Skip to main content
NCBI home page

This is a preprint.

It has not yet been peer reviewed by a journal.

The National Library of Medicine is running a pilot to include preprints that result from research funded by NIH in PMC and PubMed.

bioRxiv logoLink to bioRxiv
[Preprint]. 2023 Jul 25:2023.07.25.550588. [Version 1] doi: 10.1101/2023.07.25.550588

Uropathogenic Escherichia coli wield enterobactin-derived catabolites as siderophores

Zongsen Zou 1,2,#, John I Robinson 1,2, Lindsey K Steinberg 1,2, Jeffrey P Henderson 1,2,*
PMCID: PMC10402112  PMID: 37546885

Abstract

Uropathogenic E. coli (UPEC) secrete multiple siderophore types to scavenge extracellular iron(III) ions during clinical urinary tract infections, despite the metabolic costs of biosynthesis. Here we find the siderophore enterobactin and its related products to be prominent components of the iron-responsive extracellular metabolome of a model UPEC strain. Using defined enterobactin biosynthesis and import mutants, we identify lower molecular weight, dimeric exometabolites as products of incomplete siderophore catabolism, rather than prematurely released biosynthetic intermediates. In E. coli, iron acquisition from iron(III)-enterobactin complexes requires intracellular esterases that hydrolyze the siderophore. Although UPEC are equipped to consume the products of completely hydrolyzed enterobactin, we find that enterobactin and its derivatives may be incompletely hydrolyzed to yield products with retained siderophore activity. These results are consistent with catabolic inefficiency as means to obtain more than one iron ion per siderophore molecule. This is compatible with an evolved UPEC strategy to maximize the nutritional returns from metabolic investments in siderophore biosynthesis.

INTRODUCTION

Urinary tract infections (UTI) are among the most common outpatient and inpatient infections encountered by physicians (1–4). Escherichia coli is the bacterial species most commonly associated with UTI, accounting for about 70% ~ 95% of clinical cases (5,6). Clinical E. coli isolates associated with UTI that exhibit polymorphisms in conserved genes (7–9) and carry accessory genes associated with increased pathogenic potential are designated as uropathogenic E. coli (UPEC) (2,4,10). Prominent among these virulence-associated adaptions are iron uptake systems, such as siderophores, which use distinctive chemical groups to competitively bind iron and render it selectively bioavailable to support bacterial growth (2,3,10–14). In UPEC, siderophore iron-acquisition systems have been identified as both colonization factors and virulence factors during UTI pathogenesis (15–19). The enterobactin, salmochelin, yersiniabactin, and aerobactin siderophore systems have all been associated with E. coli strains that causing extraintestinal infections (20–22).

Siderophores are specialized, secreted metabolites (exometabolites) that are synthesized by non-essential bacterial pathways and competitively chelate extracellular iron(III) during the iron-limited growth conditions characteristic of infection microenvironments (16,17,23,24). The resulting iron(III)-siderophore complexes are selectively imported by bacterial transporters as an iron source. E. coli and many other gram-negative bacteria actively transport iron-siderophore complexes through outer membrane receptors using the cytoplasmic membrane localized TonB/ExbB/ExbD complex, which transduces energy from the proton motive force (25–27). Siderophore biosynthesis and transport systems are regulated by the ferric uptake regulator (Fur), a transcriptional repressor that downregulates siderophore gene transcription in conditions associated with high cytosolic iron (28).

All UPEC carry the conserved enterobactin system and may encode up to three additional siderophore systems, each associated with chemically distinctive exometabolomes (29–31). Biosynthesis of these additional exometabolites incurs additional metabolic demands (32), suggesting that their sustained presence in clinical populations is associated with siderophore-specific payoffs. For example, the salmochelin system, encoded by genes in the iroA cassette, glucosylates enterobactin to improve its aqueous solubility and evade sequestration by the host immune protein lipocalin-2/siderocalin/NGAL (14,33,34). The yersiniabactin system in UPEC supports multiple non-siderophore functions not associated with enterobactin or salmochelin (35,36). Yersiniabactin production incurs metabolic costs, which appear to be mitigated by an ability to recycle the intact siderophore to support multiple rounds of metal ion import (37) and an additional quorum-sensing regulatory input that emphasizes biosynthesis in diffusionally restricted or crowded environments where the siderophore is more likely to remain nearby (38).

Enterobactin (Ent) is detectable in the urine of patients with urinary tract infections, where its synthesis is required to evade growth inhibition by Lipocalin-2 (13,14). Ent achieves exceptional iron (III) affinity (Kd ≈ 10-52 M) with three catechol (1,2-dihydroxybenzene) groups that provide all six coordination sites for iron(III) (10,39). Ent is synthesized by a nonribosomal peptide synthetase (NRPS) system encoded by entABCDEF. This NRPS system is a molecular assembly line that synthetizes Ent by repeatedly forming enzyme-bound N-(2,3-dihydroxybenzoyl)serine (DHBS) and linking them via ester bonds until a cyclic trilactone core composed of three DHBS is released (40,41). In UPEC expressing the iroA cassette, the glucosyltransferase IroB further modifies Ent catechols with up to three distinctive C-linked glucoses (10,42). Iron retrieval from imported iron(III) Ent complexes (with or without C-glucose modifications) requires dissociation through both esterase-catalyzed Ent hydrolysis (by Fes and/or IroD) and iron (III) reduction to iron(II) (43–45).

In this study we examined the enterobactin biosynthetic pathway’s contribution to the iron-dependent UPEC exometabolome. We measured used targeted mutant strains and chemical complementation with purified products to assess the catabolic origins of short-length catechol exometabolites. To assess the nutritional potential of siderophore catabolism, we used reverse stable isotope labeling to find that 2,3-dihydroxybenoic acid from outside the biosynthetic pathway could be used for Ent biosynthesis. Finally, we used a siderophore-dependent growth condition to evaluate the siderophore potential of non-trimeric enterobactin metabolites found in the UPEC exometabolome. Our findings are consistent with a catabolic network that has evolved to maximize the iron delivery potential of Ent biosynthesis.

RESULTS

Enterobactin and the iron-responsive exometabolome in uropathogenic E. coli.

