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

Abstract

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.

FIG. 1

Effect of CNF2 on the electrophoretic mobility of GST-RhoA. GST-RhoA was coexpressed with (lane 2) or without (lane 1) CNF2 in E. coli. GST fusion protein was purified from the E. coli homogenate by using glutathione-Sepharose beads according to the manufacturer's instructions. Proteins were analyzed by SDS-PAGE in a 10% gel and by subsequent staining with Coomassie brilliant blue. The molecular masses of the standard are indicated by arrows.

FIG. 2

Effect of the CNF2-induced modification on GTPase activity of Rho. (A) Effect of CNF2-induced modification on GTPase activity of GST-RhoA. GST-RhoA coexpressed with (▴) or without (●) CNF2 in E. coli was purified and assayed for GTPase activity as described in Materials and Methods. (B) Effect of CNF2-induced modification on GTPase activity of FLAG-RhoA Q63 and FLAG-RhoA E63. FLAG-RhoA Q63 (wild type) coexpressed with (▴) or without (●) CNF2 and FLAG-RhoA E63 coexpressed with (▵) or without (○) CNF2 in E. coli were purified and assayed for GTPase activity as described in Materials and Methods. The results shown are representative of three independent experiments.

FIG. 3

SDS-PAGE and Western blotting analyses of FLAG-RhoA. FLAG-RhoA Q63 (wild type) coexpressed with (lane 2) or without (lane 1) CNF2 and FLAG-RhoA E63 coexpressed with (lane 4) or without (lane 3) CNF2 in E. coli were purified as described in Materials and Methods. Purified proteins were analyzed by SDS-PAGE with Coomassie brilliant blue staining (A) or by Western blotting with anti-E63 antibody (dilution, 1,000-fold) (B).

FIG. 4

In vitro modification of FLAG-RhoA by CNF2. Results of Western blotting analysis of FLAG-RhoA with anti-E63 antibody are shown. Purified FLAG-RhoA was coincubated with nothing (control), purified His-tag–CNF2 (C term.), His-tag–CNF2 (N term.), or His-tag–CNF2 (full) at a molar ratio of 2:3 in PBS at 37°C for 30 min. The treated samples were subjected to SDS-PAGE and probed with anti-E63 antibody (dilution, 1,000-fold).

FIG. 5

SDS-PAGE and Western blotting analyses of GST-fused small GTPases. GST-RhoA (RhoA), GST-Rac1 (Rac1), and GST-Cdc42 (Cdc42) coexpressed without (control) or with CNF1 or CNF2 in E. coli were purified. Purified proteins were analyzed by SDS-PAGE with Coomassie brilliant blue staining (lower panel) or by Western blotting with anti-E63 antibody (dilution, 1,000-fold) (upper panel).

FIG. 6

Western blotting analysis of CNF2-treated Swiss 3T3 cell homogenate with anti-E63 antibody. Homogenates (10 μg) of Swiss 3T3 cells treated with (lane 2) or without (lane 1) CNF2 (50 ng/ml) for 30 min were subjected to SDS-PAGE and probed with anti-E63 antibody (dilution, 1,000-fold) as the secondary antibody.

FIG. 7

Effect of CNF2 on accumulation of the GTP-bound form of Rho subfamily GTPases in Cos-7 cells. FLAG-RhoA (●), FLAG-RhoA E63 (▾), FLAG-Rac1 (▴), and FLAG-Cdc42 (■) were transiently expressed in Cos-7 cells. The cells were then treated with CNF2 at the indicated concentrations for 12 h and labeled with ³²P_i. The cell lysates were subjected to immunoprecipitation with anti-FLAG monoclonal antibody. Immunoprecipitates were developed by TLC, and the radioactivity was analyzed with a BAS2000. The ratio of GTP to small GTPase-bound guanine nucleotides was indicated as a percentage.