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% b-sheet, 20% b-turn, 10% a-helix and 20% remaining structure. Contact with detergent micellae or liposomes induced a reorganization of b-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 b-structure, that needs to oligomerize and inserts into membranes; it is very similar to the Staphylococcus aureus a-toxin.Keywords: attenuated total reflection±Fourier transform infrared spectroscopy; oligomerization; pore formation; protein secondary structure; Pseudomonas aeruginosa cytotoxin.Several strains of Pseudomonas aeruginosa produce the phageencoded 31.7-kDa procytotoxin, which is processed by removal of the C terminus during bacterial autolysis. The mature, watersoluble, acidic 29-kDa protein attacks plasma membranes of a great variety of eukaryotic cells resulting in a channel of < 1±2 nm in diameter [1] and forms ion channels in planar lipid bilayers [2]. The toxicity of cytotoxin on Ehrlich ascites tumour cells is increased by raising the incubation temperature up to 37 8C and lowering the medium pH from 8.0 to 5.1 [3]. Oligomer formation of cytotoxin after incubation with SDS was detected by Lutz et al. [4]. The process of cytotoxin oligomerization, initiated by incubation with erythrocyte membranes and liposomes at 37 8C and pH 6.0, was described by Ohnishi et al. [5]. Analysis of the cytotoxin primary structure [6,7] indicates predominant b-sheet and a very amphiphilic character of the toxin similar to other bacterial pore formers.In this work the different mechanisms and conditions of nonspecific or functional oligomerization of the pore-forming cytotoxin were examined and the subunit stoichiometry of the functional oligomer was determined more precisely. The secondary structure composition of cytotoxin was determined by attenuated total...

No abstract