Louis Alex Julien

Florence Baron

Sylvie Bonnassie

Francoise Nau

Catherine Guérin-Dubiard

Sophie Jan

Simon Colin Andrews
email: s.c.andrews@reading.ac.uk

Louis Alex

Julien Florence

Baron Sylvie

Bonnassie Franc

Nau Catherine

Gue ´rin Sophie

Jan Simon

Colin Andrews

Á S C Andrews

Á F Baron

Á S Bonnassie

Á F Nau

Á C Gue

Á S Jan

The anti-bacterial iron-restriction defence mechanisms of egg white; the potential role of three lipocalin-like proteins in resistance against Salmonella

Keywords: Salmonella Enteritidis, Salmochelin, Enterobactin, Ex-FABP, Cal-c, Alpha-1ovoglycoprotein

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Survival of Salmonella within egg white and the role of iron restriction

The powerful antibacterial defence mechanisms of egg white In the EU, Salmonella enterica is the bacterium most frequently (93%) detected in egg white, with S. enterica serovar Enteriditis (SE) being the major (67%) strain associated with outbreaks caused by eggs and egg products (EFSA 2014). Thus, SE appears to be very well suited to infection of, and survival within, eggs [START_REF] Clavijo | Identification of genes associated with survival of Salmonella enterica serovar Enteritidis in chicken egg albumen[END_REF][START_REF] Vylder | Salmonella enteritidis is superior in egg white survival compared with other Salmonella serotypes[END_REF][START_REF] Gantois | A comparative study on the pathogenesis of egg contamination by different serotypes of Salmonella[END_REF]). Egg white is noted for its strong antimicrobial activity which indicates that SE has powerful egg-white resistance mechanisms. Indeed, the various antimicrobial activities exhibited by egg white can be considered to present a unique set of challenges for bacterial invaders. These include physico-chemical factors, in particular high pH (inhibiting growth; [START_REF] Sharp | The relation of the hydrogen ion concentration of egg white to its germicidal action[END_REF] and high viscosity (limiting motility; [START_REF] Schneider | Effect of viscosity on bacterial motility[END_REF][START_REF] Yadav | Mechanism of egg white resistance to bacterial growth[END_REF]; in addition, high osmolarity (causing osmotic stress) has been suggested [START_REF] Clavijo | Identification of genes associated with survival of Salmonella enterica serovar Enteritidis in chicken egg albumen[END_REF]. The pH of egg white shifts from * 7.6 (upon oviposition) to 9.3 (a few days later) as a result of CO 2 release [START_REF] Sharp | Increase in the pH of the white and yolk of hens' eggs[END_REF]. The viscosity of egg white (with a shear rate of 400 s -1 : 5 mPa.s -1 at 20 °C; [START_REF] Lang | Apparent shear viscosity of native egg white[END_REF] is mainly caused by the presence of ovomucin, a glycoprotein contributing 3.5% w/w of the total egg albumin protein (egg white has a total protein content of * 10% w/w; [START_REF] Kovacs-Nolan | Advances in the value of eggs and egg components for human health[END_REF].

In addition to these physico-chemical factors, egg white possesses an array of proteins that provide further defence against pathogens [see review by [START_REF] Baron | Egg white versus Salmonella Enteritidis! A harsh medium meets a resilient pathogen[END_REF] for more detail], notably:

• ovotransferrin (oTf), involved in iron deprivation [START_REF] Garibaldi | Role of microbial iron transport compounds in bacterial spoilage of eggs[END_REF]) and bacterial membrane damage [START_REF] Aguilera | Transferrins selectively cause ion efflux through bacterial and artificial membranes[END_REF]); • lysozyme [START_REF] Derde | Hen egg white lysozyme permeabilizes the Escherichia coli outer and inner membranes[END_REF]) and defensins [START_REF] Herve | Purification and characterization of avian b-defensin 11, an antimicrobial peptide of the hen egg[END_REF][START_REF] Gong | Gallin; an antimicrobial peptide member of a new avian defensin family, the ovodefensins, has been subject to recent gene duplication[END_REF], that would be expected to disrupt bacterial membrane integrity; • ovalbumin X, a heparin-binding protein exhibiting antimicrobial activity (Re ´hault-Godbert et al. 2013); • ovostatin [START_REF] Nagase | Ovostatin: a novel proteinase inhibitor from chicken egg white[END_REF]) and cystatin [START_REF] Wesierska | Antimicrobial activity of chicken egg white cystatin[END_REF], presumed to inhibit exogenous proteases; and • avidin [START_REF] Banks | Natural antimicrobial systems and their potential in food preservation of the future[END_REF]), a biotin sequestration protein.

