Abstract
Bordetella adenylate cyclase toxin-hemolysin (CyaA, AC-Hly, or ACT) permeabilizes cell membranes by forming small cation-selective (hemolytic) pores and subverts cellular signaling by delivering into host cells an adenylate cyclase (AC) enzyme that converts ATP to cAMP. Both AC delivery and pore formation were previously shown to involve a predicted amphipathic alpha-helix(502-522) containing a pair of negatively charged Glu(509) and Glu(516) residues. Another predicted transmembrane alpha-helix(565-591) comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA. Substitutions of Glu(516) increased specific hemolytic activity of CyaA by two different molecular mechanisms. Replacement of Glu(516) by positively charged lysine residue (E516K) increased the propensity of CyaA to form pores, whereas proline (E516P) or glutamine (E516Q) substitutions extended the lifetime of open single pore units. All three substitutions also caused a drop of pore selectivity for cations. Substitutions of Glu(570) and Glu(581) by helix-breaking proline or positively charged lysine residue reduced (E570K, E581P) or ablated (E570P, E581K) AC membrane translocation. Moreover, E570P, E570K, and E581P substitutions down-modulated also the specific hemolytic activity of CyaA. In contrast, the E581K substitution enhanced the hemolytic activity of CyaA 4 times, increasing both the frequency of formation and lifetime of toxin pores. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in ion filtering inside or close to pore mouth. The pairs of glutamate residues in the predicted transmembrane segments of CyaA thus appear to play a key functional role in membrane translocation and pore-forming activities of CyaA.
Highlights
Bordetella pertussis, the etiological agent of whooping cough, secretes an adenylate cyclase toxin (CyaA,3 adenylate cyclase-hemolysin (AC-Hly), or ACT) that is a key virulence factor of the bacteria during early phases of respiratory tract colonization [1,2,3,4]
Substitutions of Glutamate 516 Increase Hemolytic Activity of CyaA by Extending Lifetime or by Enhancing Formation Frequency of CyaA Pores—In previous work, we showed that AC membrane translocation, as well as formation and cation selectivity of CyaA pores, depend on the structure and net charge of a potential amphipathic ␣-helical transmembrane segment [34]
The mutant CyaA proteins were expressed in E. coli and purified by a combination of ion exchange and hydrophobic chromatography (Fig. 2), and their specific cell-binding, cell-invasive, and hemolytic activities were compared with those of intact CyaA, using sheep erythrocytes as target cells
Summary
CyaA, adenylate cyclase toxin; AC, adenylate cyclase; Hly, hemolysin; BSA, bovine serum albumin; pS, picosiemens. We demonstrated that both of these activities involve a transmembrane ␣-helix predicted between residues 502 and 522 of the poreforming domain of CyaA This harbors a pair of negatively charged glutamate residues, Glu509 and Glu516. Combination of the substitutions in CyaA-E509K/E516K strongly decreased the cation selectivity of formed pores, indicating that Glu509 and Glu516 are located within or close to the membrane pore [34] On this basis, we suggested that prior to interaction with target membrane, two conformational isomers of CyaA might form, one being a translocation precursor allowing AC domain delivery into cell cytosol and the other being a pore precursor, whose insertion into membranes yields formation of oligomeric membrane pores [34]. We show that AC membrane translocation and poreforming (hemolytic) activity of CyaA further depends on an additional predicted transmembrane amphipathic ␣-helix, localized between residues 565 and 591 of the pore-forming domain and comprising yet another pair of glutamate residues, Glu570 and Glu581. We show for the first time that different amino acid substitutions of the glutamate residue at position 516 can increase the hemolytic activity of CyaA by two different molecular mechanisms
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