To define the iron-responsive exometabolome of uropathogenic E. coli and its relationship to the ent-encoded biosynthetic pathway, we compared small molecule profiles in conditioned media from the model UPEC strain UTI89 and its isogenic biosynthesis-deficient mutant, UTI89∆entB (21), in low and high iron conditions (32) using LC-MS. Sparse principle components analysis (sPCA) of these data demonstrated discrete groupings along principle component 1 (PC1, Fig. 1A) for the experimental groups that account for 26.8% of total variation between specimens (Fig. 1A, supplemental Fig. S1A). The greatest separation was evident between wild type UTI89 grown in low iron medium and the other experimental groups, which were fully separated along PC1. Logistic regression of PC1 values to classify these two PC1 exometabolome clusters yielded a prediction accuracy of 1.0 (SD = 0, supplemental Fig. S1B) and an AUC of 1.0 (SD = 0, supplemental Fig. S1C) with four-fold cross validation. PC1 differences did not correspond to inter-group differences in growth density. (supplemental Fig. S2). The distinctive PCA grouping of wild type UTI89 grown in low iron corresponds with detection of enterobactin, the canonical, eponymous product of the enterobactin biosynthesis pathway (Fig. 1B). Together, these results are consistent with a prominent role for the enterobactin biosynthetic pathway in defining the iron-responsive UTI89 exometabolome.

Figure 1. The enterobactin biosynthetic pathway is a prominent contributor to the iron-responsive uropathogenic E. coli UTI89 exometabolome.

Figure 1.

Open in a new tab

(A) Score plot from sparse PCA (sPCA) analysis of media conditioned by UTI89 grown in low and high iron media (wild type and wild type+Fe, respectively) and the enterobactin-null mutant UTI89∆entB in low and high iron media (entB and entB +Fe, respectively). High iron medium is achieved by addition of 100 µM FeCl3 (B) LC-MS/MS chromatograms corresponding to the precursor-product ions from enterobactin for each experimental group. Chromatograms are displayed in identical ion current unit scales. (C) PC1-loadings plot demonstrates that multiple ions contribute to PC1. The top 13 metabolites with greater abundance in wild type UTI89 (lower PC1 value) are identified as red data points.

Multiple enterobactin-associated products define the UTI89 exometabolome.

The exometabolites that define principal components in these data may be identified by loadings analysis. Loadings analysis of PC1, corresponding to the greatest variance in exometabolites, identified multiple molecules enterobactin alone (Fig. 1C). Detailed mass spectrometric and chromatographic analyses of the 13 molecular features with the largest PC1 loadings associated with the UTI89 exometabolome under iron-restricted conditions (supplemental Fig. S3-12) identified a series of 10 N-(2,3-dihydroxybenzoyl)serine (DHBS) polymers (Fig. 2A) consistent with enterobactin (Ent) and salmochelin biosynthesis (46–48) (Table S1). These include cyclic and linear DHBS trimers with 0, 1, or 2 C-glucosylations, DHBS dimers with 0, 1, or 2 C-glucosylations, and monomeric DHBS previously reported in an avian pathogenic E. coli (APEC) strain. Unlike the APEC strain, UTI89 did not produce tri-glucosylated Ent products (54), consistent with inter-strain differences in the Ent exometabolome that are not explained by iroA alone. To more precisely quantify these exometabolites, we constructed a high-resolution targeted LC-MS/MS multiplexed selected reaction monitoring (LC-MRM) method (Table 1). We confirmed that all 10 products were present in low iron media conditioned by wild type UTI89, were significantly diminished in high iron media conditioned by UTI89, and were undetectable in any media conditioned by UTI89∆entB (Fig. 2B, supplemental Fig. S13, supplemental Fig. S14, P < 0.001). In an iroA-null strain (UTI89ΔybtSΔiroA) that lacks the C-glucosylation pathway, C-glucosylated exometabolites were absent, while non-glucosylated exometabolites were elevated, (Fig. 2B, supplemental Fig. S14, P < 0.001), consistent with the precursor-product relationship between these exometabolites. Together, these results connect iron-associated biosynthetic activity in uropathogenic E. coli to multiple enterobactin-related exometabolites extending beyond the canonical trimeric DHBS products.

Figure 2. Exometabolites associated with the iron-responsive UTI89 exometabolome.

Figure 2.

Open in a new tab

(A) Chemical structures of the ten enterobactin-associated exometabolites identified by comparative metabolomic analysis, including enterobactin (Ent), monoglucosylated enterobactin (MGE), diglucosyalted enterobactin (DGE), linear enterobactin (lin-Ent), linear monoglucosylated enterobactin (lin-MGE), linear diglucosylated enterobactin (lin-DGE), N-(2,3-dihydroxybenzoyl)serine dimer [(DHBS)2], monoglucosylated N-(2,3-dihydroxybenzoyl)serine dimer [G1-(DHBS)2], diglucosyalted N-(2,3-dihydroxybenzoyl)serine dimer [G2-(DHBS)2], and N-(2,3-dihydroxybenzoyl)serine monomer (DHBS). The positions of C-glucosylated DHBS units within linear polymers have not been definitively identified. (B) Heatmap showing enterbactin-associated exometabolite concentrations in media with iron supplementation or defined biosynthetic mutants of UTI89. Intensity represents concentration expressed as ratio of LC-MS/MS peak area to that of internal standard. Individual biological replicates are shown for each condition.

Table 1.

Targeted LC-MS/MS protocols for detecting and quantifying ten ent siderophores and short-length products.

Identity Multiple reaction RT (min) Reference
Q1 Q3
Ent 668 222 8.47 (21,46,49–52)
lin-Ent 686 222 7.21 (21,46,47,49)
MGE 832 222 6.94 (46,47,49)
lin-MGE 848 222 5.89 (46,47,49)
DGE 993 222 5.95 (46,47,49,53)
lin-DGE 1010 222 4.98 (21,46,47,49,53)
(DHBS)2 464 222 5.92 (46,49–52)
G1-(DHBS)2 625 222 4.69 (46,47,49,53)
G2-(DHBS)2 787 402 3.64 (46,47,49)
DHBS 240 153 2.34 (46,49–52)
Open in a new tab

Outer membrane importers differentially affect enterobactin-associated exometabolites.

