Mutation of specific acidic residues of the CNF1 T domain into lysine alters cell membrane translocation of the toxin

The Rho-GTPases-activating toxin CNF1 (cytotoxic necrotizing factor 1) deli vers its catalytic activity into the cytosol of eukaryotic cells by a low p H membrane translocation mechanism reminiscent of that used by diphtheria t oxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two p redicted hydrophobic helices (H1-2) (aa 350-412) separated by a short pepti dic loop (CNF1-TL) (aa 373-386) with acidic residues. In the DT loop, the l oss of charge of acidic amino acids, as a result of protonation at low pH, is a critical step in the transfer of the DT catalytic activity into the cy tosol. To determine whether the CNF1 T domain operates similarly to the DT T domain, we mutated several ionizable amino acids of CNF1-TL to lysine. Si ngle substitutions such as D373K or D379K strongly decreased the cytotoxic effect of CNF1 on HEp-2 cells, whereas the double substitution D373K/D379K induced a nearly complete loss of cytotoxic activity. These single or doubl e substitutions did not modify the cell-binding, enzymatic or endocytic act ivities of the mutant toxins. Unlike the wild-type toxin, single- or double -substituted CNF1 molecules bound to the HEp-2 plasma membrane could not tr anslocate their enzymatic activity directly into the cytosol following a lo w pH pulse.