Enterobactin induces the chemokine, interleukin-8, from intestinal epithelia by chelating intracellular iron.

Iron is an indispensable nutrient for both mammals and microbes. Bacteria synthesize siderophores to sequester host iron, whereas lipocalin 2 (Lcn2) is the host defense protein that prevent this iron thievery. Enterobactin (Ent) is a catecholate-type siderophore that has one of the strongest known affinities for iron. Intestinal epithelial cells (IECs) are adjacent to large microbial population and are in contact with microbial products, including Ent. We undertook this study to investigate whether a single stimulus of Ent could affect IEC functions. Using three human IEC cell-lines with differential basal levels of Lcn2 (i.e. HT29 < DLD-1 < Caco-2/BBe), we demonstrated that iron-free Ent could induce a dose-dependent secretion of the pro-inflammatory chemokine, interleukin 8 (IL-8), in HT29 and DLD-1 IECs, but not in Caco-2/BBe. Ent-induced IL-8 secretion was dependent on chelation of the labile iron pool and on the levels of intracellular Lcn2. Accordingly, IL-8 secretion by Ent-treated HT29 cells could be substantially inhibited by either saturating Ent with iron or by adding exogenous Lcn2 to the cells. IL-8 production by Ent could be further potentiated when co-stimulated with other microbial products (i.e. flagellin, lipopolysaccharide). Water-soluble microbial siderophores did not induce IL-8 production, which signifies that IECs are specifically responding to the lipid-soluble Ent. Intriguingly, formyl peptide receptor (FPR) antagonists (i.e. Boc2, cyclosporine H) abrogated Ent-induced IL-8, implicating that such IEC response could be, in part, dependent on FPR. Taken together, these results demonstrate that IECs sense Ent as a danger signal, where its recognition results in IL-8 secretion.

Enterobactin, an iron chelating bacterial siderophore, arrests cancer cell proliferation.

Iron is essential for many biological functions, including being a cofactor for enzymes involved in cell proliferation. In line, it has been shown that cancer cells can perturb their iron metabolism towards retaining an abundant iron supply for growth and survival. Accordingly, it has been suggested that iron deprivation through the use of iron chelators could attenuate cancer progression. While they have exhibited anti-tumor properties in vitro, the current therapeutic iron chelators are inadequate due to their low efficacy. Therefore, we investigated whether the bacterial catecholate-type siderophore, enterobactin (Ent), could be used as a potent anti-cancer agent given its strong iron chelation property. We demonstrated that iron-free Ent can exert cytotoxic effects specifically towards monocyte-related tumor cell lines (RAW264.7 and J774A.1), but not primary cells, i.e. bone marrow-derived macrophages (BMDMs), through two mechanisms. First, we observed that RAW264.7 and J774A.1 cells preserve a bountiful intracellular labile iron pool (LIP), whose homeostasis can be disrupted by Ent. This may be due, in part, to the lower levels of lipocalin 2 (Lcn2; an Ent-binding protein) in these cell lines, whereas the higher levels of Lcn2 in BMDMs could prevent Ent from hindering their LIP. Secondly, we observed that Ent could dose-dependently impede reactive oxygen species (ROS) generation in the mitochondria. Such disruption in LIP balance and mitochondrial function may in turn promote cancer cell apoptosis. Collectively, our study highlights Ent as an anti-cancer siderophore, which can be exploited as an unique agent for cancer therapy.

The bacterial siderophore enterobactin confers survival advantage to Salmonella in macrophages.

Enterobactin (Ent), a prototypical bacterial siderophore known for its unparalleled affinity for iron, is widely conserved among members of the Enterobacteriaceae family of Gram-negative bacteria. In this study, we demonstrated that, aside from mediating iron acquisition, Ent also dampened the macrophages (MΦs) antimicrobial responses against intracellular infection by Salmonella enterica serovar Typhimurium. Accordingly, the loss of Ent expression (ΔentB) in Salmonella demoted their survivability against MΦs. Addition of exogenous Ent not only rescued the survival of ΔentB Salmonella, but also augmented WT Salmonella to better withstand the microbicidal activity of MΦs. The protection conferred to WT Salmonella was observed only when Ent was administered as iron-free, thus indicating the requirement of iron chelation in this context. In contrast, the exogenous iron-bound Ent retained its ability to promote the survival of ΔentB Salmonella, albeit modestly. Assessment on MΦs labile iron pool (LIP) revealed that iron-free Ent is able to permeate into MΦs, chelate the intracellular LIP, and regulate the expression of several key iron-regulatory proteins, i.e., divalent metal transporter 1, ferroportin, and hepcidin. Chelation of iron by Ent was also observed to promote the MΦs towards M2 polarization. Collectively, our findings demonstrated that Ent not only facilitates bacterial iron uptake but also disrupts MΦs iron homeostasis and M1/M2 polarization to safeguard intracellular bacteria against the anti-bacterial effects of their host.