While trimer products are consistent with the enterobactin biosynthetic pathway, the specific origin of short-length dimeric and monomeric products, (DHBS)2, (G2-(DHBS)2, G1-(DHBS)2, and DHBS is unclear. We considered that these truncated products could reflect premature release from the biosynthetic pathway (anabolic production) (11,55), spontaneous extracellular hydrolysis, or intracellular esterolysis of imported, ferric catechol siderophores (catabolic production) (46,47,49). To distinguish these possibilities, we compared UTI89 with UTI89ΔtonB, an isogenic mutant with a deficiency in siderophore import at the outer membrane. In E. coli and related gram-negative bacteria, the TonB/ExbB/ExbD complex energizes outer membrane transporters to import ferric siderophores (56). Relative to UTI89, UTI89ΔtonB cultures exhibit a strikingly dichotomous effect on enterobactin-associated exometabolites, with elevated trimer concentrations and diminished dimer and monomer concentrations (Fig. 3). These differences were not associated with differential growth density between groups (supplemental Fig. S15). These results are consistent with intracellular dimer and monomer production in events that are downstream from extracellular trimer import.

Figure 3. Outer membrane import differentially affects trimeric and non-trimeric enterobactin-associated exometabolites in culture.

Figure 3.

Open in a new tab

Enterobactin-associated exometabolite concentrations in media conditioned by UTI89 (wild type), an import-deficient UTI89 mutant (ΔtonB), or co-culture of enterobactin-null and import-deficient UTI89 mutants (ΔtonB+ΔentB). Y-axis is concentration expressed as ratio of LC-MS/MS peak area to that of internal standard. (A) Ent. (B) lin-Ent. (C) MGE. (D) lin-MGE. (E) DGE. (F) lin-DGE. (G) (DHBS)2 (H) G1-(DHBS)2. (I) G2-(DHBS)2. (J) DHBS. Statistics were performed using 1-way ANOVA with Dunnett’s multiple-comparison test with P ≤ 0.05 considered as statistically significant. ns: not significant. *: P <= 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001.

Co-culture with import-proficient UTI89 complements the UTI89ΔtonB phenotype.

To further test the hypothesis that monomer and dimer exometabolites are products of siderophore catabolism, we devised a co-culture system in which UTI89ΔtonB is poised to serve as a siderophore-producer and enterobactin-deficient UTI89ΔentB as a siderophore consumer. We hypothesized that UTI89ΔentB import of UTI89ΔtonB-derived exometabolites would counteract the UTI89ΔtonB dimer and monomer deficiency phenotype. Compared to UTI89ΔtonB-conditioned media, media conditioned by the UTI89ΔtonB + UTI89ΔentB co-culture contained significantly greater monomer and dimer concentrations, and variably lower trimer concentrations (Fig. 3). As such, the combined ent exometabolome of UTI89ΔtonB + UTI89ΔentB more closely resembled that of wild type UTI89 than either mutant alone. Different levels of enterobactin-associated products were not associated with growth density differences between groups (supplemental Fig. S15). These results are consistent with extracellular UTI89ΔtonB-derived trimers as public goods that are imported by UTI89ΔentB, which partially catabolizes them and releases esterolysis products to the extracellular space (47,49).

Monomer and dimer production during trimer-dependent growth.

Enterobactin-associated trimers contain two or three serine-serine ester bonds and three serine-DHB peptide bonds (Fig. 2A) with potential for hydrolysis to yield free DHB and serine, which may become new metabolic substrates in the cytoplasm. Despite this catabolic potential, UTI89 releases incompletely hydrolyzed trimer catabolites. To determine whether this occurs during siderophore-dependent growth, we measured the enterobactin-associated exometabolomes of siderophore-null UTI89 (UTI89∆entB∆ybtS) cultures with trimer supplementation. Growth of this strain was rendered siderophore-dependent by addition of bovine serum albumin, a biologically relevant, non-specific binder of labile iron ions (57,58). Compared to siderophore-free controls, Ent, MGE, or DGE addition stimulated UTI89∆entB∆ybtS growth (Fig. 4) and were progressively consumed during culture (Fig. 5A, D, G), consistent with their canonical siderophore activity. Dimer and monomer production varied with the specific trimer provided. Ent supplementation yielded neither dimer nor monomer (Fig. 5B, C), MGS supplementation yielded (DHBS)2, G1-(DHBS)2, and DHBS (Fig. 5E, F), and DGS yielded G1-(DHBS)2, G2-(DHBS)2, and DHBS (Fig. 5H, I). Dimer C-glucosylation products are structurally consistent with the C-glucosylation structure of each trimeric substrate. These results are consistent with dimer and monomer production from esterolysis following cyclic trimer-mediated iron-delivery. The lack of dimer or monomer generation from Ent is unexpected based on production by Ent-producing UTI89ΔybtSΔiroA (Fig. 2B). The nature of this discrepency is unclear and may arise from unappreciated catabolic differences, regulatory pathways, or intracellular trafficking connected to these different strains, the different cultures conditions, or combinations thereof.

Figure 4. Trimer supplementation supports siderophore-null UTI89 mutant growth in siderophore-dependent growth medium.

Figure 4.

Open in a new tab

Growth of the siderophore-null strain UTI89∆entB∆ybtS was measured by optical density at 600 nm (OD600) in siderophore-dependent medium following supplementation with Ent, MGE, or DGE and compared to unsupplemented control (ctrl). Statistics were performed using 1-way ANOVA with Dunnett’s multiple-comparison test with P ≤ 0.05 considered as statistically significant. ns: not significant. *: P <= 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001.

Figure 5. Enterobactin-associated exometabolites during trimer-dependent growth.

Figure 5.

Open in a new tab

Siderophore-null strain UTI89∆entB∆ybtS was cultured in siderophore-dependent medium containing purified Ent, MGE, or DGE. The enterobactin-associated metabolome in the medium was measured using LC-MS/MS at time points during culture. (A & B & C) Ent (A) is imported and catabolized by UTI89∆entB∆ybtS without producing any dimer (B) or monomer (C) ent catechol compounds. (D & E & F) MGS (D) is imported and catabolized by UTI89∆entB∆ybtS, which produces (DHBS)2 and G1-(DHBS)2 dimers (E) and DHBS monomer (F). (G & H & I) DGS (G) is imported and catabolized by UTI89∆entB∆ybtS, which produces G1-(DHBS)2 and G2-(DHBS)2 dimers (H) and DHBS monomer (I). Statistics were performed using unpaired t test with P ≤ 0.05 considered as statistically significant. ns: not significant. *: P <= 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001.