The major role of iron restriction and ovotransferrin in egg-white defence

It is generally accepted that the major factor limiting bacterial growth in egg white is iron restriction. This results from the presence of oTf, a powerful ironbinding protein [START_REF] Garibaldi | Role of microbial iron transport compounds in bacterial spoilage of eggs[END_REF][START_REF] Lock | Persistence of contamination of hens' egg albumen in vitro with Salmonella serotypes[END_REF][START_REF] Baron | Factors involved in the inhibition of growth of Salmonella Enteritidis in liquid egg white[END_REF]. The iron restriction of egg white was first discovered by [START_REF] Schade | Raw hen egg white and therole of iron in growth inhibition of Shigella dysenteriae, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae[END_REF] who found that exposure to egg white inhibits the growth of Shigella dysenteriae. Among 31 growth factors added to egg white, only iron overcame the observed egg white-imposed growth inhibition. Two years later, [START_REF] Alderton | Identification of the bacteria-inhibiting iron-binding protein of egg-white as conalbumin[END_REF] identified the eggwhite factor responsible as 'conalbumin', which is now known to be a member of the transferrin family and is more commonly referred as oTf. Since these early studies, subsequent work has confirmed the role of oTf as an egg-white iron-restriction agent preventing growth of a range of microbial species, including Salmonella [START_REF] Schade | Raw hen egg white and therole of iron in growth inhibition of Shigella dysenteriae, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae[END_REF][START_REF] Valenti | Studies of the antimicrobial activity of ovotransferrin[END_REF][START_REF] Valenti | Antifungal activity of ovotransferrin towards genus Candida[END_REF][START_REF] Hr | Insights into the structure-function relationships of ovalbumin, ovotransferrin, and lysozyme[END_REF][START_REF] Baron | Factors involved in the inhibition of growth of Salmonella Enteritidis in liquid egg white[END_REF][START_REF] Baron | Behaviour of Salmonella Enteritidis in industrial egg white: Egg naturally contains factors inhibitory to salmonella growth[END_REF]. Indeed, iron-acquisition mutants of SE display decreased survival and/or growth in egg white [START_REF] Kang | Survival characteristics of Salmonella enterica serovar Enteritidis in chicken egg albumen[END_REF]. Such studies confirm the antibacterial role of iron restriction in egg white. A recent global transcriptomic study [START_REF] Baron | Global gene-expression analysis of the response of Salmonella Enteritidis to egg white exposure reveals multiple egg white-imposed stress responses[END_REF]) revealed a major iron-starvation response of SE upon exposure to egg white which was caused by relief of Fur-(the global transcriptional regulator of iron-dependent gene expression; [START_REF] Rabsch | Role of receptor proteins for enterobactin and 2, 3-dihydroxybenzoylserine in virulence of Salmonella enterica[END_REF]) mediated repression. Likewise, a quantitative proteomic analysis (isobaric tags for relative and absolute quantitation; iTRAQ) showed that iron-acquisition-system-related proteins are induced by egg white [START_REF] Qin | Quantitative proteomics reveals the crucial role of YbgC for Salmonella enterica serovar Enteritidis survival in egg white[END_REF]). These findings confirm that SE suffers from iron limitation in egg white. The low iron availability in egg white exerts a strong bacteriostatic influence [START_REF] Bullen | Role of iron in bacterial infection[END_REF][START_REF] Baron | Factors involved in the inhibition of growth of Salmonella Enteritidis in liquid egg white[END_REF]) because iron is essential for growth of nearly all organisms, including bacteria [START_REF] Andrews | Bacterial iron homeostasis[END_REF]. In many ways, the antibacterial iron-restriction strategy of egg white is comparable to the iron-dependent 'nutritional immunity' defence mechanisms observed in mammals, where serum transferrin maintains concentrations of extracellular free iron at levels (10 -18 M) well below those that support bacterial growth [START_REF] Bullen | Iron and infection, the heart of the matter[END_REF].

OTf is believed to be the critical iron-restriction component in egg white. Like other members of the transferrin family, its structure consists of two 'lobes', each with a strong affinity for a single Fe 3? ion (apparent binding constant of around 10 32 M -1 , with of 1.5 9 10 18 M -1 and 1.5 9 10 14 M -1 for the C-and N-terminal lobes, respectively, at pH 7.5; [START_REF] Guha-Thakurta | Structure of diferric hen serum transferrin at 2.8 A resolution[END_REF][START_REF] Chart | Iron restriction and the growth of Salmonella Enteritidis[END_REF][START_REF] Schneider | Evidence for synergistic anion binding to iron in ovotransferrin complexes from resonance raman and extended X-ray absorption fine structure analysis[END_REF]. The iron-restriction-based bacteriostatic activity of oTf is enhanced by bicarbonate (which is likely related to the apparent dependence of metal binding on the presence of a suitable anion; [START_REF] Valenti | Studies of the antimicrobial activity of ovotransferrin[END_REF]) and high pH [START_REF] Valenti | Influence of bicarbonate and citrate on the bacteriostatic action of ovotransferrin towards staphylococci[END_REF][START_REF] Antonini | Effetto delle transferrine sulla patogenicita delle Enterobacteriaceae[END_REF][START_REF] Lin | Calorimetric studies of serum transferrin and ovotransferrin. Estimates of domain interactions, and study of the kinetic complexities of ferric ion binding[END_REF]. Interestingly, egg white contains levels of iron (* 0.1 mg iron per 100 g which is equivalent to * 18 lM; USDA 2010; Nys and 123 [START_REF] Nys | Valeur nutritionnelle des oeufs[END_REF]) that would normally be sufficient for bacterial growth. However, oTf is present in such high abundance in egg white (170 lM; 13% of total protein content, second most abundant egg white protein after ovalbumin; Sauveur 1988) that oTf iron-binding capacity exceeds iron availability by 17-fold. Since egg white is aerobic and has a high pH (after laying), iron in egg white would be expected to be largely in the ferric state (the form bound by oTf) so it can be assumed that virtually all iron in egg white is bound to oTf such that very little is freely available [START_REF] Sauveur | Structure, composition et valeur nutritionnelle de l'oeuf[END_REF]). However, some bacteria are more susceptible to growth inhibition by oTf than others. Indeed, in vitro studies showed that the most sensitive species are Pseudomonas and Escherichia coli, and the most resistant are Staphylococcus aureus, Proteus and Klebsiella [START_REF] Valenti | Studies of the antimicrobial activity of ovotransferrin[END_REF]. Unsurprisingly, the effects of oTf can be relieved by iron-mobilising agents (e.g. citrate) [START_REF] Valenti | Studies of the antimicrobial activity of ovotransferrin[END_REF]. OTf also appears to possess additional antibacterial activities since its effects are diminished when separated from direct contact with bacteria through location in a dialysis bag or immobilisation on beads [START_REF] Valenti | Antifungal activity of ovotransferrin towards genus Candida[END_REF]. Indeed, it has been shown that oTf can partially penetrate and permeabilise bacterial membranes, acting as an uncoupling agent [START_REF] Aguilera | Transferrins selectively cause ion efflux through bacterial and artificial membranes[END_REF]). This activity is likely related to the presence of a Cysrich antibacterial-peptide-like motif located on the surface of the oTf molecule which confers the ability to kill Gram-negative bacteria [START_REF] Ibrahim | Identification of a distinct antibacterial domain within the N-lobe of ovotransferrin[END_REF][START_REF] Ibrahim | Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism[END_REF].

