Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN

Abstract

Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized by Fourier transform ion cyclotron resonance-MS͞MS and GC-MS. The tentative structure of salmochelin 1 contained two 2,3-dihydroxybenzoylserine moieties bridged by a glucose residue, bound to the serine hydroxyl group of one moiety and the carboxylate of the second moiety. Salmochelin 2 contained in addition a second glucose residue linked to a third 2,3-dihydroxybenzoylserine moiety. Salmochelins were not produced by an iroBC mutant, which indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases. Uptake experiments with radiolabeled 55 Fe-salmochelin and growth promotion tests with salmochelins showed that the IroN outer membrane receptor, encoded in the iroA locus of S. enterica and uropathogenic Escherichia coli strains, was the main receptor for ferric salmochelin transport.I n iron-poor environments, many bacteria secrete ironcomplexing agents called siderophores to satisfy their iron needs. For some pathogenic bacteria, siderophores are important virulence factors because iron is bound to transferrin and lactoferrin in body fluids. These proteins reduce the free Fe 3ϩ concentration to about one molecule per liter. Enterobacteria, including Escherichia coli and Salmonella enterica, often produce the catecholate siderophore enterochelin (also called enterobactin) (1, 2). It has been postulated that enterochelin is an inferior siderophore in serum because it adsorbs to hydrophobic sites in serum proteins, such as albumin (3). This fact has always been puzzling because iron supply for many pathogens plays a decisive role in the infection process, and enterochelin, a major siderophore synthesized by S. enterica, does not seem to enhance pathogenicity.Recently, the iroA locus, consisting of the two convergent operons iroN and iroBCDE, has been defined in S. enterica serotype Typhi and also in most other S. enterica serotypes (4, 5). The outer membrane siderophore receptor, IroN, is involved in the transport of several catecholate siderophores in S. enterica (5, 6). In the present investigation, we show that the presence of the iro gene cluster in S. enterica leads to glycosylation of the enterochelin building block 2,3-dihydroxybenzoylserine (DHBS), which makes the hydrophobic enterochelin molecules more hydrophilic, thereby possibly contributing to the observed pathogenicity of Salmonella strains. This siderophore was named salmochelin because it appears to be a characteristic siderophore of Salmonella strains. Interestingly, certain E. coli strains, e.g., the uropathogenic E. coli 563, also possess a very similar iro gene cluster on pathogenicity island III (7). The production and the tentative structural elucidation of salmochelins and their specific uptake via the outer membrane receptor IroN are described. In addition, it is shown that salmo...

Interferon‐γ limits the availability of iron for intramacrophage Salmonella typhimurium

“…In accordance with the results obtained on cellular iron efflux, the incorporation of 59 Fe into ferritin multimeric shells was significantly reduced upon stimulation with IFN-c and upon Salmonella infection (Fig. 4B, lower panel).…”

“…We employed three strains of S. typhimurium, namely wild-type S. typhimurium C5RP4, wildtype S. typhimurium H5542 and strain H5709, the latter characterized by a deficiency in its enzyme entC, which renders it unable to synthesize the siderophores enterochelin and salmochelins, consequently. Salmonella wild-type strains C5RP4 and H5542 imported similar amounts of 59 Fe in comparison to each other from both TBI and NTBI sources ( Fig. 5A and B).…”

“…We first examined the putative changes in iron acquisition by Salmonella-infected RAW264.7 macrophages in response to IFN-c by investigating the regulation of TfR1 and Dmt1 expression and by measuring cellular 59 Fe-uptake. TfR1 mRNA expression was not significantly different between Salmonella-infected macrophages and control cells, which is consistent with our previous report [40].…”

“…To test whether this observed induction of iron export as well as of Hmox1 and Lcn2 expression following Salmonella infection and IFN-c activation may serve as a mechanism to withdraw this essential nutrient from internalized bacteria, we directly measured the acquisition of 59 Fe, delivered as either TBI or NTBI, by intramacrophage Salmonella. We employed three strains of S. typhimurium, namely wild-type S. typhimurium C5RP4, wildtype S. typhimurium H5542 and strain H5709, the latter characterized by a deficiency in its enzyme entC, which renders it unable to synthesize the siderophores enterochelin and salmochelins, consequently.…”

“…H5542 is identical to S. typhimurium strain ATCC14028 and was grown under sterile conditions in LB broth, its entC-deficient derivative H5709 is identical to H5542 but fepA:Tn10dTc ent. S. typhimurium strain H5709 was generated as described [59] and grown in LB broth containing 50 lg/mL tetracycline (Sigma-Aldrich). For macrophage infection experiments employing S. typhimurium strains H5542 and H5709, complete DMEM without antibiotics was used.…”