UTI89 uses exogenous dihydroxybenzoic acid (DHB) to synthesize enterobactin.

It is unclear why UTI89 foregoes complete catabolic reclamation of intracellular trimer constituents to instead release incompletely hydrolyzed trimer catabolites to the extracellular space. Bonacorsi et al. have connected enhanced bacterial DHB production for siderophore biosynthesis as a virulence-associated activity in neonatal meningitis-associated E. coli, suggesting that UPEC could similarly benefit from DHB reclamation (59). To determine whether UPEC can use exogenously derived DHB to support trimer biosynthesis, we derived an experimental system to monitor its incorporation. Specifically, we used a reverse isotope-labelling strategy to detect incorporation of unlabeled carbon atoms from exogenous DHB during culture with 13C3-glycerol as the carbon source. We found that addition of 200 μM DHB led to the appearance of a new Ent isotopologue with a m/z value 21 atomic mass units lower than 13C-substituted enterobactin, consistent with 12C7-DHB incorporation at all three catechol sites (Fig. 6, Table S2). DHB supplementation also yielded lower levels of singly and doubly substituted isotopologues that are 7 and 14 atomic mass units lower, respectively (supplemental Fig. S16, Table S2). These results demonstrate the capacity for exogenous DHB incorporation into Ent biosynthesis by UTI89, demonstrating the potential value of complete Ent hydrolysis to UPEC.

Figure 6. Exogenous 2,3-dihydrobenzoic acid (DHB) supports enterobactin biosynthesis.

Figure 6.

Open in a new tab

(A) Chemical structure of cyclic enterobactin (Ent) with the three DHB-derived groups, containing seven carbon atoms, highlighted in green. (B) LC-MS/MS detection of fully 13C30-substituted Ent ([M-H]-, m/z 698) in UTI89-conditioned 13C3-glycerol culture medium without (-DHB), or with (+DHB), 200 µM unlabeled DHB. (C) LC-MS/MS detection of 13C9-substituted Ent ([M-H]-, m/z 677) into which three DHB molecules have been incorporated without (-DHB), or with (+DHB), 200 µM unlabeled DHB.

Siderophore activity of purified dimers.

We hypothesized that UPEC forego complete trimer hydrolysis because the resulting dimers retain valuable siderophore activity. This would enable biosynthesis of one trimer molecule to support multiple rounds of iron import. To test this, we evaluated the siderophore activity of purified dimers in the siderophore-dependent growth condition described above. We observed that supplementation with either of two dimer metabolites, (DHBS)2 or G2-(DHBS)2, restored bacterial growth in iron-deficient conditions, with slower growth kinetics for G2-(DHBS)2 dimer than those observed for (DHBS)2 dimer and trimers (Fig. 7A, Fig. 4). DHBS production was generated from (DHBS)2 supplementation and catabolism only (Fig. 7B-E). Glucosylated N-(2,3-dihydroxybenzoyl)serine (G1-DHBS), that was expected to be generated from G2-(DHBS)2 hydrolysis, was poorly resolved in the LC-MS/MS conditions used here, likely because its high hydrophilicity renders it poorly resolved in reversed phase liquid chromatography. Together, these data are consistent with siderophore activity by both C-glucosylated and non-glycosylated dimers.

Figure 7. Enterobactin-associated dimers support siderophore-dependent growth.

Figure 7.

Open in a new tab

(A) Growth of the siderophore-null strain UTI89∆entB∆ybtS was measured by optical density at 600 nm (OD600) in siderophore-dependent medium following supplementation with the enterobactin-associated dimer exometabolites (DHBS)2 or G2-(DHBS)2, or siderophore-free control (ctrl). (B-E) The enterobactin-associated metabolome in the medium was measured using LC-MS/MS at time points during culture with dimer and monomer results shown for (DHBS)2-supplemented cultures (B,C) and G2-(DHBS)2.-supplemented cultures (D,E). Statistics were performed using unpaired t test with P ≤ 0.05 considered as statistically significant. ns: not significant. *: P <= 0.05. **: P < 0.01. ***: P < 0.001. ****: P < 0.0001.

DISCUSSION

Multiple bacterial siderophore systems release exometabolites in addition to their canonical biosynthetic end products. Here, we find that UPEC have the potential to hydrolyze Ent trimers to recover raw materials for new biosynthesis, yet limit this process to instead generate and secrete incompletely hydrolyzed Ent (dimer), which is released as a siderophore. This suggests a bacterial “choice” between complete hydrolysis to maximize catabolic reclamation of biosynthetic substrates and incomplete hydrolysis to generate a dimeric catabolite that acts as a siderophores with lower iron(III) affinity. The former lowers the biosynthetic cost of new trimer biosynthesis while the latter yields another siderophore. The balance between these fates (complete or partial hydrolysis) may reflect evolutionary adaptation or, possibly, active regulation.

Siderophore function, as classically understood, is a metabolically costly process in which siderophore biosynthesis and secretion occurs because there is a chance some of these siderophores will diffuse back as iron complexes to support nutritional demands. For Ent and related siderophores, iron release requires hydrolysis by intracellular esterases, suggesting a diminished return on biosynthetic investment compared to siderophores that are non-destructively “recycled” and re-secreted (37,47). The results above suggest that a more nuanced situation has evolved in which trimer hydrolysis proceeds only to the extent necessary for iron release so that a catabolite may be secreted for additional rounds of siderophore-mediated iron delivery. The growth-promoting siderophore activity of dimers observed here is supported by a previous report of (DHBS)2-mediated 55Fe localization to E. coli (60). Additional supportive evidence was reported for iron-dependent growth of Campylobacter jejuni, an Ent non-producer that uses (DHBS)2 from E. coli as a siderophore in an example of siderophore “piracy” by this organism (61). The siderophore activity of dimers is thus associated with another example of metabolic cost-avoidance.

