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.
Highlights
The bacterium Bordetella pertussis causes human whooping cough which has re-emerged and continued to be a major health problem worldwide, possibly due to the waning of vaccine-induced immunity and/or pathogen adaptation [1]
We have further shown that polarity and/or charges at Glu570 and Glu581 of the α3 are important for hemolytic activity of adenylate cyclase-hemolysin toxin (CyaA)-Hly [17]
Our results demonstrate that the CyaA-Hly domain is the major pore-forming determinant that forms oligomeric lytic pores on susceptible erythrocyte membranes as well as single channels on planar lipid bilayers
Summary
The bacterium Bordetella pertussis causes human whooping cough which has re-emerged and continued to be a major health problem worldwide, possibly due to the waning of vaccine-induced immunity and/or pathogen adaptation [1]. CyaA can exhibit hemolytic activity through its C-terminal pore-forming or hemolysin (Hly) domain (~126 kDa) against sheep erythrocytes which lack the integrin receptor [6,7,8]. The RTX toxins share Gly-Asp rich nonapeptide repeats (X-U-X-Gly-Gly-X-Gly-X-Asp, X for any amino acid and U for large hydrophobic residues) that fold into a stable β-roll structure upon exposure to extracellular calcium ions at concentrations in the millimolar range [12,13] This β-roll motif could mediate receptor recognition by which variation in number and organization of the RTX repeats would cause differences in target cell specificity of the RTX cytolysins [14]. Our results demonstrate that the CyaA-Hly domain is the major pore-forming determinant that forms oligomeric lytic pores on susceptible erythrocyte membranes as well as single channels on planar lipid bilayers
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