Functional significance of the highly conserved Glu570 in the putative pore-forming helix 3 of the Bordetella pertussis haemolysin toxin

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.

► Glu570 of B. pertussis CyaA is highly conserved in the RTX cytotoxin family. ► Glu570 in the proposed lumen-lining helix 3 is critical for toxin haemolysis. ► Glu570 could serve as a general pore-lining residue involved in ion conduction.