Iron acquisition by Salmonella

The role of the two siderophores of Salmonella in iron uptake and pathogenicity

The most important mechanism used by bacteria to circumvent iron restriction involves the synthesis of siderophores that bind exogenous ferric iron with high affinity and specificity, and enable acquisition of iron from host sources [START_REF] Andrews | Bacterial iron homeostasis[END_REF]. The siderophores employed by Salmonella are catecholates called enterobactin (or enterochelin) and salmochelin. Enterobactin was first identified in E. coli [START_REF] O'brien | The structure of enterochelin and related 2,3-dihydroxy-N-benzoylserine conjugates from Escherichia coli[END_REF] and Salmonella Typhimurium (ST) (Pollack and Neilands 1970). Although enterobactin synthesis was shown to be required for survival of ST in low-iron in vitro environments (Pollack et al. 1970), its role in pathogenesis is limited for reasons that were, initially, unclear [START_REF] Benjamin | The ability of Salmonella Typhimurium to produce the siderophore enterobactin is not a virulence factor in mouse typhoid[END_REF][START_REF] Rabsch | Role of receptor proteins for enterobactin and 2, 3-dihydroxybenzoylserine in virulence of Salmonella enterica[END_REF]. Salmochelin (which is closely similar to enterobactin) was not identified until more than three decades after enterobactin when it was found to be a product of pathogenic enterobacteria, such as Salmonella [START_REF] Hantke | Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN[END_REF]. It was designated 'salmochelin' as it appeared at first to be a characteristic of Salmonella strains. However, salmochelins have now been reported in avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC), ST and Klebsiella pneumoniae where they contribute to virulence [START_REF] Caza | Specific roles of the iroBCDEN genes in virulence of an avian pathogenic Escherichia coli O78 strain and in production of salmochelins[END_REF][START_REF] Gao | Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model[END_REF][START_REF] Crouch | Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium[END_REF][START_REF] Bachman | Interaction of lipocalin 2, transferrin, and siderophores determines the replicative niche of Klebsiella pneumoniae during pneumonia[END_REF]). It should be noted that the ferrous-iron transport systems of Salmonella (FeoABC and SitABCD) can also contribute to pathogenicity and/or gut colonisation (see review by [START_REF] Carpenter | Response to oxygen and iron availability[END_REF].

Enterobactin: a powerful siderophore, but with limited effect in vivo Enterobactin is a serine macrotrilactone (Fig. 1) that has a far higher affinity for iron than oTf (formation constants of 10 52 and 10 32 M -1 , respectively), which allows siderophore-producing bacteria to use oTf as a source of iron [START_REF] Chart | Iron restriction and the growth of Salmonella Enteritidis[END_REF]. Although the metabolism of enterobactin is best studied in E. coli, Salmonella possesses a highly similar set of enterobactin-related genes which are assumed to play similar roles. The enterobactin precursor, 2,3-dihydroxybenzoate (DHB), is synthesized from chorismate by enzymes encoded by the entC, entB and entA genes. In a second step, DHB and serine are combined, polymerized and cyclized to form enterobactin by enzymes encoded by the entE, entB and entF genes [START_REF] Gehring | Reconstitution and characterization of the Escherichia coli enterobactin synthetase from EntB, EntE, and EntF[END_REF]. EntS is required for enterobactin export through the cytosolic membrane [START_REF] Furrer | Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter[END_REF] whereas TolC is involved in enterobactin efflux across the outer membrane [START_REF] Bleuel | TolC is involved in enterobactin efflux across the outer membrane of Escherichia coli[END_REF]) (Fig. 2). Once complexed with ferric iron, uptake of ferric-enterobactin into the periplasm is mediated by the iron-regulated outer-membrane proteins, FepA and Cir (and IroN in Salmonella) [START_REF] Rabsch | Salmonella typhimurium IroN and FepA proteins mediate uptake of enterobactin but differ in their specificity for other siderophores[END_REF][START_REF] Rabsch | Role of receptor proteins for enterobactin and 2, 3-dihydroxybenzoylserine in virulence of Salmonella enterica[END_REF]; the energytransducing TonB-ExbBD complex is also required for this step [START_REF] Skare | Energy transduction between membranes: tonB, a cytoplasmic membrane protein, can be chemically crosslinked in vivo to the outer membrane receptor FepA[END_REF]Fig. 2). Ferricenterobactin is then imported into the cytoplasm by the ATP-binding cassette transporter, FepBDGC 123 [START_REF] Langman | Enterochelin system of iron transport in Escherichia coli: mutations affecting ferric-enterochelin esterase[END_REF][START_REF] Chenault | Identification of hydrophobic proteins FepD and FepG of the Escherichia coli ferrienterobactin permease[END_REF]. Finally, the imported ferric-enterobactin complex is processed by the Fes esterase which cleaves the cyclic ring of the siderophore, lowering affinity for the bound iron which enables dissociation [START_REF] O'brien | Enterochelin hydrolysis and iron metabolism in Escherichia coli[END_REF].

