Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin.
Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa pore-forming protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on intermolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pentamers that were resistant to reducing conditions, indicating an additional oligomerization mechanism. Functional oligomerization after incubation with different membranes resulted in an oligomer of approximately 145 kDa that was identified as the pentamer by comparison with the SDS-induced oligomers. Covalent modification with diethylpyrocarbonate showed that histidine residues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indicating that rising membrane fluidity did not increase oligomerization. The secondary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of beta-structure associations, as observed by attenuated total reflection-Fourier transform infrared spectroscopy. Electron microscopy and principle component analysis of the cytotoxin monomer demonstrated a tapered molecule of 11 nm in length and a maximum width of 3.5 nm. These results classify the cytotoxin as a pore-forming toxin, rich in antiparallel beta-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus alpha-toxin.
Sliwinski-Korell
A
A
Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universität Giessen, Germany.
Engelhardt
H
H
Kampka
M
M
Lutz
F
F
D000818:Animals; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004910:Erythrocyte Membrane; D007473:Ion Channels / Q000737:chemistry* / Q000648:ultrastructure; D007956:Leukocidins / Q000737:chemistry*; D008081:Liposomes; D008823:Micelles; D019814:Myristic Acid; D010713:Phosphatidylcholines; D020836:Protein Structure, Quaternary / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D051381:Rats; D017550:Spectroscopy, Fourier Transform Infrared
D007473:Ion Channels; D007956:Leukocidins; D008081:Liposomes; D008823:Micelles; D010713:Phosphatidylcholines; D019814:Myristic Acid; D004047:Diethyl Pyrocarbonate