Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization

Abstract

The siderophore salmochelin is produced under iron-poor conditions by Salmonella and many uropathogenic Escherichia coli strains. The production of salmochelin, a C-glucosylated enterobactin, is dependent on the synthesis of enterobactin and the iroBCDEN gene cluster. An E. coli IroD protein with an N-terminal His-tag cleaved cyclic salmochelin S4 to the linear trimer salmochelin S2, the dimer salmochelin S1, and the monomers dihydroxybenzoylserine and C-glucosylated dihydroxybenzoylserine (salmochelin SX, pacifarinic acid). The periplasmic IroE protein was purified as a MalE-IroE fusion protein. This enzyme degraded salmochelin S4 and ferric-salmochelin S4 to salmochelin S2 and ferric-salmochelin S2, respectively. In E. coli, uptake of ferric-salmochelin S4 was dependent on the cleavage by IroE, and independent of the FepBDGC ABC transporter in the cytoplasmic membrane. IroC, which has similarities to ABC-multidrug-resistance proteins, was necessary for the uptake of salmochelin S2 from the periplasm into the cytoplasm. IroE did not function as a classical binding protein since salmochelin S2 was taken up in the absence of a functional IroE protein. IroC mediated the uptake of iron via enterobactin in a fepB mutant. IroE was also necessary in this case for the uptake of ferric-enterobactin, which indicated that only the linear degradation products of enterobactin were taken up via IroC. PfeE, the Pseudomonas aeruginosa IroE homologue, was cloned, and its enzymic activity was shown to be very similar to that of IroE. It is suggested that homologues in other bacteria are also periplasmic IroE-type esterases of siderophores.

Siderophore-Based Iron Acquisition and Pathogen Control

“…Although the similarity of IroC to ABC-type efflux proteins is significant, growth promotion studies indicated that IroC might be responsible for the uptake of linearized forms of Fe-salmochelin and Fe-enterobactin (359). Thus, two conflicting models of salmochelin utilization currently exist, one assigning IroC as a salmochelin exporter (95,191) and the other one assigning IroC as an importer of the iron-charged linearized salmochelin trimer (359). IroC-dependent Fe-salmochelin transport studies or iroC mutant phenotype analyses might help to clarify the opposing interpretations.…”

“…Since it was observed that E. coli fepB mutants that possess the iroA locus were still able to utilize salmochelin as an iron source, it was concluded that IroC, the only CM permease encoded in the iroA cluster, might be responsible for Fe-salmochelin uptake (359). Direct evidence for IroC function, however, for its role in uptake or in secretion does not exist so far.…”

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2

“…1B), which is also found in pathogenic strains of E. coli, Salmonella spp., and K. pneumoniae (9)(10)(11)(12). The iroA gene cluster encodes five proteins involved in modification and transport of Ent: IroB Cglucosylates Ent (13), IroE hydrolytically linearizes Ent (14,15), IroD degrades ferric complexes of the siderophore to release iron in the bacterial cytoplasm (14,15), and IroN and IroC transport Ent and its derivatives (16). Rather than Ent, iroAharboring bacteria secrete a mixture of salmochelins S4 and S2 ( Fig.…”

Iron metabolism at the host pathogen interface: Lipocalin 2 and the pathogen-associated iroA gene cluster

“…The transport of salmochelin is still under investigation, and the functional role of IroC is disputed. The IroC protein has been implicated as a inner membrane exporter (Baumler et al, 1996) and an inner membrane importer (Zhu, Valdebenito, Winkelmann, & Hantke, 2005). We have illustrated the former model where salmochelin is exported through the inner membrane protein IroC, and then shuttled across the periplasmic space and secreted through the outer membrane via an unknown mechanism.…”