & Gen. Genet. 1995; 247: 670-679Crossref PubMed Scopus (32) Google Scholar, 10Eichel-Streiber C. Sauerborn M. Gene (Amst.). 1990; 96: 107-113Crossref PubMed Scopus (85) Google Scholar). The N-terminal part carries the biological activity (11Eichel-Streiber C. Sebald M. Genetics and Molecular Biology of Anaerobic Bacteria. Springer-Verlag, New York1993: 264Crossref Google Scholar). The common property of these intracellularly acting protein toxins is their cytotoxic activity, which leads to preferential destruction of the microfilament system of cell monolayers.Recently, C. difficile ToxA and ToxB have been identified as monoglucosyltransferases that selectively modify the low molecular mass GTP-binding proteins of the Rho subfamily (12Just I. Selzer J. Wilm M. Von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar, 13Just I. Wilm M. Selzer J. Rex G. Von Eichel-Streiber C. Mann M. Aktories K. J. Biol. Chem. 1995; 270: 13932-13936Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). The target proteins Rho, Rac, and Cdc42 are involved in the regulation of the actin cytoskeleton. Whereas Rho controls the formation of focal adhesions and stress fibers (14Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar), Rac participates in membrane ruffling (15Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3050) Google Scholar) and Cdc42 in formation of filopodia (16Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar, 17Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar). Furthermore, the Rho subfamily proteins have been identified as being involved in the activation of transcription factors via the Ras-regulated pathway (18Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar) and via a Ras-independent signal cascade (19Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 20Coso O.A. Chiariello M. Yu J.-C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 21Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar).ToxA/ToxB-catalyzed glucosylation, which occurs at Thr-37 in RhoA, causes functional inactivation leading to depolymerization of the actin filament system (12Just I. Selzer J. Wilm M. Von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar). Glucosylation of Rho in Thr-37 blocks subsequent ADP-ribosylation by Clostridium botulinum C3 exoenzyme in Asn-41 (22Just I. Fritz G. Aktories K. Giry M. Popoff M.R. Boquet P. Hegenbarth S. Von Eichel-Streiber C. J. Biol. Chem. 1994; 269: 10706-10712Abstract Full Text PDF PubMed Google Scholar, 23Just I. Selzer J. Von Eichel-Streiber C. Aktories K. J. Clin. Invest. 1995; 95: 1026-1031Crossref PubMed Scopus (98) Google Scholar).Here we report the identification of C. novyi α-toxin as a N-acetyl-glucosaminyltransferase that modifies the Rho subtype proteins). 2Preliminary data were presented at the 7th European Workshop Conference on Bacterial Protein Toxins in Hindsgavl, Denmark (July 2-7, 1995). INTRODUCTIONClostridium novyi type A strains have been identified as the causative organisms of gas gangrene infections of humans and animals (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar). Type A strains produce an exotoxin, termed α-toxin, that exhibits in vivo both lethal and edematizing activity (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar). In tissue culture, α-toxin is cytotoxic, causing cell shape changes that are accompanied by disruption of the microfilament cytoskeleton and by minor effects at the vimentin and tubulin system (2Bette P. Oksche A. Mauler F. Eichel-Streiber C. Popoff M.R. Habermann E. Toxicon. 1991; 29: 877-887Crossref PubMed Scopus (51) Google Scholar, 3Müller H. Von Eichel-Streiber C. Habermann E. Infect. Immun. 1992; 60: 3007-3010Crossref PubMed Google Scholar, 4Oksche A. Nakov R. Habermann E. Infect. Immun. 1992; 60: 3002-3006Crossref PubMed Google Scholar). Recently, α-toxin has been cloned and sequenced (5Hofmann F. Herrmann A. Habermann E. Von Eichel-Streiber C. Mol. & Gen. Genet. 1995; 247: 670-679Crossref PubMed Scopus (32) Google Scholar). As deduced from these data, α-toxin has a molecular mass of 250,166 Da and shows 48% homology with Clostridium difficile toxin A (ToxA) 1The abbreviations used are: ToxAC. difficile toxin AToxBC. difficile toxin BGalgalactoseGlcglucoseGlcNAcN-acetyl-galactosaminePAGEpolyacrylamide gel electrophoresisGTPγSguanosine 5ʹ-3-O-(thio)triphosphate. and toxin B (ToxB). ToxA and ToxB are the major virulence factors of pathogenic C. difficile strains and have been identified as the causative agents of the antibiotic-associated diarrhea and the fatal form, the pseudomembranous colitis (6Lyerly D.M. Krivan H.C. Wilkins T.D. Clin. Microbiol. Rev. 1988; 1: 1-18Crossref PubMed Scopus (431) Google Scholar, 7Kelly C.P. Pothoulakis C. LaMont J.T. N. Engl. J. Med. 1994; 330: 257-262Crossref PubMed Scopus (1041) Google Scholar). Furthermore, α-toxin shows 34% homology to the lethal toxin from Clostridium sordellii (5Hofmann F. Herrmann A. Habermann E. Von Eichel-Streiber C. Mol. & Gen. Genet. 