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

Sabine Löhner^*, Iwan Walev^*, Fatima Boukhallouk^*, Michael Palmer, Sucharit Bhakdi^*,1 and Angela Valeva^*

^* Institute of Medical Microbiology and Hygiene, University of Mainz, Mainz, Germany; and

Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada

¹ Correspondence: Institute of Medical Microbiology and Hygiene, University of Mainz, Augustusplatz, D55131 Mainz, Germany. E-mail: sbhakdi{at}uni-mainz.de

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 monomers in these hybrid oligomers was still observed, but the functional pore diameter was reduced. These findings indicate that formation of an oligomeric prepore precedes membrane insertion of the pore-forming amino acid sequence and demonstrate that pore formation by VCC follows the same archetypical pathway as β-barrel cytolysins of gram-positive organisms such as staphylococcal -toxin.—Löhner, S., Walev, I., Boukhallouk, F., Palmer, M., Bhakdi, S., Valeva, A. Pore formation by Vibrio cholerae cytolysin follows the same archetypical mode as β-barrel toxins from gram-positive organisms.

Key Words: prepore • assembly intermediate • membrane perforation • microbial pathogenicity