Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment
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ORIGINAL PAPER
EVects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment Busaba Powthongchin · Chanan Angsuthanasombat
Received: 24 September 2007 / Revised: 21 July 2008 / Accepted: 22 July 2008 / Published online: 20 August 2008 © Springer-Verlag 2008
Abstract The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis was employed to investigate the contributions to haemolysis of Wve predicted helices within the N-terminal hydrophobic region of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1500– 522, 2529–550, 3571–593 and 5678–698, respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in the predicted helix 4602–627 (A616P). MALDI–TOF–MS and LC–MS–MS analyses veriWed the palmitoylation at Lys983 of all Wve mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modiWcation via this acylation was not aVected by the mutations. Altogether, these results
Communicated by Axel Brakhage. B. Powthongchin · C. Angsuthanasombat (&) Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand e-mail: [email protected] B. Powthongchin Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakornpathom 73000, Thailand
suggest that structural integrity of the predicted helices 1, 2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and 3 that might conceivably be involved in pore formation of the CyaA-PF fragment. Keywords Adenylate cyclase-haemolysin · Bordetella pertussis · Haemolytic activity · Pore-forming toxin · Palmitoylation · Transmembrane helices Abbreviations AC Adenylate cyclase CyaA Adenylate cyclase-haemolysin toxin CyaA-PF CyaA pore-forming IPTG Isopropyl--D-thiogalactopyranoside PCR Polymerase chain reaction PMSF PhenylmethylsulfonylXuoride RTX Repeat-in-toxin SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
Introduction Among the known bacterial RTX (Repeat-in-ToXin) poreforming toxins, adenylate cyclase-haemolysin toxin (CyaA), a key virulence component of Bordetella pertussis (the causative agent of whooping cough in humans), is the only member that exhibits both cell-invasive adenylate cyclase and pore-forming activities (Carbonetti et al. 2005). CyaA is synthesi
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