Contributions of the Hydrophobic Helix 2 of the Bordetella pertussis CyaA-hemolysin to Membrane Permeabilization.

BACKGROUND

Adenylate cyclase (CyaA) is one of the major virulence factors of Bordetella pertussis that plays a key role in whooping cough pathogenesis. A putative transmembrane helical hairpin (α2-loop-α3), encompassing residues 529-594 of CyaA hemolysin (CyaA-Hly) domain, was previously proposed to be crucially involved in hemolytic activity against target erythrocytes.

OBJECTIVE

The main objective of this study was to gain more insight into membrane permeabilization of this toxin. Membrane-permeabilizing abilities of the purified 130-kDa CyaA-Hly and synthetic peptides corresponding to the helical component of interest, were evaluated.

METHODS

Synthetic peptides corresponding to the critical helical component, i.e. α2 (W528-G550), α3 (G568-R594) and α2-loop-α3 (W528-R594), were examined on various membrane models in comparison with the purified 130-kDa CyaA-Hly. The peptides were commercially synthesized and the purified toxin was obtained from recombinant plasmid construction and expression in Escherichia coli, followed by purification via immobilized-metal affinity chromatography. Membrane permeabilization or hemolysis of the peptides or the purified toxin were determined by liposomal leakage, hemolysis assays and atomic force microscopy (AFM) imaging.

RESULTS

Our results showed that the truncated 130-kDa CyaA-Hly, the synthetic peptides α2, α3 and the α2-loop-α3 hairpin exhibited distinct membrane-permeabilizing capacities in different membrane models. We demonstrated that the CyaA-Hly toxin and the peptides, especially the α2 peptide, caused nonspecific liposomal leakage as monitored by fluorescence dequenching of sulforhodamine B-loaded lipid vesicles. Notably, α2 peptide showed a predominant effect of membrane permeabilization when compared to α2-loop-α3 hairpin and α3 peptides. In addition, AFM imaging demonstrates lipid membrane disruption induced by the CyaA-Hly toxin or the peptidic α2-loop-α3 hairpin.

CONCLUSION

Overall, the study provides the supporting evidence that the putative helical α2-loop-α3 hairpin could interact with the lipid membranes while the helical α2 peptide strongly induced liposomal leakage and hemolysis, as compared with the helical α3 or the α2-loop-α3 peptides, suggesting that the helix 2 from the hydrophobic region of CyaA-Hly is a crucial component that contributes to membrane permeabilization.