Role of receptor proteins for enterobactin and 2,3-dihydroxybenzoylserine in virulence of Salmonella enterica.

Single, double, and triple mutants of an enterobactin-deficient mutant strain of Salmonella enterica serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The fepA, iroN, and cir alleles were transduced to enterobactin-proficient strains of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the S. enterica serovar Typhimurium strains indicated that the parental strain and the fepA iroN double mutant were similarly virulent but that the fepA iroN cir triple mutant was significantly attenuated. Moreover, in mixed infections, the fepA iroN mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with S. enterica serovar Enteritidis strains also indicated that mutation of the fepA iroN genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of S. enterica serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the S. enterica serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of S. enterica.