Despite its high affinity for iron, enterobactin is not as effective as other siderophores in vivo [START_REF] Konopka | Aerobactin mediated utilization of transferrin iron[END_REF][START_REF] Montgomerie | Association of hydroxamate siderophore (aerobactin) with Escherichia coli isolated from patients with bacteremia[END_REF], and this poor performance appears to be related to its rapid clearance from the serum [START_REF] Konopka | Effect of serum albumin on siderophore-mediated utilization of transferrin iron[END_REF]). An unknown factor in serum was found to impede transfer of iron from transferrin to enterobactin, and from 55 Fe-enterobactin to E. coli [START_REF] Konopka | Effect of serum albumin on siderophore-mediated utilization of transferrin iron[END_REF]. However, serum has little impact on iron chelation by the aerobactin siderophore [START_REF] Konopka | Effect of serum albumin on siderophore-mediated utilization of transferrin iron[END_REF]. Aerobactin was also shown to provide a significant selective advantage for E. coli growth in vitro [START_REF] Williams | Iron, siderophores, and the pursuit of virulence: independence of the aerobactin and enterochelin iron uptake systems in Escherichia coli[END_REF], and in a cutaneous infection model [START_REF] Demir | Production by Escherichia coli isolates of siderophore and other virulence factors and their pathogenic role in a cutaneous infection model[END_REF], even though its affinity for iron is weaker than that of enterobactin (formation constants of 10 23 and 10 52 M -1 , respectively; [START_REF] Neilands | Microbial iron compounds[END_REF]. Similar findings were found for S. enterica, as enterobactin is not a virulence factor for ST or SE in mouse and chicken infection models [START_REF] Benjamin | The ability of Salmonella Typhimurium to produce the siderophore enterobactin is not a virulence factor in mouse typhoid[END_REF][START_REF] Rabsch | Role of receptor proteins for enterobactin and 2, 3-dihydroxybenzoylserine in virulence of Salmonella enterica[END_REF]. Later, the serum factor responsible for limiting the action of enterobactin was identified as an acute-phase protein, called LCN2 (lipocalin 2 or neutrophil gelatinase-associated lipocalin) [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF], that previously had an unclear specific purpose. LCN2 was subsequently found to be induced and secreted in response to activation of Toll-like innate immune receptors [START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF]), bind to enterobactin [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF] and inhibit enterobactin activity partly through rapid clearance from the serum [START_REF] Devireddy | A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake[END_REF], and thus shown to function as a 'siderocalin' (siderophorebinding lipocalin).

Salmochelin: a glucosylated siderophore, promoting Salmonella pathogenicity through LCN2 evasion Salmochelin S4 is a diglucosyl-C enterobactin (Fig. 1). The affinity of salmochelin for Fe 3? is not reported [START_REF] Valdebenito | Environmental factors influence the production of enterobactin, salmochelin, aerobactin, and yersiniabactin in Escherichia coli strain Nissle 1917[END_REF][START_REF] Watts | Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli[END_REF]), however, it is assumed that glucosylation does not significantly impact Fe 3? ligation or affinity [START_REF] Luo | Enzymatic tailoring of enterobactin alters membrane partitioning and iron acquisition[END_REF]. The genetic locus responsible for this glucosylation of enterobactin in Salmonella is the iro-gene cluster (or 'iroA locus') [START_REF] Hantke | Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN[END_REF]. This locus consists of two convergent transcription units: iroBCDE and iroN [START_REF] Ba ¨umler | IroN, a novel outer membrane siderophore receptor characteristic of Salmonella enterica[END_REF]. Salmochelin synthesis involves the di-glucosylation of enterobactin [START_REF] Bister | The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica[END_REF]; the resulting salmochelin is then exported across the cytosolic membrane by IroC [START_REF] Crouch | Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium[END_REF]; Fig. 2). Once complexed with ferric iron, ferric-salmochelin is taken up across the outer membrane via IroN [START_REF] Hantke | Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN[END_REF] and is subsequently linearized to the S2 form by the periplasm IroE esterase [START_REF] Lin | In vitro characterization of salmochelin and enterobactin trilactone hydrolases IroD, IroE, and Fes[END_REF][START_REF] Zhu | Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization[END_REF]. The S2 form is then transported into the cytoplasm via FepBCDG (which also imports enterobactin) [START_REF] Crouch | Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium[END_REF]). The imported S2 salmochelin is further esterified by IroD into monomeric and/or dimeric forms, which is presumed to facilitate iron release [START_REF] Lin | In vitro characterization of salmochelin and enterobactin trilactone hydrolases IroD, IroE, and Fes[END_REF][START_REF] Zhu | Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization[END_REF]. The resulting degradation products, S1 and SX (monoglucosylated dimer and monomer, respectively), are exported from the cytoplasm into the medium where they potentially contribute to iron acquisition [START_REF] Lin | In vitro characterization of salmochelin and enterobactin trilactone hydrolases IroD, IroE, and Fes[END_REF][START_REF] Zhu | Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization[END_REF]. Initially, the reason for the glucosylation of enterobactin (generating salmochelin) was unclear. Subsequently, it was discovered that although LCN2 has high affinity for enterobactin (K d of 0.41 ± 0.11 nM) and its derivatives/precursors (DHB, K d of 7.9 ± 1.8 nM), as well as other catecholate-type ferric siderophores (e.g. parabactin, cepabactin and carboxymycobactins; [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF][START_REF] Holmes | Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration[END_REF], it does not effectively bind to salmochelin S4 [START_REF] Fischbach | The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2[END_REF][START_REF] Valdebenito | Special conditions allow binding of the siderophore salmochelin to siderocalin (NGAL-lipocalin)[END_REF]). Furthermore, the LCN2 receptor, 24p3, was shown to mediate the import of ferri-siderophorebound LCN2 into mammalian cells, removing both the iron and enterobactin from circulation [START_REF] Devireddy | A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake[END_REF]. Thus, glucosylation of enterobactin is considered to be a strategy employed by pathogens to prevent siderophore sequestration and removal from circulation by LCN2.

