Pore formation by Vibrio cholerae cytolysin follows the same archetypical mode as β-barrel toxins from gram-positive organisms

Vibrio cholerae cytolysin (VCC) forms SDS-stable heptameric β-barrel transmembrane pores in mammalian cell membranes. In contrast to structurally related pore formers of gram-positive organisms, no oligomeric prepore stage of assembly has been detected to date. In the present study, disulfide bonds were engineered to tie the pore-forming amino acid sequence to adjacent domains. In their nonreduced form, mutants were able to bind to rabbit erythrocytes and to native erythrocyte membranes suspended in PBS solution and form SDS-labile oligomers. These remained nonfunctional and represented the long-sought VCC prepores. Disulfide bond reduction in these oligomers released the pore-forming sequence from its locked position, and subsequent membrane insertion led to formation of SDS-stable pores and hemolysis. Addition of increasing amounts of an inactive mutant to wild-type toxin resulted in the formation of mixed oligomers with progressively reduced SDS stability on membranes. Membrane insertion of active mono...