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Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment

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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 five 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 verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation was not affected by the mutations. Altogether, these results 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.

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Abbreviations

AC:

Adenylate cyclase

CyaA:

Adenylate cyclase-haemolysin toxin

CyaA-PF:

CyaA pore-forming

IPTG:

Isopropyl-β-d-thiogalactopyranoside

PCR:

Polymerase chain reaction

PMSF:

Phenylmethylsulfonylfluoride

RTX:

Repeat-in-toxin

SDS-PAGE:

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

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Acknowledgments

We thank the Proteomic service center, Bio Service Unit, BIOTEC, Thailand for MALDI–TOF–MS and LC–MS–MS analyses. B.P. was financially supported in part by Faculty of Graduate Studies, Mahidol University and Faculty of Pharmacy, Silpakorn University. This work was funded by the Thailand Research Fund in cooperation with the Commission of Higher Education (to C.A.).

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Authors and Affiliations

  1. Laboratory of Molecular Biophysics and Structural Biochemistry, Institute of Molecular Biology and Genetics, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand

    Busaba Powthongchin & Chanan Angsuthanasombat

  2. Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakornpathom, 73000, Thailand

    Busaba Powthongchin

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  1. Busaba Powthongchin
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  2. Chanan Angsuthanasombat
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Correspondence to Chanan Angsuthanasombat.

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Communicated by Axel Brakhage.

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Powthongchin, B., Angsuthanasombat, C. Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment. Arch Microbiol 191, 1–9 (2009). https://doi.org/10.1007/s00203-008-0421-3

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