Chapter 16 : Glucosylating and Deamidating Bacterial Protein Toxins

Scheme of the primary structure of C. difficile toxin B, the prototype of large clostridial cytotoxins. Toxin B consists of three domains: (i) N-terminal enzyme domain harboring glucosyltransferase activity; (ii) small hydrophobic domain in the middle of the toxin, which is most likely involved in toxin translocation; and (iii) C-terminal part consisting of polypeptide repeats suggested to be involved in receptor binding. The minimal length of the glucosyltransferase enzyme domain includes the 546 N-terminal amino acids. The conserved DXD motif is essential for transferase activity and likely to be involved in divalent cation and/or sugar nucleotide binding. The conserved Trp-102 is also important for nucleotide binding.

Cytotoxic effects of C. difficile toxin B on NIH3T3 fibroblasts.

Rho GTPases are modified by various bacterial protein toxins. Large clostridial cytotoxins glucosylate Rho subfamily proteins at Thr-37 (RhoA) or at the equivalent Thr-35 (Rac, Cdc42). C3 transferases, including Clostridium botulinum C3 exoenzyme, ADP-ribosylate RhoA at Asn-41. CNFs from Escherichia coli and the DNT from Bordetella sp. deamidate and transglutaminate, respectively, Gln-63 of RhoA and Gln-61 of Rac and Cdc42 (2).

Toxins A and B from C. difficile modify GDP-bound RhoA. In GTPbound RhoA, the hydroxyl group of Thr-37 coordinates a divalent cation directed into the protein and is not available for glucosylation by the toxins. In GDP-bound RhoA, the hydroxyl group of Thr-37 is directed to the solvent and is accessible for glucosylation by the toxins.

Functional consequences of the glucosylation of Rho GTPases. Inhibition of the interaction of glucosylated Rho with effectors is the most important effect of glucosylation. In addition, glucosylation inhibits Rho activation by guanine nucleotide exchange factors (GEF), the Rho cycling because glucosylated Rho locates at the membrane and does not bind to the guanine nucleotide dissociation inhibitor (GDI), and the stimulation of the GTP hydrolase activity by GTPase-activating proteins (GAP).

Structural comparison of CNF1 from E. coli with the DNT from Bordetella species. Alignment analysis identified a short region of high sequence identity at the C terminus. Indicated are the catalytical amino acids cysteine (C; Cys-1292 of DNT and Cys-866 of CNF1) and histidine (H, His-1307 of DNT and His-881 of CNF1). The minimal domain, possessing enzyme activity, is given as ΔCNF and ΔDNT, respectively.

Cytotoxic effects of CNF1 on fibroblasts. CNF1 increases formation of lamellipodia, filopodia, and stress fibers.

CNF treatment of RhoA induces a change in the migration of the GTPase on SDS-PAGE to an apparent larger molecular size. Deamidation of RhoA by CNF results in an increase in size of 1 Da. This can be analyzed by MALDI-TOF mass spectrometry. RhoA modified by CNF1 is digested by trypsin and subsequently the peptides formed are analyzed by mass spectrometry. One peptide, corresponding to amino acids 52 to 68, shows an increase in size of 1 Da, caused by deamidation of Gln-63.

Deamidation or transglutamination of RhoA at Glu-63 by CNF and DNT, respectively, blocks the GTP hydrolysis stimulated by GAP. Rho-Q63E is constitutively active and induces activation of effectors, which eventually cause formation of stress fibers and a large array of other effects.