Obesity-promoting and anti-thermogenic effects of neutrophil gelatinase-associated lipocalin in mice.

Neutrophil gelatinase-associated lipocalin (NGAL, lipocalin 2 or LCN2) is an iron carrier protein whose circulating level is increased by kidney injury, bacterial infection and obesity, but its metabolic consequence remains elusive. To study physiological role of LCN2 in energy homeostasis, we challenged female Lcn2 knockout (KO) and wild-type (WT) mice with high fat diet (HFD) or cold exposure. Under normal diet, physical constitutions of Lcn2 KO and WT mice were indistinguishable. During HFD treatment, Lcn2 KO mice exhibited larger brown adipose tissues (BAT), consumed more oxygen, ate more food and gained less body weights as compared to WT mice. When exposed to 4 °C, KO mice showed higher body temperature and more intense 18F-fluorodeoxyglucose uptake in BAT, which were cancelled by ß3 adrenergic receptor blocker or iron-loaded (but not iron-free) LCN2 administration. These findings suggest that circulating LCN2 possesses obesity-promoting and anti-thermogenic effects through inhibition of BAT activity in an iron-dependent manner.

Bacterial Siderophores Hijack Neutrophil Functions.

Neutrophils are the primary immune cells that respond to inflammation and combat microbial transgression. To thrive, the bacteria residing in their mammalian host have to withstand the antibactericidal responses of neutrophils. We report that enterobactin (Ent), a catecholate siderophore expressed by Escherichia coli, inhibited PMA-induced generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) in mouse and human neutrophils. Ent also impaired the degranulation of primary granules and inhibited phagocytosis and bactericidal activity of neutrophils, without affecting their migration and chemotaxis. Molecular analysis revealed that Ent can chelate intracellular labile iron that is required for neutrophil oxidative responses. Other siderophores (pyoverdine, ferrichrome, deferoxamine) likewise inhibited ROS and NETs in neutrophils, thus indicating that the chelation of iron may largely explain their inhibitory effects. To counter iron theft by Ent, neutrophils rely on the siderophore-binding protein lipocalin 2 (Lcn2) in a "tug-of-war" for iron. The inhibition of neutrophil ROS and NETs by Ent was augmented in Lcn2-deficient neutrophils compared with wild-type neutrophils but was rescued by the exogenous addition of recombinant Lcn2. Taken together, our findings illustrate the novel concept that microbial siderophore's iron-scavenging property may serve as an antiradical defense system that neutralizes the immune functions of neutrophils.

Neutrophil gelatinase-associated lipocalin increases HLA-G(+)/FoxP3(+) T-regulatory cell population in an in vitro model of PBMC.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is emerging as a mediator of various biological and pathological states. However, the specific biological role of this molecule remains unclear, as it serves as a biomarker for many conditions. The high sensitivity of NGAL as a biomarker coupled with relatively low specificity may hide important biological roles. Data point toward an acute compensatory, protective role for NGAL in response to adverse cellular stresses, including inflammatory and oxidative stress. The aim of this study was to understand whether NGAL modulates the T-cell response through regulation of the human leukocyte antigen G (HLA-G) complex, which is a mediator of tolerance. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMCs) were obtained from eight healthy donors and isolated by centrifugation on a Ficoll gradient. All donors gave informed consent. PBMCs were treated with four different concentrations of NGAL (40-320 ng/ml) in an iron-loaded or iron-free form. Changes in cell phenotype were analyzed by flow cytometry. NGAL stimulated expression of HLA-G on CD4+ T cells in a dose- and iron-dependent manner. Iron deficiency prevented NGAL-mediated effects, such that HLA-G expression was unaltered. Furthermore, NGAL treatment affected stimulation of regulatory T cells and in vitro expansion of CD4(+) CD25(+) FoxP3(+) cells. An NGAL neutralizing antibody limited HLA-G expression and significantly decreased the percentage of CD4(+) CD25(+) FoxP3(+) cells. CONCLUSIONS/SIGNIFICANCE: We provide in vitro evidence that NGAL is involved in cellular immunity. The potential role of NGAL as an immunomodulatory molecule is based on its ability to induce immune tolerance by upregulating HLA-G expression and expansion of T-regulatory cells in healthy donors. Future studies should further evaluate the role of NGAL in immunology and immunomodulation and its possible relationship to immunosuppressive therapy efficacy, tolerance induction in transplant patients, and other immunological disorders.

