ABSTRACT:
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
.