Functional significance of the highly conserved Glu in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin |
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| Authors: | Chattip Kurehong Busaba Powthongchin Niramon Thamwiriyasati Chanan Angsuthanasombat |
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| Institution: | a Laboratory of Molecular Biophysics and Structural Biochemistry, Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand;b Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakornpathom 73000, Thailand;c Department of Medical Technology, Faculty of Allied Health Sciences, Burapha University, Chonburi 20131, Thailand |
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| Abstract: | Adenylate cyclase-haemolysin toxin (CyaA) is a virulence factor secreted from the etiologic agent of whooping cough, Bordetella pertussis. Previously, the haemolysin or pore-forming domain (CyaA-PF) has been shown to cause cell lysis of sheep erythrocytes independently, and the predicted helix 3(570?593) within the PF-hydrophobic stretch could be a pore-lining constituent. Here, a plausible involvement in haemolytic activity of polar or charged residues (Glu570, Gln574, Glu581, Ser584 and Ser585) lining the hydrophilic side of CyaA-PF helix 3 was investigated via single-alanine substitutions. All the 126-kDa mutant proteins over-expressed in Escherichia coli were verified for toxin acylation as the results are corresponding to the wild-type toxin. When haemolytic activity of E. coli lysates containing soluble mutant proteins was tested against sheep erythrocytes, the importance of Glu570, which is highly conserved among the pore-forming RTX cytotoxin family, was revealed for pore formation, conceivably for a general pore-lining residue involved in ion conduction. |
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| Keywords: | Adenylate cyclase-haemolysin Bordetella pertussis Haemolytic activity Pore-forming toxin Transmembrane helix |
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