Mucosal lipocalin 2 has pro-inflammatory and iron-sequestering effects in response to bacterial enterobactin.

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.

Oxinobactin, a siderophore analogue to enterobactin involving 8-hydroxyquinoline subunits: synthesis and iron binding ability.

Oxinobactin, a siderophore analogue to enterobactin but possessing 8-hydroxyquinoline instead of catechol complexing subunits, has been synthesized starting from L-serine and 8-hydroxyquinoline. Comparative iron binding studies showed that oxinobactin is as effective as enterobactin for the complexation of Fe(III) at physiological pH but with improved complexing ability at acidic pH.

Synthesis and biological activity of analogues of vanchrobactin, a siderophore from Vibrio anguillarum serotype O2.

Several analogues of vanchrobactin, a catechol siderophore isolated from the bacterial fish pathogen Vibrio anguillarum serotype O2 strain RV22, have been synthesized. The biological evaluation of these novel compounds showed that most of them are active as siderophores, as determined by growth promotion assays using the producer strain, as well as V. anguillarum serotype O1, Salmonella enterica, and Erwinia chrysanthemi. These compounds also gave a positive chrome azurol-S (CAS) test. On the basis of these results, we were able to deduce some structure-activity relationships. Furthermore, we found an analogue with siderophore activity that has appropriate functionality (an amino group) for use as an antibiotic vector to be employed in a "Trojan horse strategy".

Interleukin-8 secretion in response to aferric enterobactin is potentiated by siderocalin.

Siderophores are low-molecular-weight iron chelators secreted by microbes to obtain iron under deprivation. We hypothesized that the catecholate siderophore enterobactin, produced by Enterobacteriaceae, serves as a proinflammatory signal for respiratory epithelial cells. Respiratory tract responses were explored, since at this site siderocalin, an enterobactin-binding mammalian gene product, is expressed inducibly at high levels and enterobactin-secreting respiratory flora is rare, suggesting selection against a dependence on enterobactin. Addition of aferric, but not iron-saturated, enterobactin elicits a dose-dependent increase in secretion of the proinflammatory chemokine interleukin-8 by human respiratory epithelial cells in culture. This response to purified enterobactin is potentiated by recombinant siderocalin at physiologically relevant concentrations. Conditioned media from genetically modified Escherichia coli strains expressing various levels of enterobactin induce an enterobactin-mediated proinflammatory response. Siderocalin has been shown to deliver enterobactin to other mammalian cell types, exogenously supplied siderocalin can be detected within epithelial cells, and siderocalin increases delivery of enterobactin to the intracellular compartment. Although many siderophores perturb labile cellular iron pools, only enterobactin elicits interleukin-8 secretion, suggesting that iron chelation is necessary but not sufficient. Thus, aferric enterobactin may be a proinflammatory signal for respiratory epithelial cells, permitting detection of microbial communities that have disturbed local iron homeostasis, and siderocalin expression by the host amplifies this signal. This may be a novel mechanism for the mucosa to respond to metabolic signals of expanding microbial communities.

Two iron-regulated putative ferric siderophore receptor genes in Bordetella bronchiseptica and Bordetella pertussis.

Two iron-regulated genes with deduced homology to TonB-dependent ferric siderophore receptors were cloned from Bordetella bronchiseptica by screening a library of TnphoA insertion mutants. bfrB and bfrC were iron-repressed in B. bronchiseptica by a Fur-dependent mechanism, and were expressed from promoters overlapped by potential Fur-binding sites. Both genes were highly conserved among Bordetella species and were also iron-regulated in Bordetella pertussis. bfrB and bfrC mutants of both species and a bfrB-bfrC double mutant of B. bronchiseptica had no discernible defects in utilization of known iron sources for Bordetella.

Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria.

Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo.

The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist.

Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae. Different growth conditions of the producer strain affect microcin activity. The production of a microcin antagonist is responsible for the changes in microcin activity. The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity. The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin. The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron. The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli. The microcin antagonist was identified as enterochelin through thin-layer chromatography.

Iron supply to Escherichia coli by synthetic analogs of enterochelin.

