FIG. 1.

Cytotoxicity associated with ExoU proteins containing amino acid substitutions within the MLD. MLD variants were expressed in HeLa cells by transient transfection, and LDH release into the medium was measured 20 h later. Results are presented as cytotoxicity relative to that of wild-type ExoU. For proteins with multiple substitutions, the cumulative cytotoxicity is depicted for each substituted residue. If substantial loss of cytotoxicity occurred and this defect could not be attributed to a single-amino-acid substitution, site-directed mutagenesis was employed to define which residue was critical for cytotoxicity, and the level of cytotoxicity associated with the single-amino-acid substitution is shown. All experiments were performed in triplicate. Data represents mean values. For clarity, standard deviations are not shown, but they were usually within ±10%.