LNC2 was originally identified as a component of neutrophil granules but is also expressed in epithelial cells in response to inflammatory signals [START_REF] Kjeldsen | Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase[END_REF]. [START_REF] Nielsen | Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases[END_REF] revealed that LCN2 might bind lipophilic inflammatory mediators like platelet-activating factor, leukotriene B4 and lipopolysaccharide. This led to the initial suggestion that LCN2 acts as immune-modulatory factor through transport of lipophilic molecules to inflammation sites [START_REF] Goetz | Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin[END_REF]. As eluded to above, a clearer 123 purpose for LCN2 became apparent when the protein was produced heterologously in E. coli and was isolated bound, surprisingly, to a red chromophore, which was subsequently identified as enterobactin [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF]. This finding led to further studies demonstrating a role for LCN2 in host-pathogen interactions [START_REF] Bachman | Interaction of lipocalin 2, transferrin, and siderophores determines the replicative niche of Klebsiella pneumoniae during pneumonia[END_REF][START_REF] Fischbach | The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2[END_REF][START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF]). These further studies showed that the iro-gene cluster confers resistance to the growth inhibitory effects of LCN2 in vitro and that mice rapidly succumb to infection by E. coli H9049 harbouring the iro-gene cluster, but not its iro-free counterpart [START_REF] Fischbach | The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2[END_REF]. Other studies showed that salmochelin contributes to virulence of both avian pathogenic and uropathogenic E. coli (APEC and UPEC) through its iron-binding activity [START_REF] Gao | Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model[END_REF]. Indeed, salmochelin-defective mutants of APEC E058 and UPEC U17 showed significantly decreased pathogenicity compared to the wild-type strains in a chicken infection model [START_REF] Gao | Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model[END_REF]. Likewise, the efficient glucosylation (IroB), transport (IroC and IroN) and processing (IroD and IroE) of salmochelins were shown to be required for APEC virulence [START_REF] Caza | Specific roles of the iroBCDEN genes in virulence of an avian pathogenic Escherichia coli O78 strain and in production of salmochelins[END_REF]. The role of glucosylation in S. enterica pathogenicity was further illustrated by the observation that the iro locus confers a competitive advantage to ST in colonizing the inflamed intestine of wild-type, but not of LCN2deficient, mice [START_REF] Raffatellu | Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype typhimurium for growth and survival in the inflamed intestine[END_REF]). It should be noted that the glucosylation and linearisation of enterobactin was suggested to enhance the activity of salmochelin through increasing its hydrophilic nature, which might be advantageous for iron scavenging in a membrane-rich microenvironment [START_REF] Luo | Enzymatic tailoring of enterobactin alters membrane partitioning and iron acquisition[END_REF].

Egg-white 'lipocalins': role in enhancing iron restriction through sequestration of bacterial siderophores?

Evidence for the presence of lipocalin-like proteins in egg white LCN2 belongs to the 'lipocalin superfamily' which includes a variety of proteins involved in transport of hydrophobic ligands, such as purpurin, retinol-binding protein, a-1-glycoprotein, apolipoprotein, probasin, a-1-microglobulin and prostaglandin D synthetase. Although the family members display low overall sequence identity [START_REF] Greene | Conserved signature proposed for folding in the lipocalin superfamily[END_REF], lipocalins share a common three-dimensional structure characterised by an eight-stranded b-barrel (with a small C-terminal helix) that forms a chalice, at the bottom of which the hydrophobic ligand is bound (Franc ¸oise 1994). Due to their diversity, lipocalin-like proteins have various functions e.g. in immune response, pheromone transport, biological prostaglandin synthesis, retinoid binding and cancer cell interactions [START_REF] Flower | The lipocalin protein family: structure and function[END_REF]. Lipocalins can be divided into two major subfamilies (see the Pfam database; El-Gebali et al. 2019). One subfamily (PF00061) consists of * 4000 Pfam entries that are mostly (88%) from Metazoan species, and includes LCN2, whereas the other subfamily (PF08212) consists of * 3000 entries, mostly from (67%) Bacteria. Lipocalins are predominantly (92%) single domain proteins and multiple homologues are found in vertebrates (e.g. there are 37 lipocalins identified in the human genome; [START_REF] Du | A systematic analysis of human lipocalin family and its expression in esophageal carcinoma. Sci Rep 5:1-14 EFSA Panel on Biological Hazards[END_REF].

Since lipocalins are found throughout most of the living kingdom, it may not be surprising to find that they are present in egg white. Extracellular fatty-acidbinding protein (Ex-FABP) was the first lipocalin-like protein identified in egg white and was discovered by a proteomic analysis of hen egg white using 2-dimensional gel electrophoresis (2-DE) followed by liquid chromatography-mass spectroscopy (LC-MS/MS) [START_REF] Desert | Comparison of different electrophoretic separations of hen egg white proteins[END_REF]. Further work by Gue ´rin-Dubiard et al. ( 2006) using 2-DE, LC-MS/MS and MALDI-TOF identified a total of 16 proteins in hen egg white, including Ex-FABP as well as two other lipocalin-like proteins: chondrogenesis-associated lipocalin (Cal-c or prostaglandin D synthase) and a-1-ovoglycoprotein. The presence of the three lipocalin-like proteins in egg white was later confirmed by further proteomic analyses involving 1-DE with LC-MS/MS, 2-DE combined with protein-enrichment (peptide ligand libraries) technology, and a dual-pressure linear-iontrap Orbitrap instrument (LTQ Orbitrap Velos) [START_REF] Mann | The chicken egg white proteome[END_REF][START_REF] D'ambrosio | Exploring the chicken egg white proteome with combinatorial peptide ligand libraries[END_REF][START_REF] Mann | In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos[END_REF].

