Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affact cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined. Trp-433-Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-Phe zas least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1nS) conductance steps. Small and nedium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-Phe formed large channels that were insensitive to caiton-induced closure. Poly-saccharides of molecular mass more tham 15 kDa ingibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-Phe. Electron microscopy revealed that Trp-433-Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-Phe variant. We conclude that the change Trp-433-Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels
A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin and planar lipid bilayers
Pederzolli, Cecilia;
1998-01-01
Abstract
Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affact cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined. Trp-433-Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-Phe zas least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1nS) conductance steps. Small and nedium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-Phe formed large channels that were insensitive to caiton-induced closure. Poly-saccharides of molecular mass more tham 15 kDa ingibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-Phe. Electron microscopy revealed that Trp-433-Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-Phe variant. We conclude that the change Trp-433-Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channelsI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
