Abstract
The Bordetella pertussis CyaA-hemolysin (CyaA-Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion-channel formation in planar lipid bilayers (PLBs). Here, net-charge variations in the pore-lining helix of thirteen related RTX cytolysins including CyaA-Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net-charge alterations on hemolytic activity and channel formation of CyaA-Hly, specific mutations were made at Gln574 or Glu581 in its pore-lining α3 of which both residues are highly conserved Lys in the three highly active RTX cytolysins (i.e., Escherichia coli α-hemolysin, Actinobacillus pleuropneumoniae toxin, and Aggregatibacter actinomycetemcomitans leukotoxin). All six constructed CyaA-Hly mutants that were over-expressed in E. coli as 126 kDa His-tagged soluble proteins were successfully purified via immobilized Ni2+-affinity chromatography. Both positive-charge substitutions (Q574K, Q574R, E581K, E581R) and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin-induced hemolysis while the substitution with a negatively-charged side-chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions (1.0 M KCl, pH 7.4), all five mutant toxins with the increased hemolytic activity produced clearly-resolved single channels with higher open probability and longer lifetime than the wild-type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA-Hly pore model comprising three α2-loop-α3 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively-charged side-chains substituted at positions Gln574 and Glu581 in the pore-lining α3 to the enhanced hemolytic activity and ion-channel opening of CyaA-Hly that actually mimics the highly-active RTX (repeat-in-toxin) cytolysins.
Highlights
Bordetella pertussis is a causative agent of human whooping coughwhich has re-emerged globally as a consequence of pathogen adaptation to vaccination and/or waning protection from acellular pertussis vaccines [1,2,3]
We previously demonstrated that two putative transmembrane helices (α2 and α3) in the CyaA-Hly domain are crucially involved in hemolytic activity [23,24,25]
As revealed from the Kyte-Doolittle-hydropathy analysis [28], four potential transmembrane segments corresponding to α2–α5 of CyaA-Hly can be identified in the HP region of among thirteen related RTX cytolysins including three highly active members, i.e., Escherichia coli α-hemolysin (HlyA), Actinobacillus pleuropneumoniae toxin (ApxIA), and Aggregatibacter actinomycetemcomitans leukotoxin (LtxA)
Summary
Bordetella pertussis is a causative agent of human whooping cough ( known as “pertussis”). CyaA and/or CyaA-Hly needs a palmitoyl group to be added at Lys983 by acyltransferase (CyaC, 21 kDa) [13,14] and is subsequently secreted into the surrounding environment where it selectively binds to the αM β2 integrin receptor ( known as CD11b/CD18) on human macrophages via the RTX segment [15]. Upon such specific receptor-binding, the ~130 kDa CyaA-Hly domain would mediate translocation of the ~40 kDa catalytic AC domain into the cytosol of target cells causing apoptotic cell death [16]. Amino acids are colored according to their charged/polar properties (red is negatively-charged, blue is positively-charged, and light-blue is N-containing polar uncharged) with H atoms omitted
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