Although the concentrations of the three lipocalinlike proteins have not been reported, in the Gue ´rin-Dubiard et al. ( 2006) study the intensity of Cal-c and Ex-FABP 2-DE spots were weak indicating a very low concentration. In other work [START_REF] Mann | The chicken egg white proteome[END_REF][START_REF] Mann | In-depth analysis of the chicken egg white proteome using an LTQ Orbitrap Velos[END_REF], the exponentially-modified-protein abundance index (emPAI) was used to provide an estimate of the absolute abundance of each egg-white protein, which indicated that the lipocalin-like 123 proteins belong to the 'minor proteins' set (such as avidin, cystatin, apolipoprotein D, HEP21, Defensin-11) rather than the 'major proteins' set (such as ovalbumin, ovotransferrin, lysozyme, ovomucoid and ovoinhibitor). However, as a-1-ovoglycoprotein is glycosylated its detection might be obscured [START_REF] Mann | The chicken egg white proteome[END_REF]. In summary, although several studies have shown that three lipocalin-like are present in egg white, their exact concentrations remain unclear. Therefore, their biological significance in egg white remains to be established.

Sequestration of bacterial siderophores by the lipocalin-like Ex-FABP protein found in egg white

Ex-FABP was first discovered as a fatty-acid-binding protein with a role in hen-embryo development [START_REF] Cancedda | Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes[END_REF] were the first to report and identify Ex-FABP (Ch21) as a protein expressed and secreted by in vitro differentiating hen chondrocytes at a late stage of development. Ex-FABP was later shown to be a 21 kDa protein in cartilage [START_REF] Cancedda | Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes[END_REF], muscle tissue [START_REF] Gentili | Expression of the extracellular fatty acid binding protein (Ex-FABP) during muscle fiber formation in vivo and in vitro[END_REF]) and granulocytes [START_REF] Dozin | Expression, regulation, and tissue distribution of the Ch21 protein during chicken embryogenesis[END_REF]) of chicken embryos. This protein was classified as a member of the superfamily of lipocalins and thus was considered to have a likely role in the transport of small hydrophobic molecules [START_REF] Cancedda | The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins[END_REF]). The protein was renamed (from CH21) 'extracellular fatty acid-binding protein' because of its ability to selectively bind and transport fatty acids (i.e. oleic, linoleic, and arachidonic acid) in extracellular fluids and serum [START_REF] Cancedda | The developmentally regulated avian Ch21 lipocalin is an extracellular fatty acid-binding protein[END_REF]. It was shown to be expressed during muscle-fibre formation [START_REF] Gentili | Expression of the extracellular fatty acid binding protein (Ex-FABP) during muscle fiber formation in vivo and in vitro[END_REF] and later shown to have involvement in endochondral-bone formation [START_REF] Cermelli | Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: 'physiological' acute phase response in endochondrol bone formation[END_REF][START_REF] Gentili | Acute phase lipocalin Ex-FABP is involved in heart development and cell survival[END_REF]. Transfection of proliferating chondrocytes and myoblasts with an expression vector expressing antisense Ex-FABP cDNA led to a decreased cell viability. Therefore, Ex-FABP seems to play a part in cell differentiation and cell survival (Di [START_REF] Marco | Inhibition of cell proliferation and induction of apoptosis by Ex-FABP gene targeting[END_REF][START_REF] Gentili | Acute phase lipocalin Ex-FABP is involved in heart development and cell survival[END_REF]. It was more recently shown that Ex-FABP binds the C16 and C18 isoforms of lysophosphatidic acid (LPA, 1-or 2-acyl-sn-glycerol-3-phosphate) [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF]. LPAs are phospholipids mediating differentiation, inflammation, immune function, oxidative stress, cell migration, smooth muscle contraction, apoptosis and development [START_REF] Zhao | Lysophophatidic acid (LPA) and its receptors: role in airway inflammation and remodeling[END_REF]. It is likely that the functions of Ex-FABP reported above depend on its role in sensing or transporting phospholipids [START_REF] Sia | Siderocalins: siderophore binding proteins evolved for primary pathogen host defense[END_REF].

Ex-FABP also binds siderophores and inhibits bacterial growth

More recent reports indicate that Ex-FABP functions in pathogen defence through an ability to bind siderophores, in a manner analogous to that of LCN2 [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF][START_REF] Gare ´naux | Avian lipocalin expression in chickens following Escherichia coli infection and inhibition of avian pathogenic Escherichia coli growth by Ex-FABP[END_REF]. This suggests that Ex-FABP may have two distinct purposes, one in fatty acid/LPA binding and another as a siderophore-binding factor. Work of [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF] shows that Ex-FABP sequesters ferric-enterobactin, as well as its mono-glucosylated (Fe-MGEnt) form with a K d of 0.22 and 0.07 nM, respectively; but not its di-glucosylated form (Fe-DGEnt; K d- [ 600 nM). Furthermore, Ex-FABP at 5 lM caused growth inhibition of both E. coli and Bacillus subtilis under iron-limited in vitro conditions. Growth was restored by supplementing the cultures with stoichiometric amounts of FeCl 3 [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF]). Thus, Ex-FABP might act to reduce bacterial growth in egg white by enhancing iron restriction (Fig. 3). Ex-FABP did not inhibit Pseudomonas aeruginosa growth under iron limitation, which correlates with the observation that Ex-FABP does not bind the corresponding siderophores. Indeed, both enterobactin and bacillibactin produced by E. coli and B. subtilis (respectively) were found to be sequestrated by Ex-FABP (K d of 0.5 and 30 nM, respectively), while pyochelin and pyoverdine produced by P. aeruginosa were not [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF]. These findings are also in accordance with those from Gare ´naux et al. (2013) showing that E. coli K-12 is subject to a 10 5 -fold growth reduction when exposed to 2.5 lM Ex-FABP or LCN2. However, when transformed with a plasmid harbouring the iroBCDEN cluster, no growth defect was observed by 2.5 lM Ex-FABP or LCN2. Exposure of six poultry APEC isolates to 2.5 lM Ex-FABP or LCN2 inhibited the growth of strains producing enterobactin as sole siderophore, but not those producing additional siderophores (salmochelin, aerobactin and/or yersinabactin) [START_REF] Gare ´naux | Avian lipocalin expression in chickens following Escherichia coli infection and inhibition of avian pathogenic Escherichia coli growth by Ex-FABP[END_REF]). Therefore, it can be concluded that Ex-FABP is an avian siderocalin-type lipocalin with a function similar to that of LCN2.