Enterobactin-associated dimers may have lower iron affinity than trimers, given the role of catechols in iron (III) affinity. A 1:1 dimer-iron complex provides catechol hydroxyl ligands for four of the six iron (III) coordination sites. This tetra-dentate coordination is observed for other siderophores such as pyochelin from Pseudomonas aeruginosa (62,63) and azotochelin from Azotobacter vinelandii (64). Despite this potential drop in affinity, we observed comparable iron acquisition capability by (DHBS)2 dimers and trimers, though differences could emerge in more stringent competitive iron binding conditions. As with DGE, G2-(DHBS)2 may similarly resist sequestration by Lipocalin-2 (14,33,34), rendering it more capable of delivering iron to UPEC during infections.

Incomplete trimer hydrolysis by UPEC suggests that siderophore esterase and peptidase systems have evolved to be inefficient as a means to support dimer-associated iron acquisition. The extent of hydrolysis may vary with the specific trimer and the hydrolases recruited during iron recovery. We found that siderophore-null UTI89 consumed purified Ent without significant monomer or dimer secretion, while purified glucosylated Ent trimers (MGE and DGE) resulted in abundant monomer and dimer formation. It remains unclear whether these differences reflect higher order metabolic interactions or unappreciated regulatory process affecting hydrolase activity, possibly responsive to Ent C-glucosylation. Complete hydrolysis of C-glucosylated trimers could be evolutionarily disfavored due to the likely inability to use C-glucosylated DHB as an Ent biosynthetic pathway. Lin, et al. previously reported that purified Fes and IroD can hydrolyze Ent trimer to produce DHBS monomer and (DHBS)2 dimer, and IroE can hydrolyze Ent to produce (DHBS)2 dimer (47).

We found that generation of short-length enterobactin-associated metabolites from trimers was dependent on TonB-dependent transport, consistent with their catabolic origin. It is likely that dimers similarly require TonB-dependent transporters, which may include transporters different from the Ent or glucosylated Ent transporters (Fep and IroN, respectively) associated with trimer transport. Fiu and Cir have been proposed to import Ent metabolites and catechols (65–68), with Cir serving as an important drug target for the catechol-modified cephalosporin cefiderocol (69–71), consistent with its activity in human infections. The substrate specificity for these transporters and their relationship to the network of enterobactin-associated exometabolites described here is incompletely understood and may yield deeper functional insights into enterobactin system function.

In conclusion, the exometabolite network described here is consistent with a series of regulatory and functional adaptations that minimize costs of Ent-mediated iron delivery in E. coli cells. Enterobactin biosynthesis, a metabolically costly process, is activated under iron-restricted conditions by Fur repressor regulation. At low bacterial density, E. coli have the ability to render Ent a private good, available only to the producing organism, and minimizing diffusional loss (72). Sub-maximal siderophore hydrolysis in UPEC to release dimers extends the iron delivery potential of Ent and its derivatives. Together, these activities results are consistent with a biochemical network connecting intracellular and extracellular E. coli metabolomes to cost-effectively support iron-dependent growth. These findings may help explain why enterobactin expression can be sustained as the universal siderophore system in urinary E. coli isolates. Aspects of this network may be useful in devising new antimicrobial therapeutics for uropathogenic E. coli and related bacteria.

METHODS AND MATERIALS

Bacterial strains and culture conditions

We examined exometabolite production, consumption, and use with the well-characterized, cystitis-derived model UPEC strain UTI89 and its previously described isogenic mutants UTI89ΔentB, UTI89ΔtonB, and UTI89ΔentB∆ybtS (Table S3) (11,13,21,73). UPEC strain CFT073 was used for bacterial secondary metabolite production due to its high yield of C-glucosylated products (Table S3) (11, 13). Bacterial cultures were grown from single colonies in LB broth for overnight under 37, washed with PBS, back-diluted 1:1000 into filter-sterilized M63 minimum media, inoculated with 200 µL into 96-well microplates, and incubated under 37 for in the indicated assays. Experimental cultures were conducted in M63 minimum media containing 0.2% glycerol as a carbon source and 10 µg/mL nicotinic acid (low iron), with 100 µM FeCl3 (high iron), or with 10 µM bovine serum albumin addition (siderophore-dependent)(21, 32). Bacterial growth was quantified by the optical density at 600 nm (OD600) using a Spectrophotometer (Beckman Coulter, DU-800) or an incubated microplate reader (Tecan Spark).

Untargeted liquid chromatography mass spectrometry (LC-MS)

Untargeted full scan LC-MS profiling was performed to characterize the extracellular metabolome (exometabolome) in media conditioned by UTI89 and UTI89∆entB under low and high iron conditions. Conditioned medium was collected by centrifugation and filtration through 0.22 µM filters with storage at −80. Samples were thawed on ice for liquid chromatography mass spectrometry (LC-MS) analysis with a Shimadzu Prominence UFLC-coupled AB Sciex 4000 QTrap mass spectrometer with a Turbo V electrospray ionization (ESI) source. LC separation was performed on an Ascentis Express phenyl-hexyl column (100 × 2.1 mm, 2.7 μm; Sigma-Aldrich) with solvent A (HPLC-grade water + 0.1% Formic acid; Sigma-Aldrich, Fluka) and B (90% Acetonitrile + 0.1% Formic acid; Sigma-Aldrich, Fluka) at 0.35 ml/min in a 36 minute gradient as follows: solvent B increased from 2% to 35% by 23 min, then increased to 98% by 33 min, and finally held steady at 98% for another 3 min. ESI-MS was performed in negative ion-enhanced MS mode, scanning from 50 to 1,500 m/z. A quality control sample was injected first and every ten samples thereafter to assess instrument stability. MarkerView, version 1.2.0 (Sciex) was used for peak alignment, generating the list of peaks for computational metabolome comparison analysis in the next section (13,14,32).

Computational metabolomic comparison

Exometabolome comparisons between four groups of samples, including UTI89 grown in low and high iron media (wild type and wild type+Fe, respectively) and the enterobactin-null mutant UTI89∆entB in low and high iron media (entB and entB +Fe, respectively), was performed on a combined computational model consisting of a sparse principal component analysis (sPCA) followed by a logistic regression (LR) classification. The computation was performed in R and Python, using the scikit-learn module and mixOmics package, respectively (74–77). Of note, sparsity penalization was enforced in the PCA dimensionality reduction step to prevent overfitting for this metabolome metadata consisting of much higher component dimensions than the number of samples (78,79). The iron-responsive sub-metabolome in UTI89 extracellular space were identified by the loadings analysis of all identified metabolites.

