Oligomerization and structural changes of the pore-forming Pseudomonas aeruginosa cytotoxin

Pseudomonas aeruginosa produces a pathogenic factor, the 29-kDa port-formin g protein cytotoxin. Nonspecific oligomers of cytotoxin up to the hexamer, induced by oxidative crosslinking or detergent micellae, were based on inte rmolecular disulfide bridges. SDS induced tetramer, hexamer and mainly pent amers 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 res idues are indispensable for functional pentamerization. Pentamer formation was not influenced by the lipid composition of the liposomes tested, indica ting that rising membrane fluidity did not increase oligomerization. The se condary structure of cytotoxin determined by spectroscopy is characterized by approximately 50% beta-sheet, 20% beta-turn, 10% alpha-helix and 20% rem aining structure. Contact with detergent micellae or liposomes induced a re organization of beta-structure associations, as observed by attenuated tota l reflection-Fourier transform infrared spectroscopy. Electron microscopy a nd principle component analysis of the cytotoxin monomer demonstrated a tap ered molecule of 11 nm in length and a maximum width of 3.5 nm. These resul ts classify the cytotoxin as a pore-forming toxin, rich in antiparallel bet a-structure, that needs to oligomerize and inserts into membranes; it is ve ry similar to the Staphylococcus aureus alpha-toxin.