Functional significance of the highly conserved Glu 570 in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin

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 (Glu 570 , Gln 574 , Glu 581 , Ser 584 and Ser 585 ) 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 Glu 570 , 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. © 2011 Elsevier Ltd.