Product ion scan and targeted LC-MS/MS

Product ion scan measurements were conducted to characterize chemical structures of the 10 enterobactin-associated molecules. The LC separation as above but with a flow of 0.5 ml/min and a 16 minute gradient as follows. Solvent B increased from 5% to 56% by 10 min, then increased to 98% by 12 min, and finally held steady at 98% for another 4 min. MS/MS product ion spectra of each negative ion was obtained in the enhanced product ion mode (80,81). Targeted LC-MS/MS multiplexed selected reaction monitoring (MRM) analyses were performed to validate the identities of 10 enterobactin-associated metabolites that were determined by the full-scan comparative metabolomic analysis as described above. MRM parameters protocols (Table 1) were established based on the results of product ion scan for each of the 10 targeted enterobactin-associated metabolites (11,13,14,21).

Exometabolite purifications

Enterobactin-associated exometabolites were generated by growing CFT73 in M63/0.2% glycerol medium supplemented with 2,3-dihydroxybenzoic acid (DHB; Sigma-Aldrich) and 100 µM Dipyridyl at 37 for 18 hours. Culture supernatant was collected and separated by separated by four consecutive steps, including a DEAE-sepharose resin (Sigma), an Amberlite XAD16N resin (20 ~ 60 mesh, Sigma), an Kromasil Eternity 5-PhenylHexyl column (250 × 4.6 mm, 5 μm; Nouryon), and an Ascentis Express Phenyl-Hexyl column (100 × 4.6 mm, 2.7 μm; Sigma-Aldrich) to achieve the purification of five enterobactin-associated molecules, including Ent, MGE, DGE, [(DHBS)2, and G2-(DHBS)2, as previously described (11,47). Culture supernatant was first applied to a methanol (20%)-conditioned DEAE-sepharose column (Sigma). The column was washed with water and then eluted with 7.5 M ammonium formate. The DEAE eluate was supplemented with 120 mM sodium dithionite, incubated with methanol-conditioned Amberlite resin (XAD16N, Sigma-Aldrich) overnight, and eluted with 100% methanol. The eluate was concentrated in a rotatory evaporator (R-100 Rotavapor, BUCHI), lyophilized (Labconco), resuspended in HPLC-grade water plus 0.1% formic acid, and further purified on a Bio-Rad BioLogic DuoFlow 10 system equipped with a QuadTec UV-Vis detector and a Kromasil Eternity-5-PhenylHexyl column (Sigma-Aldrich). The Kromasil column was run at 0.30 ml/min with HPLC-grade water plus 0.1% formic acid (solvent A) and acetonitrile plus 0.1% formic acid (solvent B) using gradient as follows. Solvent B held steady at 2% for 1.0 ml, then increased to 15% over 1 ml, then increased to 52% over 40 ml, and finally increased to 100% over 1 ml. The DuoFlow elute was finally separated by another Ascentis Express Phenyl-Hexyl column (Sigma-Aldrich) in a Shimadzu Prominence UFLC system coupled with an SPD-M20A Prominence Diode Array (PDA) Detector. In order to purify the compounds with different properties, the LC separation was performed by injecting solvent A (HPLC-grade water + 0.1% Formic acid; Sigma-Aldrich, Fluka) and B (90% Acetonitrile + 0.1% Formic acid; Sigma-Aldrich, Fluka) at 0.5 ml/min with an 44 minute gradient under two scenarios as follows. Solvent B increased from 2% to 35% or 44% by 35 min, then increased to 98% B by 38 min, and finally held steady at 98% for another 6 min. Fractions containing purified molecules were measured via UV-Vis detection at 319 nm, pooled together, dried down by lyophilization, and stored in −80 freezer. On day of use, samples were resuspended in HPLC-grade water plus 0.1% formic acid, and concentrations were calculated by Beer-Lambert law using UV-Vis absorbances at 319 nm with an extinction coefficient of 11200 M-1cm-1. Purity was confirmed by targeted LC-MS/MS measurements (11,50,83,84).

Exogeneous DHB for synthesizing enterobactin in isotope-labelling assay

To determine whether UPEC can synthesize Ent from DHB that is not immediately generated by endogenous DHB biosynthesis (40,41,85), we grew UTI89 from single colonies in LB broth for 12 h at 37 was washed with PBS, back-diluted 1:1000 into 13C3-glycerol M63 minimum media with or without the supplement of 200 uM 12C-DHB in a 96-well plate, and grown at 37 for 24 hours. Targeted LC-MS/MS of Ent was conducted to monitor the incorporation of 12C from incorporation of unlabeled DHB (Table S2) by comparing 13C-substituted enterobactin isotopologues into which 0, 1, 2, or 3 12C7-DHB were incorporated.

Data availability

The computer codes for the analyses in this study are available in Github (https://github.com/QL5001/EntMetabolome_script; branch name: main; commit ID, 82317f0). All other data generated and analyzed in this study are included in the published article and supplementary materials.

Statistical methods

GraphPad Prism 9.0 (GraphPad software) was used to generate graphs and perform statistical analysis in this study. We used the unpaired, two-tailed t test for comparisons between two groups, and one-way ANOVA for multigroup comparisons. P < 0.05 was considered statistically significant.

Supplementary Material

Supplement 1
media-1.docx (2.8MB, docx)

ACKNOWLEDGEMENTS

The authors acknowledge funding from the Centers for Disease Control Prevention Epicenters Program Grant (CU54 CK 000162). JPH acknowledges National Institutes of Health grants R01DK099534 and R01DK111930. The content is solely the responsibility of the authors and does not necessarily represent the official view of the CDC or NIH.

Footnotes

COMPETING INTERESTS

The authors declare no competing interests.