123

The pleiotropic function (i.e. siderophore and LPA binding) of Ex-FABP might be explained by the large binding site of the molecule [START_REF] Sia | Siderocalins: siderophore binding proteins evolved for primary pathogen host defense[END_REF]). Ex-FABP has a three-dimensional fold common to that of lipocalin family proteins but has an extra a-helix (residues 22-30) and short helical element (residues 139-141). This results in an extended calyx that encompasses upper and lower cavities (Fig. 4). The upper cavity comprises a siderophore-binding site with three catechol-binding pockets involving basic residues (K82, R101 and R112) key to ligand binding [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF]. K82 forms hydrogen bonds with the 3-OH of the catechol groups, while R101 and R112 provide significant electrostatic contributions to ligand-binding. The lower cavity acts as a hydrophobic binding site that can bind C16 and C18 LPA. Modelling of the complex shows that the side-chains Y50, K82, R112 and Y114 of Ex-FABP make hydrogen bonds with LPA [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF].

The other lipocalins of egg white

Phylogenic relationship

A multiple-sequence alignment and phylogenetic analysis of the three lipocalin-like proteins of egg white is presented in Figs. 5 and6. The tree can be organised into three lobes, as described by [START_REF] Flower | The lipocalin protein family: structural and sequence overview[END_REF]: proteins in the green lobe include prostaglandin D synthase (PTGDS), neutrophil lipocalin and a-1-microglobulin (a1 M); the blue lobe includes bilin-binding protein (BBP), retinol-binding protein (RBP) and apolipoprotein D (apoD); and the orange lobe is formed of major-urinary protein (MUP) and b-lactoglobulin (b-lg). This phylogenetic analysis indicates that both Cal-c and a-1-ovoglycoprotein have a human orthologue. This is in accordance with a comparative analysis of the chicken genome that showed that 60% of chicken protein-coding genes have a single human orthologue (Consortium ICGS 2004). This tree also indicates that LCN2 from H. sapiens is more closely related to Cal-c than to the other two lipocalin-like proteins found in chicken egg white, and that a-1-ovoglycoprotein could be considered as an outlier among the lipocalin family. Yet, despite their limited sequence identities (26%; NCBI 2019), hen Ex-FABP and human LCN2 have similar ligand-binding affinities [START_REF] Correnti | Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities[END_REF]). According to Fig. 6, there is a close homologue of known function to Ex-FABP found in quail: the Q83 lipocalin (88% identity; NCBI 2019). Q83 was originally identified based on its overexpression in quail embryo fibroblasts transformed with the v-myc oncogene. Q83 sequesters enterobactin with a mode of binding equivalent to that of LCN2 [START_REF] Coudevylle | The v-myc-induced Q83 lipocalin is a siderocalin[END_REF]. This resembles Ex-FABP's function, as described above, in siderophore inhibition, and is consistent with its presence in egg white. Surprisingly, 2019). The Uniprot accession number of each protein is in brackets. Three motifs are shown (*) centred on the conserved tryptophan, threonine and arginine residues. These residues are preserved in lipocalins and seal the bottom end of the barrel, along with the 3 10 -helix [START_REF] Bao | The Ligands of neutrophil gelatinase-associated lipocalin[END_REF]. Two cysteine residues (#) playing a role in disulphide bridging are also conserved. Residues (putatively) involved in siderophore binding are circled in yellow for LCN2: R81, K125, K134 [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF] and blue for Q83: K83, R102, R113 [START_REF] Coudevylle | The v-myc-induced Q83 lipocalin is a siderocalin[END_REF]. For Ex-FABP, residues involved in siderophore (K82, R101, R112) and LPA (Y50, K82, R112, Y114) binding are circled in orange. The b-strands of the eightstranded b-barrel of the LCN2, Q83 and Ex-FABP are underlined in yellow, orange and blue, respectively 123 sequence identity, respectively; NCBI 2019). This suggests that the siderophore-binding activities of these two proteins have evolved independently, in related proteins, in order to fulfil similar functional requirements in innate immunity.

