Secretion, but not overall synthesis, of catecholate siderophores contributes to virulence of extraintestinal pathogenic Escherichia coli

P>Extraintestinal pathogenic Escherichia coli (ExPEC) use siderophores to sequester iron during infection. Enterobactin and salmochelins are catecholate siderophores produced by some ExPEC strains and other pathogenic enterobacteria. Siderophore export and synthesis mutants of avian ExPEC strain chi 7122 were tested in a chicken infection model. In single-strain infections, siderophore-negative (Delta entD Delta iuc), Delta entS and Delta entS Delta iroC export mutants were attenuated in tissues and blood, whereas the Delta iroC export mutant was only attenuated in blood. Interestingly, the Delta entD mutant, producing only aerobactin, retained full virulence, and loss of entD in the Delta entS Delta iroC mutant restored virulence. LC-MS/MS quantification of siderophores in export mutants demonstrated that loss of entS impaired enterobactin and mono-glucosylated enterobactin secretion, whereas loss of iroC impaired di- and tri-glucosylated enterobactin secretion. Loss of entS and/or iroC resulted in intracellular accumulation and increased secretion of siderophore monomers. Catecholate siderophore export mutants also demonstrated decreased fitness in a co-challenge infection model. By contrast, catecholate siderophore synthesis mutants (Delta entD and Delta iroB) competed as well as the wild-type strain. Results establish that EntS and IroC mediate specific export of catecholate siderophores and the role of these exporters for ExPEC virulence is contingent on enterobactin synthesis, which is not required when other siderophores like aerobactin are functional.