B-148. The Role of PFO D4 L1-3 Loops in Recognition of Cholesterol-Rich Membranes and Pore Formation

A. Farrand, R. Tweten;
Oklahoma City Hlth. Sci. Ctr., Oklahoma City, OK.

Perfringolysin O (PFO), a virulence factor produced by Clostridium perfringens, is known to affect the anti-inflammatory response during gas gangrene. PFO is a prototypic member of the cholesterol-dependent cytolysin (CDC) family of pore forming toxins. These multi-domain toxins require membrane cholesterol for pore formation. It has been thought for many years that the domain 4 (D4) undecapeptide was responsible for the interaction with cholesterol-rich membranes, but it was recently shown this interaction was dependent on the three D4 hydrophobic loops, not the undecapeptide. We therefore hypothesized that there were residues in these loops that contribute to this cholesterol-specific recognition. Alanine was substituted for each residue in the D4 loops and the resulting mutants were assayed for membrane binding by surface plasmon resonance, oligomer formation on cholesterol-rich liposomes by sodium dodecyl-sulfate agarose gel electrophoresis, and pore formation by hemolytic assay using erythrocytes. All mutants were able to bind to and oligomerize on cholesterol-rich liposomes, but varied significantly in their pore-forming activity on erythrocytes. Mutants in loop 1 had the most dramatic loss of activity, while those in loops 2 and 3 were less affected. These data suggest that individual mutants do not significantly disrupt binding to cholesterol-rich membranes, but that specific residues in D4 must interact with the membrane in order to facilitate the downstream conversion of the prepore to pore complex. A structural transition that follows binding and is necessary for prepore to pore conversion is membrane insertion of the undecapeptide. Using fluorescence spectroscopy to measure changes in the intrinsic tryptophan fluorescence it was determined that all mutants inserted their undecapeptide. These data suggest that membrane interaction of specific D4 residues affected subsequent conformational changes that follow membrane insertion of the undecapeptide and are necessary for the assembly and/or insertion of the D3 transmembrane β-barrel.