Alpha-1-ovoglycoprotein and Cal-c: their potential functions in egg white

Alpha-1-ovoglycoprotein (or 'orosomucoid') shares a closely-related common ancestor with other a1-acid glycoproteins found in various animals (Fig. 6). Despite its induction as an acute-phase protein, and Fig. 6 Neighbour-joining tree of the lipocalin-like proteins found in egg white and their closest homologues. The alignment of lipocalin-like proteins (Fig. 5) extracted from the Pfam family PF00061 and NCBI (2019) was used to build the phylogeny tree with CLC Genomics Workbench (protein distance was estimated from the Jukes-Cantor model). Three lobes are identified with coloured half-circles (green for NGAL, PTGDS and a1M; orange for b-lg and MUP; blue for RBP, BBP and ApoD). Red stars show the lipocalin homologues found in hen egg white. The blue star indicates human lipocalin-2 its role in cellular inflammation and transport of drugs in human serum, its biological purpose is unclear [START_REF] Huang | Effect of alpha-1-acid glycoprotein binding on pharmacokinetics and pharmacodynamics[END_REF]. In man, this protein is found in the serum where it is heavily glycosylated and highly acidic due to the presence of sialic acid. Human a1acid-glycoprotein (a1-AGP) is a highly glycosylated protein (approximatively 45%) of 43 kDa with a pI of 2.7 [START_REF] Schmid | The plasma proteins (structure, function and genetic control)[END_REF]. There are at least two genes encoding a1-AGP identified, thus it has been suggested that the protein found in plasma is a mixture of the products of these two distinct genes [START_REF] Dente | Structure of the human alpha 1-acid glycoprotein gene: sequence homology with other human acute phase protein genes[END_REF]. The normal plasma concentration in man is between 0.7 and 1.0 g/L. However, as an acute-phase protein, its concentration can increase to 3 g/L under inflammatory conditions [START_REF] Kremer | Drug binding to human alpha-1-acid glycoprotein in health and disease[END_REF]. It is known to bind lipopolysaccharide and can stimulate the activation of inflammatory cell lines [START_REF] Boutten | a 1 -Acid glycoprotein potentiates lipopolysaccharide-induced secretion of interleukin-1b, interleukin-6 and tumor necrosis factor-a by human monocytes and alveolar and peritoneal macrophages[END_REF]. Interestingly, a1-AGP has a protective effect in a mouse meningococcal shock model, suggesting a potential antibacterial role [START_REF] Moore | Alpha-1 acid glycoprotein (orosomucoid): interaction with bacterial lipopolysaccharide and protection from sepsis[END_REF]. In egg white, the a-1-ovoglycoprotein has an average molecular weight of 30 kDa, an isoelectric point of 4.37-4.51 and a sugar content of about 25% [START_REF] Matsunaga | Identification of disulfide bonds and site-specific glycosylation in chicken? 1-acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry[END_REF]). While little is known about its function, this ovoglycoprotein is often used for its chiral properties to separate drug enantiomers [START_REF] Sadakane | Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition[END_REF][START_REF] Haginaka | Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein[END_REF]. However, its biochemical, functional and biological properties in egg white remain unknown.

The phylogenetic analysis (Fig. 6) indicates that the closest homologue of Cal-c is lipocalin-like prostaglandin synthase (PTGDS). In mammals, PTGDS is secreted into various body fluids. This protein catalyses the isomerization of prostaglandin H2 to prostaglandin D2, and was reported to bind a variety of lipophilic molecules such as biliverdin, bilirubin and retinoic acid. In humans, this protein is likely to be involved in both maturation and maintenance of the central-nervous system and male reproductive system [START_REF] Saito | Prostaglandin D 2 and Reproduction[END_REF][START_REF] Urade | Biochemical, structural, genetic, physiological, and pathophysiological features of lipocalin-type prostaglandin D synthase[END_REF]. Two isoforms, of 22 kDa, can be separated by 2D-electrophoresis of egg white, thanks to their different isoelectric points (pI of 5.6 and 6.0) (Gue ´rin- [START_REF] Gue ´rin-Dubiard | Proteomic analysis of hen egg white[END_REF]. [START_REF] Pagano | A chondrogenesis-related lipocalin cluster includes a third new gene[END_REF] have shown that Cal-c expression correlates with endochondral bone formation and the inflammatory response. As for Ex-FABP, Cal-c mRNA is increasingly synthesized during chondrocyte differentiation both in vivo and in vitro. Although Ex-FABP and Cal-c may both play a part in bone formation and the inflammatory response, any possible role for Cal-c in siderophore sequestration remains to be explored.

Conclusion

The antibacterial iron-restriction activity of egg white, as mediated by oTf, is well established and it is now apparent that the egg-white lipocalin, Ex-FABP, can inhibit bacterial growth via an enhanced iron-restriction effect that is mediated by its siderophore-binding capacity. However, the siderophore-sequestering activity of Ex-FABP has neither been studied in egg white, nor with appropriate SE or hen infection models. Furthermore, how and in what quantity this protein is incorporated into egg white remains unknown. As yet, it is unclear whether the other lipocalins of egg white (Cal-c and a-1-glycoprotein) might also sequester siderophores. Although many egg-white proteins have been shown to be components of the arsenal of defence factors within egg white, the contribution (if any) of the three lipocalins as new eggwhite defence factors remains an open question, although this now appears highly likely for Ex-FABP. As matters stand, it is unclear whether the capacity of salmochelin to assists SE virulence in mammalian models can be extended to include support of SE survival in egg white. Thus, there remains much scope for further understanding of the role of lipocalin proteins in the defence of egg white against bacteria.

Fig. 1

 1 Fig. 1 Enterobactin (Ent), Salmochelin (S4) and related structures. a Structure of enterobactin. b Schematic representation of Ent, S4 and its derivatives. Salmochelin S4 and S2 are both di-glucosylated forms of enterobactin (DGEnt) but the latter is linear. MGEnt is a 2,3-dihydroxybenzoyl serine

Fig. 2

 2 Fig. 2 Summary of synthesis, export, import and utilisation, of enterobactin and salmochelin. Enterobactin (synthesised by EntABCDEF) is mono or di-glucosylated by IroB. Enterobactin is exported from the cytoplasm by EntS whereas salmochelin is exported by IroC. TolC is then involved in enterobactin efflux across the outer membrane. Once complexed with ferric iron, ferri-salmochelin is taken up across the outer membrane via IroN and is then linearized to the S2 form by the periplasm IroE esterase, whereas ferri-enterobactin is taken up via CirA, or

Fig. 3

 3 Fig. 3 Potential interactions between SE siderophores and Ex-FABP. In the egg white, enterobactin (Ent) and MGEnt might be sequestrated by Ex-FABP (represented by black semi-circles), while DGEnt would remain free to chelate iron from

Fig. 4 a

 4 Fig. 4 a X-ray diffraction (1.8 A ˚resolution) of Ex-FABP; two ligands are represented, ferric-DHB in the upper cavity and LPA in the lower cavity. Structure extracted from Protein Data

Acknowledgements This work was supported by the Strategic Fund of the University of Reading (UK) and the Regional Council of Britanny (allocations de recherche doctorale, ARED; France).

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