Effects on haemolytic activity of single proline substitutions in the Bordetella pertussis CyaA pore-forming fragment
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 co...
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| Format: | Article |
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2018
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| Online Access: | https://repository.li.mahidol.ac.th/handle/123456789/27312 |
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| Institution: | Mahidol University |
| Summary: | 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, 3571-593and 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 Lys983of 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. © 2008 Springer-Verlag. |
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