Synthetic analogs of enterochelin (enterobactin) were tested for their ability to support the growth of Escherichia coli K-12 under iron-limiting conditions. The cyclic compound MECAM [1,3,5-N.N'; N"-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene] and its N-methyl derivative Me3MECAM promoted growth, whereas the 2,3-dihydroxy-5-sulfonyl derivatives MECAMS and Me3MECAMS were inactive. The same results were obtained with TRIMCAM [1,3,5-tris(2,3-dihydroxybenzoylcarbamido)-benzene] and TRIMCAMS (the 2,3-dihydroxy-5-sulfonyl derivative of TRIMCAM). However, the sulfonic acid-containing linear compound LICAMS [1,5,10-N,N', N"-tris(5-sulfo-2,3-dihydroxybenzoyl)-triaza-decane] supported growth. In contrast, LIMCAMC, in which the sulfonyl groups at the five position of LICAMS are replaced by carboxyl groups at the four position, was inactive. The uptake of the active analogs required the functions specified by the fepB, fesB, and tonB genes. Surprisingly, growth promotion of mutants lacking the enterochelin receptor protein in the outer membrane was observed. Only MECAM protected cells against colicin B (which kills cells after entering at the enterochelin uptake sites) and transported Fe3+ at about half the enterochelin rate.

Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli.

Using biological iron chelators to control specifically iron availability to Escherichia coli K-12 in conjunction with radioactive pulse-labels, we examined the biosynthesis of six iron-regulated membrane proteins. Iron deprivation induced the synthesis of five proteins, which had molecular weights of 83,000 (83K), 81K (Fep), 78K (TonA), 74K (Cir), and 25K. The kinetics of induction were the same in entA and entA(+) strains, but were affected by the initial iron availability in the media. Iron-poor cells induced rapidly (half-time, 10 min), whereas iron-rich cells began induction after a lag and showed a slower induction half-time (30 min). Within this general pattern of induction after iron deprivation, several different kinetic patterns were apparent. The 83K, 81K, and 74K proteins were coordinately controlled under all of the conditions examined. The 78K and 25K proteins were regulated differently. The synthesis of a previously unrecognized 90K inner membrane protein was inhibited by iron deprivation and stimulated by iron repletion. Both ferrichrome and ferric enterobactin completely repressed 81K and 74K synthesis when the siderophores were supplied at concentrations of 5 muM in vivo (half-time, 2.5 min). At concentrations less than 5 muM, however, both siderophores repressed synthesis only temporarily; the duration of repression was proportional to the amount of ferric siderophore added. The half-lives of the 81K and 74K mRNAs, as measured by rifampin treatment, were 1.2 and 1.6 min, respectively. The results of this study suggest that enteric bacteria are capable of instantaneously detecting and reacting to fluctuations in the extracellular iron concentration and that they store iron during periods of iron repletion for utilization during periods of iron stress. Neither iron storage nor iron regulation of envelope protein synthesis is dependent on the ability of the bacteria to form heme.

Involvement of inner and outer membrane components in the transport of iron and in colicin B action in Escherichia coli.

Spheroplasts of Escherichia coli mutants were used to investigate the roles of the inner and outer membranes in the transport of iron. tonA mutants, known to be defective in an outer membrane component of the ferrichrome transport system, regained the ability to transport ferrichrome when converted to spheroplasts. On the other hand, the tonB mutant was unable to transport ferric enterochelin in either whole cells or spheroplasts. This implies that an element of the inner membrane is affected. fep mutants were also unable to transport ferric enterochelin, and fell into two classes, fepA and fepB. Spheroplasts of the former class transported ferric enterochelin, and those of the latter did not. This implies that the fepA mutants are defective in ferric enterochelin transport across the outer membrane, and that fepB mutants probably lack the facility to transport ferric enterochelin across the inner membrane. Colicin B action on fepA mutants was found to differ from that on fepB mutants.

Involvement of outer membrane proteins in enterochelin-mediated iron uptake in Escherichia coli.

Escherichia coli K-12 grown in iron-deficient media contained a large amount of outer membrane proteins O-2a, O-2b, and O-3, while cells grown in iron-supplemented media contained far smaller amounts of these proteins. The iron uptake by the iron-deficient cells was significantly stimulated in the presence of enterochelin, while that by the iron-rich cells was not. The outer membrane isolated from cells grown in the iron-deficient media showed enterochelin-stimulated binding of iron, while the outer membrane from iron-rich cells and cytoplasmic membranes from both types of cells did not show such binding activity. The amount of iron bound by the outer membrane was almost equivalent to the amount of O-2a, O2b, or O-3, irrespective of the amount of these proteins in the outer membrane, which is controlled by the amount of iron in the medium. Small particles rich in these proteins were prepared from cells by EDTA extraction. The particles were active in enterochelin-mediated iron binding and the amount of iron bound was equivalent to the amount of each of these proteins in the particles. Although the outer membrane of E. coli B was as active in iron binding as that of E. coli K-12, it did not possess an appreciable amount of O-2a. Gel electrophoretic analysis revealed that 9-2b and 9-3 were identical with the proteins missing mutants feuB and feuA, respectively.