In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity.
The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166-183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.
Aili
Margareta
M
Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden.
Telepnev
Maxim
M
Hallberg
Bengt
B
Wolf-Watz
Hans
H
Rosqvist
Roland
R
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000633:toxicity*; D001427:Bacterial Toxins / Q000633:toxicity*; D003601:Cytotoxicity Tests, Immunologic; D003603:Cytotoxins / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000032:analysis; D016415:Sequence Alignment; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D020690:GTPase-Activating Proteins; D011994:Recombinant Proteins; C063239:yopE protein, Yersinia; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein