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. 2015 Apr 30;7(5):1486-96.
doi: 10.3390/toxins7051486.

Membrane-Pore Forming Characteristics of the Bordetella Pertussis CyaA-Hemolysin Domain

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Membrane-Pore Forming Characteristics of the Bordetella Pertussis CyaA-Hemolysin Domain

Chattip Kurehong et al. Toxins (Basel). .
Free PMC article

Abstract

Previously, the 126-kDa Bordetella pertussis CyaA pore-forming/hemolysin (CyaA-Hly) domain was shown to retain its hemolytic activity causing lysis of susceptible erythrocytes. Here, we have succeeded in producing, at large quantity and high purity, the His-tagged CyaA-Hly domain over-expressed in Escherichia coli as a soluble hemolytically-active form. Quantitative assays of hemolysis against sheep erythrocytes revealed that the purified CyaA-Hly domain could function cooperatively by forming an oligomeric pore in the target cell membrane with a Hill coefficient of ~3. When the CyaA-Hly toxin was incorporated into planar lipid bilayers (PLBs) under symmetrical conditions at 1.0 M KCl, 10 mM HEPES buffer (pH 7.4), it produced a clearly resolved single channel with a maximum conductance of ~35 pS. PLB results also revealed that the CyaA-Hly induced channel was unidirectional and opened more frequently at higher negative membrane potentials. Altogether, our results first provide more insights into pore-forming characteristics of the CyaA-Hly domain as being the major pore-forming determinant of which the ability to induce such ion channels in receptor-free membranes could account for its cooperative hemolytic action on the target erythrocytes.

Figures

Figure 1
Figure 1
(a) (Above) Schematic diagram of CyaA showing adenylate cyclase (AC) and hemolysin (Hly) domains. Five putative helices, α1–α5, in the hydrophobic region (HP, residues 500–700) are represented by gray blocks. The palmitoylation site is indicated by Lys983, whereas the repeat in toxin (RTX) region (residues 1006–1612) is represented by multiple lines, with each line corresponding to a single nonapeptide repeat (X-U-X-Gly-Gly-X-Gly-X-Asp). (Below) Diagram of 6×His-tagged CyaA-Hly showing residues 482–1706 with the joining 6×His residues of 1707–1712; (b) SDS-PAGE (12% gel) stained with Coomassie Brilliant Blue of lysates extracted from E. coli (~106 cells) harboring: lane 1, pCyaAC-PF with IPTG induction; lanes 2 and 3, pCyaAC-PF/H6 without and with IPTG induction, respectively. Lane 4 represents the Ni-NTA purified 6×His-tagged CyaA-Hly toxin. Protein bands of CyaA-Hly (~126 kDa) and its activator CyaC-acyltransferase (~21-kDa) are arrowed; (c) Western blot analyses of the corresponding gels from (b) incubated with anti-RTX (left panel) or anti-6×His epitope tag (right panel) antibodies, showing the reacted bands of 126-kDa CyaA-Hly as arrowed.
Figure 1
Figure 1
(a) (Above) Schematic diagram of CyaA showing adenylate cyclase (AC) and hemolysin (Hly) domains. Five putative helices, α1–α5, in the hydrophobic region (HP, residues 500–700) are represented by gray blocks. The palmitoylation site is indicated by Lys983, whereas the repeat in toxin (RTX) region (residues 1006–1612) is represented by multiple lines, with each line corresponding to a single nonapeptide repeat (X-U-X-Gly-Gly-X-Gly-X-Asp). (Below) Diagram of 6×His-tagged CyaA-Hly showing residues 482–1706 with the joining 6×His residues of 1707–1712; (b) SDS-PAGE (12% gel) stained with Coomassie Brilliant Blue of lysates extracted from E. coli (~106 cells) harboring: lane 1, pCyaAC-PF with IPTG induction; lanes 2 and 3, pCyaAC-PF/H6 without and with IPTG induction, respectively. Lane 4 represents the Ni-NTA purified 6×His-tagged CyaA-Hly toxin. Protein bands of CyaA-Hly (~126 kDa) and its activator CyaC-acyltransferase (~21-kDa) are arrowed; (c) Western blot analyses of the corresponding gels from (b) incubated with anti-RTX (left panel) or anti-6×His epitope tag (right panel) antibodies, showing the reacted bands of 126-kDa CyaA-Hly as arrowed.
Figure 2
Figure 2
Time-course analysis of hemolytic activity of the purified His-tagged CyaA-Hly toxin (10 μg/mL) against sheep erythrocytes (5 × 108 cells/mL). Inset, the plot of fractional hemolytic activity at 2 h, Y (PTM/1 − PTM), versus toxin concentration, [T]. Error bars indicate SEM from two independent experiments where each toxin concentration was done in triplicate.
Figure 3
Figure 3
Ion-channel properties of CyaA-Hly on DiPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) bilayers. (a) Current traces, I (pA) versus time (s), after incorporation of the purified CyaA-Hly toxin (1 μg/mL) into both chambers of PLBs in 1.0 M KCl, 10 mM HEPES buffer (pH 7.4). Applied voltages are indicated on the right side of each trace. The closed stage levels of the channel is denoted by the letter c; (b) The I-V curves of the PLB systems performed in the symmetrical conditions (1.0 M cis: 1.0 M trans); (c) Current traces, I (pA) versus time (s), after incorporation of CyaA-Hly (1 μg/mL) into either trans or (d) cis chamber of PLBs in 1.0 M KCl, 10 mM HEPES buffer (pH 7.4); (e) The I-V curves of the CyaA-Hly channel on DiPhyPC bilayers performed in 1.0 M KCl, 10 mM HEPES buffer (pH 7.4) in the presence of toxin in either cis or trans chamber; (f) Open probability, Popen = topen/(topen + tclosed), of CyaA-Hly channels determined from the PLB current traces in the presence of toxins in either cis or trans side at various membrane potentials and its theoretical fits calculated from the Boltzmann distribution, Popen = 1/[1 + e(V−V')F/RT], where V' = −90 mV is the potential at a half maximum probability. Blue and red lines represent single and multiple modes of the toxin-induced channels, respectively.
Figure 3
Figure 3
Ion-channel properties of CyaA-Hly on DiPhyPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) bilayers. (a) Current traces, I (pA) versus time (s), after incorporation of the purified CyaA-Hly toxin (1 μg/mL) into both chambers of PLBs in 1.0 M KCl, 10 mM HEPES buffer (pH 7.4). Applied voltages are indicated on the right side of each trace. The closed stage levels of the channel is denoted by the letter c; (b) The I-V curves of the PLB systems performed in the symmetrical conditions (1.0 M cis: 1.0 M trans); (c) Current traces, I (pA) versus time (s), after incorporation of CyaA-Hly (1 μg/mL) into either trans or (d) cis chamber of PLBs in 1.0 M KCl, 10 mM HEPES buffer (pH 7.4); (e) The I-V curves of the CyaA-Hly channel on DiPhyPC bilayers performed in 1.0 M KCl, 10 mM HEPES buffer (pH 7.4) in the presence of toxin in either cis or trans chamber; (f) Open probability, Popen = topen/(topen + tclosed), of CyaA-Hly channels determined from the PLB current traces in the presence of toxins in either cis or trans side at various membrane potentials and its theoretical fits calculated from the Boltzmann distribution, Popen = 1/[1 + e(V−V')F/RT], where V' = −90 mV is the potential at a half maximum probability. Blue and red lines represent single and multiple modes of the toxin-induced channels, respectively.

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