REFERENCES

  • 1.Trautner B. W. (2021) Urinary Tract Infections as a Continuum: Implications for Diagnostic and Antibiotic Stewardship. Clin Infect Dis 72, 1339–1341 [DOI] [PubMed] [Google Scholar]
  • 2.Flores-Mireles A. L., Walker J. N., Caparon M., and Hultgren S. J. (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 13, 269–284 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Klein R. D., and Hultgren S. J. (2020) Urinary tract infections: microbial pathogenesis, host-pathogen interactions and new treatment strategies. Nat Rev Microbiol 18, 211–226 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Gonzalez-Alba J. M., Baquero F., Canton R., and Galan J. C. (2019) Stratified reconstruction of ancestral Escherichia coli diversification. BMC Genomics 20, 936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Terlizzi M. E., Gribaudo G., and Maffei M. E. (2017) UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic, and Non-antibiotic Antimicrobial Strategies. Front Microbiol 8, 1566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Shallcross L., Rockenschaub P., Blackburn R., Nazareth I., Freemantle N., and Hayward A. (2020) Antibiotic prescribing for lower UTI in elderly patients in primary care and risk of bloodstream infection: A cohort study using electronic health records in England. PLoS Med 17, e1003336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chen Swaine L, C.-S. H., Jian, Reigstad Christopher S, Magrini Vincent, Sabo Aniko, Blasiar Darin, Bieri Tamberlyn, Meyer Rekha R, Ozersky Philip, Armstrong Jon R, Fulton Robert S, Phillip Latreille J, Spieth John, Hooton Thomas M, Mardis Elaine R, Hultgren Scott J, Gordon Jeffrey I. (2006) Identification of genes subject to positive selection in uropathogenic strains of Escherichia coli- a comparative genomics approach. Proc Natl Acad Sci U S A 103, 5977–5982 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Lo Y., Zhang L., Foxman B., and Zollner S. (2015) Whole-genome sequencing of uropathogenic Escherichia coli reveals long evolutionary history of diversity and virulence. Infect Genet Evol 34, 244–250 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Nielsen K. L., Stegger M., Kiil K., Lilje B., Ejrnaes K., Leihof R. F., Skjot-Rasmussen L., Godfrey P., Monsen T., Ferry S., Hammerum A. M., and Frimodt-Moller N. (2021) Escherichia coli Causing Recurrent Urinary Tract Infections: Comparison to Non-Recurrent Isolates and Genomic Adaptation in Recurrent Infections. Microorganisms 9 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Robinson Anne E, J. R. H., Henderson Jeffrey P. (2018) The iron hand of uropathogenic Escherichia coli-the role of transition metal control in virulence. Future Microbiol 13, 745–756 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Ohlemacher S. I., Giblin D. E., d'Avignon D. A., Stapleton A. E., Trautner B. W., and Henderson J. P. (2017) Enterobacteria secrete an inhibitor of Pseudomonas virulence during clinical bacteriuria. J Clin Invest 127, 4018–4030 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Zou Z., Potter R. F., McCoy W. H. t., Wildenthal J. A., Katumba G. L., Mucha P. J., Dantas G., and Henderson J. P. (2023) E. coli catheter-associated urinary tract infections are associated with distinctive virulence and biofilm gene determinants. JCI Insight 8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Shields-Cutler R. R., Crowley J. R., Hung C. S., Stapleton A. E., Aldrich C. C., Marschall J., and Henderson J. P. (2015) Human Urinary Composition Controls Antibacterial Activity of Siderocalin. J Biol Chem 290, 15949–15960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Shields-Cutler R. R., Crowley J. R., Miller C. D., Stapleton A. E., Cui W., and Henderson J. P. (2016) Human Metabolome-derived Cofactors Are Required for the Antibacterial Activity of Siderocalin in Urine. J Biol Chem 291, 25901–25910 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Watts R. E., Totsika M., Challinor V. L., Mabbett A. N., Ulett G. C., De Voss J. J., and Schembri M. A. (2012) Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli. Infect Immun 80, 333–344 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Miethke M., and Marahiel M. A. (2007) Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev 71, 413–451 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Neilands J. B. (1995) Siderophores: structure and function of microbial iron transport compounds. J Biol Chem 270, 26723–26726 [DOI] [PubMed] [Google Scholar]
  • 18.Searle L. J., Meric G., Porcelli I., Sheppard S. K., and Lucchini S. (2015) Variation in siderophore biosynthetic gene distribution and production across environmental and faecal populations of Escherichia coli. PLoS One 10, e0117906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Rogers H. J. (1973) Iron-Binding Catechols and Virulence in Escherichia coli. Infect Immun 7, 445–456 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Brumbaugh A. R., Smith S. N., Subashchandrabose S., Himpsl S. D., Hazen T. H., Rasko D. A., and Mobley H. L. (2015) Blocking yersiniabactin import attenuates extraintestinal pathogenic Escherichia coli in cystitis and pyelonephritis and represents a novel target to prevent urinary tract infection. Infect Immun 83, 1443–1450 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Henderson J. P., Crowley J. R., Pinkner J. S., Walker J. N., Tsukayama P., Stamm W. E., Hooton T. M., and Hultgren S. J. (2009) Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli. PLoS Pathog 5, e1000305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.K G Wooldridge P. H. W. (1993) Iron uptake mechanisms of pathogenic bacteria. FEMS Microbiol Rev 12, 325–348 [DOI] [PubMed] [Google Scholar]
  • 23.Andrews S. C., Robinson A. K., and Rodriguez-Quinones F. (2003) Bacterial iron homeostasis. FEMS Microbiol Rev 27, 215–237 [DOI] [PubMed] [Google Scholar]
  • 24.Kramer J., Ozkaya O., and Kummerli R. (2020) Bacterial siderophores in community and host interactions. Nat Rev Microbiol 18, 152–163 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.G S Moeck J. W. C. (1998) TonB-dependent iron acquisition mechanisms of siderophore-mediated active transport. Mol Microbiol 28 [DOI] [PubMed] [Google Scholar]
  • 26.Zeng X., Xu F., and Lin J. (2013) Specific TonB-ExbB-ExbD energy transduction systems required for ferric enterobactin acquisition in Campylobacter. FEMS Microbiol Lett 347, 83–91 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Higgs Penelope I., R. A. L., Postle Kathleen. (2002) Quantification of known components of the Escherichia coli TonB energy transduction system. Molecular Microbiology 44, 271–281 [DOI] [PubMed] [Google Scholar]
