Prangkio Panchika, Juntapremjit Sirikran, Koehler Melanie, Hinterdorfer Peter, Angsuthanasombat Chanan
Division of Biochemistry and Biochemical Technology, Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.
College of Research Methodology and Cognitive Science, Burapha University, Chonburi 20131, Thailand.
Protein Pept Lett. 2018;25(3):236-243. doi: 10.2174/0929866525666171201120456.
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.
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.
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.
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.
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.
腺苷酸环化酶（CyaA）是百日咳博德特氏菌的主要毒力因子之一，在百日咳发病机制中起关键作用。先前有人提出，一个假定的跨膜螺旋发夹结构（α2-环-α3），包含CyaA溶血素（CyaA-Hly）结构域的529 - 594位残基，在针对靶红细胞的溶血活性中起关键作用。
本研究的主要目的是更深入地了解这种毒素的膜通透作用。评估了纯化的130 kDa CyaA-Hly和与感兴趣的螺旋成分对应的合成肽的膜通透能力。
将与关键螺旋成分相对应的合成肽，即α2（W528 - G550）、α3（G568 - R594）和α2-环-α3（W528 - R594），与纯化的130 kDa CyaA-Hly在各种膜模型上进行比较研究。这些肽通过商业合成获得，纯化的毒素通过重组质粒构建并在大肠杆菌中表达，然后通过固定金属亲和层析进行纯化。通过脂质体泄漏、溶血测定和原子力显微镜（AFM）成像来确定肽或纯化毒素的膜通透或溶血情况。
我们的结果表明，截短的130 kDa CyaA-Hly、合成肽α2、α3和α2-环-α3发夹在不同的膜模型中表现出不同的膜通透能力。我们证明，CyaA-Hly毒素和这些肽，特别是α2肽，会导致非特异性脂质体泄漏，这通过负载磺基罗丹明B的脂质囊泡的荧光猝灭来监测。值得注意的是，与α2-环-α3发夹和α3肽相比，α2肽在膜通透方面表现出主要作用。此外，AFM成像显示了CyaA-Hly毒素或肽性α2-环-α3发夹诱导的脂质膜破坏。
总体而言，该研究提供了支持性证据，表明假定的螺旋α2-环-α3发夹可与脂质膜相互作用，而螺旋α2肽与螺旋α3或α2-环-α3肽相比，强烈诱导脂质体泄漏和溶血，这表明来自CyaA-Hly疏水区域的螺旋2是促成膜通透的关键成分。
