Membrane-Pore Forming Characteristics of the Bordetella pertussis CyaA-Hemolysin Domain.
Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly) domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs) under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4), it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes.
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. g5036578@student.mahidol.ac.th.
Chattip
C
Kurehong
Laboratory of Theoretical and Computational Biophysics, Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. fscicpk@ku.ac.th.
Chalermpol
C
Kanchanawarin
Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakornpathom 73000, Thailand. p.busaba.su@gmail.com.
Busaba
B
Powthongchin
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. katzenmeier.ger@mahidol.ac.th.
Gerd
G
Katzenmeier
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. chanan.ang@mahidol.ac.th.|Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand. chanan.ang@mahidol.ac.th.
Chanan
C
Angsuthanasombat
D037361:Adenylate Cyclase Toxin / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D001886:Bordetella pertussis; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000502:physiology; D004912:Erythrocytes / Q000187:drug effects / Q000502:physiology*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000494:pharmacology; D006461:Hemolysis / Q000187:drug effects; D007473:Ion Channels / Q000502:physiology; D008051:Lipid Bilayers; D016062:Porosity; D017434:Protein Structure, Tertiary; D012756:Sheep
D037361:Adenylate Cyclase Toxin; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers