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

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

Volume: 25 Issue: 3

Author(s): Panchika Prangkio*, Sirikran Juntapremjit, Melanie Koehler, Peter Hinterdorfer and Chanan Angsuthanasombat

Affiliation:

• Division of Biochemistry and Biochemical Technology, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200,Thailand

Keywords: Adenylate cyclase toxin, Bordetella pertussis, hemolysis, lipid bilayers, membrane permeabilization, synthetic peptides.

Abstract: 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.

Cite this article as:

Prangkio Panchika *, Juntapremjit Sirikran, Koehler Melanie, Hinterdorfer Peter and Angsuthanasombat Chanan, Contributions of the Hydrophobic Helix 2 of the Bordetella pertussis CyaA-hemolysin to Membrane Permeabilization, Protein & Peptide Letters 2018; 25 (3) . https://dx.doi.org/10.2174/0929866525666171201120456