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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/41955
Title: Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation
Authors: Kanungsuk Meetum
Chompounoot Imtong
Gerd Katzenmeier
Chanan Angsuthanasombat
Mahidol University
Prince of Songkla University
Biophysics Institute for Research and Development (BIRD)
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Mar-2017
Citation: Biochimica et Biophysica Acta - Biomembranes. Vol.1859, No.3 (2017), 312-318
Abstract: © 2016 Elsevier B.V. Previously, the ~ 130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was demonstrated to be palmitoylated at Lys983 and thus activated its hemolytic activity against target erythrocytes. Here, we report the functional importance of Lys983-palmitoylation for membrane insertion and pore formation of CyaA-Hly. Intrinsic fluorescence emissions of both non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaA-Hly were indistinguishable, suggesting no severe conformational change upon acylation at Lys983. Following pre-incubation of sheep erythrocytes with NA/CyaA-Hly, there was a drastic decrease in CyaA-Hly-induced hemolysis. Direct interactions between NA/CyaA-Hly and target erythrocyte membranes were validated via membrane-binding assays along with Western blotting, suggestive of acylation-independent capability of NA/CyaA-Hly to interact with erythrocyte membranes. As compared with CyaA-Hly, NA/CyaA-Hly displayed a slower rate of incorporation into DOPC:DOPE:Ch or DiPhyPC bilayers under symmetrical conditions (1 M KCl, 10 mM HEPES, pH 7.4) and formed channels exhibiting different conductance. Further analysis revealed that channel-open lifetime in DOPC:DOPE:Ch bilayers of NA/CyaA-Hly was much shorter than that of the acylated form, albeit slightly shorter lifetime found in DiPhyPC bilayers. Sequence alignments of the Lys983-containing CyaA-segment with those of related RTX-cytolysins revealed a number of highly conserved hydrophobic residues and a Lys/Arg cluster that is predicted be important for toxin-membrane interactions. Altogether, our data disclosed that the Lys983-linked palmitoyl group is not directly involved in either binding to target erythrocyte membranes or toxin-induced channel conductivity, but rather required for efficient membrane insertion and pore formation of the acylated CyaA-Hly domain.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85006942159&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/41955
ISSN: 18792642
00052736
Appears in Collections:Scopus 2016-2017

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