Abstract
Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.
Highlights
Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens
Mutations encoding the various Y940 substitutions were introduced by allelic exchange into the cyaA genes on B. pertussis and B. bronchiseptica chromosomes and the corresponding variants of secreted CyaA toxins were extracted from bacterial cell surface and used in toxin activity assays
We show here that the aromatic ring of the side chains of the conserved tyrosine residues located in the acylated segments of four RTX toxins play a different roles in their biological activities
Summary
The aromatic ring of the tyrosine residue 940 plays a crucial role in the biological activity of Bordetella CyaA toxin. The spectra of the monomeric forms of the Y940A and Y940P protein variants eluted in the minor fraction at 32 min (black arrows over chromatograms in Fig. 2c and black curves in Fig. 2d) exhibited two negative peaks in the spectra at 206 and 218 nm, revealing the presence of a mixture of α-helices and β-sheets in their structures Taken together, these data clearly indicated that the conserved tyrosine 940 residue plays a key role in Ca2+-induced folding of the acylated domain of CyaA and its replacement by a non-aromatic residue results in misfolding and aggregation of the RTX719 construct. These data indicated that the presence of the aromatic ring in the side chain of the conserved Y643 residue of HlyA was as such not essential for the formation of the α-hemolysin pores, since the HlyA Y643A variant and the intact HlyA toxin exhibited comparable pore-forming membrane activities
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