Identification and characterization of the Pasteurella multocida toxin translocation domain.
The Pasteurella multocida toxin (PMT) is a potent mitogen which enters the cytosol of eukaryotic cells via a low pH membrane translocation event. In common with the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), the core of the PMT translocation domain is composed of two predicted hydrophobic helices (H1 - residues 402-423, H2 - 437-457) linked by a hydrophilic loop (PMT-TL - 424-436). The peptide loop contains three acidic residues (D425, D431 and E434), which may play a role equivalent to D373, D379 and E382/383 in CNF1. To test this hypothesis, a series of point mutants was generated in which acidic residues were mutated into the permanently charged positive residue lysine. Individual mutation of D425, D431 and E434 each caused a four- to sixfold reduction in toxin activity. Interestingly, mutation of D401 located immediately outside the predicted helix-loop-helix motif completely abolished toxin activity. Individual mutations did not affect cell binding nor greatly altered toxin structure, but did prevent translocation of the surface-bound proteins into the cytosol after a low pH pulse. Moreover, we demonstrate using an in vitro assay that PMT undergoes a pH-dependent membrane insertion.
Microbiology, Dental Institute, King's College London, Floor 28, Guy's Tower, Guy's Hospital, London SE1 9RT, UK.
Michael R
MR
Baldwin
Jeremy H
JH
Lakey
Alistair J
AJ
Lax
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016979:Pasteurella multocida / Q000472:pathogenicity; D017354:Point Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D041701:Swiss 3T3 Cells
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018942:Macrolides; C062258:Pasteurella multocida toxin; C040929:bafilomycin A1