Nature © Macmillan Publishers Ltd 1997 Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1 Gudula Schmidt*, Peter Sehr*, Matthias Wilm†, Jo¨ rgSelzer*, Matthias Mann† & Klaus Aktories* * Institut fu ¨r Pharmakologie und Toxikologie der Albert-Ludwigs-Universita ¨t Freiburg, Hermann-Herder-Strasse 5, 79104 Freiburg, Germany † EMBL, 69012 Heidelberg, Germany ......................................................................................................................... The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases 1,2 , which act as molecular switches in diverse signal-transduction processes 3 . Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation 1 or glucosylation 4 . Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necrotizing factors 1 and 2 (CNF1 and 2) 5,6 . These toxins induce actin assembly and multinucleation in culture cells. Here we show that treatment of RhoA with CNF1 inhibits the intrinsic GTPase activity of RhoA and completely blocks GTPase activity stimulated by the Rho-GTPase-activating protein (rhoGAP). Analysis by mass spectrometry and amino-acid sequencing of proteolytic peptides derived from CNF1-treated RhoA indicate that CNF1 induces deamidation of a glutamine residue at position 63 (Gln 63) to give constitutively active Rho protein. The cytotoxic necrotizing factors CNF1 and CNF2 (each of M r 115K) are produced by up to half of the different E. coli strains isolated from extra-intestinal infections, and by up to a fifth of E. coli strains from diarrhoea 7,8 . These toxins cause tissue damage and death of the animal host 8 . CNFs induce actin polymerization and increase the F-actin content of cells 6 . They inhibit cytokinesis, cause formation of multinucleated cells, and induce membrane ruffling 9 . Because treatment of intact cells with CNFs changes the migration of Rho on SDS–PAGE, it has been suggested that CNFs act on Rho 5,6 . We expressed and purified CNF1 as a fusion protein with glutathione S-transferase (GST) and tested the activity of the recombinant toxin in NIH3T3 cells. GST–CNF1 (at 300 ng ml - 1 ) induced multinucleation of 90% of cells after 24 h of treatment. Moreover, staining of the actin cytoskeleton with rhodamine– phalloidin revealed a dense network of actin fibres resembling that induced by CNF1 (Fig. 1a–d). Although major morphological changes in cells (multinuclea- tion) were not observed earlier than 12–16 h after intoxication, treatment of cells with GST–CNF1 at 300 ng ml - 1 for only 2–3 h altered the migration of Rho on SDS–PAGE. As shown in Fig. 2a, GST–CNF1 caused a shift in the apparent molecular mass of Rho protein labelled by C3-catalysed [ 32 P]ADP-ribosylation, indicating that the GTPase has been covalently modified. This change in migration induced by GST–CNF1 was also observed after 14 C- glucosylation of Rho by Clostridium difficile toxin B (not shown). Treatment of recombinant RhoA with GST–CNF1 (molar ratio 16 :1) for 3 h at 37 °C caused a similar shift in the apparent molecular mass of [ 32 P]ADP-ribosylated RhoA on SDS–PAGE, as letters to nature NATURE | VOL 387 | 12 JUNE 1997 725 Figure 1 Induction of multinucleation and increase in F-actin by CNF1. NIH3T3 cells were treated without (a, c) and with CNF1 (300 ng ml -1 ; b, d) for 16 h. Cells were then analysed by phase-contrast microscopy (a, b) or the actin cytoskeleton was stained with rhodamine-labelled phalloidin for fluorescence microscopy (c, d).