A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers

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 affect 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 was 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 1 nS) conductance steps. Small- and medium-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 cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited 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.

Pneumolysin generates multiple conductance pores in the membrane of nucleated cells

“…However, combination of the data in this paper, with the cryo-electron microscopy shows that a prepore complex and multiple conductance channels coexist. Our data validate previous lipid bilayer experiments that observed that a range of conductance channels was seen with pneumolysin [3] and perfringolysin [2].…”

“…These toxins bind to cholesterol-containing membrane, oligomerise and generate large pores that disrupt the membrane integrity, leading to cell death [1]. Although these pores are formed in the membranes of all eukaryotic cells tested, their electrophysiological properties have been studied only with artificial membranes [2,3]. In this work, the patch-clamp technique was used in inside out and outside out configurations to study, for the first time, the pores of one of the CDCs (pneumolysin) across the membrane of a nucleated cell.…”

The Human‐Specific Action of Intermedilysin, a Homolog of Streptolysin O, Is Dictated by Domain 4 of the Protein

“…1, ILY has three substitutions in the undecapeptide region, as found for PLO (5) although the substitution sites differ. It is most likely that these substitutions in ILY are responsible for the lack of affinity to cholesterol, because the region is suggested to be important to cholesterol binding by loss-of-function experiments in ordinary CDCs (18,31). The hemolytic action and cholesterol reactivity of the undecapeptide region ILY mutants were investigated in the present study, and it was confirmed that the lack of affinity to cholesterol in ILY can be reversed by substitutions in the ILY undecapeptide region.…”

“…The undecapeptide region of ILY has three substitutions compared with the highly conserved undecapeptide sequence of common CDCs (ECTGLAWEWWR) which has been suggested to be the cholesterol-binding site in loss-of-function experiments (18,31). To investigate this, we prepared nine undecapeptide region mutants of ILY and SLO (Fig.…”

Exploring Unique Structures: Flexibility is a Significant Factor in Biological Activity

“…12) located near the C-terminus, and that the region is important for activity and CHL binding as determined in experiments using point mutants and chemical modifications. [81][82][83] The cell lysis of CDCs is suppressed by CHL addition to the reaction mixture. Recently, a humanspecific cytolysin, intermedilysin (ILY), which is secreted from a strain of S. intermedius originally isolated from a human liver abscess, was reported.…”