Functional Contributions of Positive Charges in the Pore-Lining Helix 3 of the Bordetella pertussis CyaA-Hemolysin to Hemolytic Activity and Ion-Channel Opening

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.

Membrane Activity and Channel Formation of the Adenylate Cyclase Toxin (CyaA) of Bordetella pertussis in Lipid Bilayer Membranes

“…These substitution mutants were still capable of forming transmembrane pores, and therefore, the hemolytic capacity was not affected. The replacement of A538 by proline eliminated the voltage-activated increase of membrane conductivity of CyaA in asolectin membranes [87]. These results show that the linker segment may interact with the structure of helix II and control the formation of CyaA pores [87,88].…”

“…Kurehong et al focused on the same hydrophobic region and could confirm these results. Mutation of the polar amino acids Q574 and E581 has an influence on the pore-forming activity of CyaA [87]. Similarly, a considerable influence in the membrane penetration and pore-forming activity of CyaA was found for residues 529 to 549 in segment II [19].…”

Bioengineering of Bordetella pertussis Adenylate Cyclase Toxin for Antigen-Delivery and Immunotherapy

“…These segments are supposed to insert into the plasma membrane of the target cells and multimerize to form cation-selective pores [ 62 ]. Internal deletions and/or specific mutation within this region abolish or modulate both cytotoxicity and hemolytic activity of CyaA [ 23 , 24 , 56 , 62 , 63 , 64 ]; A region that harbors the two lysines, Lys860 and Lys983, that are acylated by CyaC. The molecular mechanisms by which the addition of a fatty acid to these residues contributes to the toxicity of CyaA are not yet clarified although recent studies suggest that the acyl chains may have a structural role in favoring the folding of the CyaA into functional active states [ 65 , 66 , 67 , 68 ]; A large C-terminal domain, the so-called RTX domain (residues 1000–1613) which comprises from about 40–50 copies of a repeating stretch of nine residues GGXGXDXLX (where X represents any amino acid).…”

“…These segments are supposed to insert into the plasma membrane of the target cells and multimerize to form cation-selective pores [ 62 ]. Internal deletions and/or specific mutation within this region abolish or modulate both cytotoxicity and hemolytic activity of CyaA [ 23 , 24 , 56 , 62 , 63 , 64 ];…”

An Introduction to the Toxins Special Issue on the Adenylate Cyclase Toxin

“…Kurehong and collaborators report the effect on the pore-forming activity of point mutations of two residues (Q574 and E581) from the hydrophobic region of CyaA. They show that mutations to positively charged residues, which mimic the local positive valence as observed in hemolytic RTX proteins, increase the pore forming activity of CyaA [ 8 ]. Hence, compared to the efficiency of other bacterial hemolysins, the hemolytic activity of CyaA is rather low; however, this loss of hemolytic activity might be explained by the fact that the main functional property of CyaA is to intoxicate cells by translocating its catalytic domain into host cells and to induce a cascade of events disrupting host immune responses.…”