Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins

In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterialrepeats intoxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ?-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities ofBordetella pertussisadenylate cyclase toxin (CyaA),Escherichia coli?-hemolysin (HlyA), andKingella kingaecytotoxin (RtxA). We found that the three protoxins are acylated in the sameE. colicell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl?acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site.