1995; 247: 670-679Crossref PubMed Scopus (32) Google Scholar, 8Green G.A. Schué V. Monteil H. Gene (Amst.). 1995; 161: 57-61Crossref PubMed Scopus (26) Google Scholar), which is causally involved in diarrhea and enterotoxaemia in domestic animals and gas gangrene in man (1Hatheway C.L. Clin. Microbiol. Rev. 1990; 3: 66-98Crossref PubMed Google Scholar, 9McGregor J.A. Soper D.E. Lowell G. Todd J.K. Am. J. Obstet. Gynecol. 1989; 161: 987-995Abstract Full Text PDF PubMed Scopus (90) Google Scholar). These clostridial toxins share common structural features. The C-terminal part of the single-chained toxins covers repetitive peptides that are most likely involved in cell receptor binding, followed by a small hydrophobic intermediate region which probably participates in the translocation of the toxins into the cytoplasm of the target cell (5Hofmann F. Herrmann A. Habermann E. Von Eichel-Streiber C. Mol. & Gen. Genet. 1995; 247: 670-679Crossref PubMed Scopus (32) Google Scholar, 10Eichel-Streiber C. Sauerborn M. Gene (Amst.). 1990; 96: 107-113Crossref PubMed Scopus (85) Google Scholar). The N-terminal part carries the biological activity (11Eichel-Streiber C. Sebald M. Genetics and Molecular Biology of Anaerobic Bacteria. Springer-Verlag, New York1993: 264Crossref Google Scholar). The common property of these intracellularly acting protein toxins is their cytotoxic activity, which leads to preferential destruction of the microfilament system of cell monolayers.Recently, C. difficile ToxA and ToxB have been identified as monoglucosyltransferases that selectively modify the low molecular mass GTP-binding proteins of the Rho subfamily (12Just I. Selzer J. Wilm M. Von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar, 13Just I. Wilm M. Selzer J. Rex G. Von Eichel-Streiber C. Mann M. Aktories K. J. Biol. Chem. 1995; 270: 13932-13936Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). The target proteins Rho, Rac, and Cdc42 are involved in the regulation of the actin cytoskeleton. Whereas Rho controls the formation of focal adhesions and stress fibers (14Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3797) Google Scholar), Rac participates in membrane ruffling (15Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3050) Google Scholar) and Cdc42 in formation of filopodia (16Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar, 17Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar). Furthermore, the Rho subfamily proteins have been identified as being involved in the activation of transcription factors via the Ras-regulated pathway (18Minden A. Lin A. Claret F.-X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1444) Google Scholar) and via a Ras-independent signal cascade (19Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 20Coso O.A. Chiariello M. Yu J.-C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1559) Google Scholar, 21Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1055) Google Scholar).ToxA/ToxB-catalyzed glucosylation, which occurs at Thr-37 in RhoA, causes functional inactivation leading to depolymerization of the actin filament system (12Just I. Selzer J. Wilm M. Von Eichel-Streiber C. Mann M. Aktories K. Nature. 1995; 375: 500-503Crossref PubMed Scopus (872) Google Scholar). Glucosylation of Rho in Thr-37 blocks subsequent ADP-ribosylation by Clostridium botulinum C3 exoenzyme in Asn-41 (22Just I. Fritz G. Aktories K. Giry M. Popoff M.R. Boquet P. Hegenbarth S. Von Eichel-Streiber C. J. Biol. Chem. 1994; 269: 10706-10712Abstract Full Text PDF PubMed Google Scholar, 23Just I. Selzer J. Von Eichel-Streiber C. Aktories K. J. Clin. Invest. 1995; 95: 1026-1031Crossref PubMed Scopus (98) Google Scholar).Here we report the identification of C. novyi α-toxin as a N-acetyl-glucosaminyltransferase that modifies the Rho subtype proteins). 2Preliminary data were presented at the 7th European Workshop Conference on Bacterial Protein Toxins in Hindsgavl, Denmark (July 2-7, 1995).