The role of a conserved tyrosine residue of acylated domain in membrane insertion and penetration of RTX toxins

Pore-forming RTX toxins are key virulence factors of many Gram-negative pathogens. These proteins share several common structural and functional features: (i) the presence of repetitive sequences rich in glycine and aspartate, which are important for calcium ion binding, (ii) transport from the bacterial cytosol through the type I secretion system (T1SS), (iii) modification by a fatty acid at specific lysines in the acylated domain by a specific acyltransferase, and (iv) the presence of an amphipathic region responsible for the formation of cation-selective pores in the target membrane. The aromatic side chain of the conserved tyrosine residue 940 in the acylated segment of the RTX adenylate cyclase toxin (CyaA, ACT or AC-Hly) of Bordetella pertussis plays a key role in the interaction of the toxin with the target cell membrane. The aim of this study was to determine whether the corresponding conserved residues Y940, Y642, Y643 and Y639 secreted by the homologous RTX toxin CyaA from Bordetella bronchiseptica, HlyA from Escherichia coli, ApxIA from Actinobacillus pleuropneumoniae and RtxA from Kingella kingae play the same critical role in membrane insertion and pore formation. The hemolytic and cytotoxic activities of these toxins were completely impaired only after replacement of the conserved..