Cytotoxic necrotizing factor type 2 produced by pathogenic Escherichia coli deamidates a gln residue in the conserved G-3 domain of the rho family and preferentially inhibits the GTPase activity of RhoA and rac1.
Cytotoxic necrotizing factor types 1 and 2 (CNF1 and -2) produced by pathogenic Escherichia coli strains have 90% conserved residues over 1,014-amino-acid sequences. Both CNFs are able to provoke a remarkable increase in F-actin structures in cultured cells and covalently modify the RhoA small GTPases. In this study, we demonstrated that CNF2 reduced RhoA GTPase activity in the presence and absence of P122(RhoGAP). Subsequently, peptide mapping and amino acid sequencing of CNF2-modified FLAG-RhoA produced in E. coli revealed that CNF2 deamidates Q63 of RhoA-like CNF1. In vitro incubation of the C-terminal domain of CNF2 with FLAG-RhoA resulted also in deamidation of the FLAG-RhoA, suggesting that this region contains the enzymatic domain of CNF2. An oligopeptide antibody (anti-E63) which specifically recognized the altered G-3 domain of the Rho family reacted with glutathione S-transferase (GST)-RhoA and GST-Rac1 but not with GST-Cdc42 when coexpressed with CNF2. In addition, CNF2 selectively induced accumulation of GTP form of FLAG-RhoA and FLAG-Rac1 but not of FLAG-Cdc42 in Cos-7 cells. Taken together, these results indicate that CNF2 preferentially deamidates RhoA Q63 and Rac1 Q61 and constitutively activates these small GTPases in cultured cells. In contrast, anti-E63 reacted with GST-RhoA and GST-Cdc42 but not with GST-Rac1 when coexpressed with CNF1. These results indicate that CNF2 and CNF1 share the same catalytic activity but have distinct substrate specificities, which may reflect their differences in toxic activity in vivo.
Sugai
M
M
Department of Microbiology, Hiroshima University School of Dentistry, Hiroshima 734-8553, Japan. sugai@ipc.hiroshima-u.ac.jp
Hatazaki
K
K
Mogami
A
A
Ohta
H
H
Pérès
S Y
SY
Hérault
F
F
Horiguchi
Y
Y
Masuda
M
M
Ueno
Y
Y
Komatsuzawa
H
H
Suginaka
H
H
Oswald
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D019556:COS Cells; D003603:Cytotoxins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*
D001427:Bacterial Toxins; D003603:Cytotoxins; D029968:Escherichia coli Proteins; D020690:GTPase-Activating Proteins; D011994:Recombinant Proteins; C086995:cytotoxic necrotizing factor type 2; C085379:rho GTPase-activating protein; C060296:cytotoxic necrotizing factor type 1; D020558:GTP Phosphohydrolases; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein