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) delivers its catalytic activity into the cytosol of eukaryotic cells by a low pH membrane translocation mechanism reminiscent of that used by diphtheria toxin (DT). As DT, CNF1 exhibits a translocation domain (T) containing two predicted hydrophobic helices (H1-2) (aa 350-412) separated by a short peptidic loop (CNF1-TL) (aa 373-386) with acidic residues. In the DT loop, the loss 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 cytosol. 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. Single 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 double substitutions did not modify the cell-binding, enzymatic or endocytic activities of the mutant toxins. Unlike the wild-type toxin, single- or double-substituted CNF1 molecules bound to the HEp-2 plasma membrane could not translocate their enzymatic activity directly into the cytosol following a low pH pulse.
Pei
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INSERM U452, Faculté de Médecine, 28 Avenue de Valombrose, 06107 Nice, France.
Doye
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Boquet
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D019943:Amino Acid Substitution; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001693:Biological Transport, Active; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003603:Cytotoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004705:Endocytosis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D003603:Cytotoxins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C060296:cytotoxic necrotizing factor type 1