  • 28.Troxell B., and Hassan H. M. (2013) Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. Front Cell Infect Microbiol 3, 59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Robinson A. E., Lowe J. E., Koh E. I., and Henderson J. P. (2018) Uropathogenic enterobacteria use the yersiniabactin metallophore system to acquire nickel. J Biol Chem 293, 14953–14961 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Martin P., Tronnet S., Garcie C., and Oswald E. (2017) Interplay between siderophores and colibactin genotoxin in Escherichia coli. IUBMB Life 69, 435–441 [DOI] [PubMed] [Google Scholar]
  • 31.Garenaux A., Caza M., and Dozois C. M. (2011) The Ins and Outs of siderophore mediated iron uptake by extra-intestinal pathogenic Escherichia coli. Vet Microbiol 153, 89–98 [DOI] [PubMed] [Google Scholar]
  • 32.Lv H., Hung C. S., and Henderson J. P. (2014) Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli. J Proteome Res 13, 1397–1404 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Guo C., Steinberg L. K., Cheng M., Song J. H., Henderson J. P., and Gross M. L. (2020) Site-Specific Siderocalin Binding to Ferric and Ferric-Free Enterobactin As Revealed by Mass Spectrometry. ACS Chem Biol 15, 1154–1160 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Abergel Rebecca J, E. G. M., Strong Roland K, Raymond Kenneth N. (2006) Microbial Evasion of the Immune System Structural Modifications of Enterobactin Impair Siderocalin Recognition. J Am Chem Soc 128, 10998–10999 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Chaturvedi K. S., Hung C. S., Crowley J. R., Stapleton A. E., and Henderson J. P. (2012) The siderophore yersiniabactin binds copper to protect pathogens during infection. Nat Chem Biol 8, 731–736 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Chaturvedi K. S., Hung C. S., Giblin D. E., Urushidani S., Austin A. M., Dinauer M. C., and Henderson J. P. (2014) Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic. ACS Chem Biol 9, 551–561 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Koh E. I., Robinson A. E., Bandara N., Rogers B. E., and Henderson J. P. (2017) Copper import in Escherichia coli by the yersiniabactin metallophore system. Nat Chem Biol 13, 1016–1021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Heffernan J. R., Katumba G. L., McCoy W. H., and Henderson J. P. (2023) Yersiniabactin is a quorum sensing autoinducer and siderophore in uropathogenic Escherichia coli. bioRxiv [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Azpiroz M. F., and Lavina M. (2004) Involvement of enterobactin synthesis pathway in production of microcin H47. Antimicrob Agents Chemother 48, 1235–1241 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Walsh Christopher T., J. L., Rusnak Frank, and Sakaitani Masahiro. (1990) Molecular studies on enzymes in chorismate metabolism and the enterobactin biosynthetic pathway. Chem. Rev. 90, 1105–1129 [Google Scholar]
  • 41.Raymond Kenneth N, E. A. D., Kim Sanggoo S. (2003) Enterobactin: An archetype for microbial iron transport. Proc Natl Acad Sci U S A 100, 3584–3588 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Fischbach Michael A., H. L., Liu David R., and Walsh Christopher T.. (2005) In vitro characterization of IroB, a pathogen-associated C-glycosyltransferase. PNAS 102, 571–576 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Josts I., Veith K., Normant V., Schalk I. J., and Tidow H. (2021) Structural insights into a novel family of integral membrane siderophore reductases. Proc Natl Acad Sci U S A 118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Cain T. J., and Smith A. T. (2021) Ferric iron reductases and their contribution to unicellular ferrous iron uptake. J Inorg Biochem 218, 111407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Miethke M., Hou J., and Marahiel M. A. (2011) The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli. Biochemistry 50, 10951–10964 [DOI] [PubMed] [Google Scholar]
  • 46.Caza M., Lepine F., Milot S., and Dozois C. M. (2008) Specific roles of the iroBCDEN genes in virulence of an avian pathogenic Escherichia coli O78 strain and in production of salmochelins. Infect Immun 76, 3539–3549 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Lin Hening, M. A. F., Liu David R, Walsh Christopher T. (2005) In Vitro Characterization of Salmochelin and Enterobactin Trilactone Hydrolases IroD, IroE, and Fes. J Am Chem Soc 127, 11075–11084 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Sorsa L. J., Dufke S., Heesemann J., and Schubert S. (2003) Characterization of an iroBCDEN gene cluster on a transmissible plasmid of uropathogenic Escherichia coli: evidence for horizontal transfer of a chromosomal virulence factor. Infect Immun 71, 3285–3293 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Caza M., Garenaux A., Lepine F., and Dozois C. M. (2015) Catecholate siderophore esterases Fes, IroD and IroE are required for salmochelins secretion following utilization, but only IroD contributes to virulence of extra-intestinal pathogenic Escherichia coli. Mol Microbiol 97, 717–732 [DOI] [PubMed] [Google Scholar]
  • 50.Berner I, M. G., Metzger J, Jung G, Winkelmann G. (1991) Identification of enterobactin and linear dihydroxybenzoylserine compounds by HPLC and ion spray mass spectrometry. Biol Met 4, 113–118 [DOI] [PubMed] [Google Scholar]
  • 51.Winkelmann Günther, A. C., Beck Werner & Jung Günther (1994) HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives. Biometals 7, 149–154 [DOI] [PubMed] [Google Scholar]
  • 52.Guo Zu-Feng, M. J., Zheng Suilan, and Guo Zhihong. (2008) Suppression of Linear Side Products by Macromolecular Crowding in Nonribosomal Enterobactin Biosynthesis. Org. Lett. 10, 649–652 [DOI] [PubMed] [Google Scholar]
  • 53.Bister Bojan, D. B., Nicholson Graeme J., Valdebenito Marianne, Schneider Kathrin, Winkelmann Günther, Hantke Klaus& Süssmuth Roderich D.. (2004) The structure of salmochelins C-glucosylated enterobactins of Salmonella enterica. Biometals 17, 471–481 [DOI] [PubMed] [Google Scholar]
  • 54.Caza M., Lepine F., and Dozois C. M. (2011) Secretion, but not overall synthesis, of catecholate siderophores contributes to virulence of extraintestinal pathogenic Escherichia coli. Mol Microbiol 80, 266–282 [DOI] [PubMed] [Google Scholar]
  • 55.Reitz Z. L., Sandy M., and Butler A. (2017) Biosynthetic considerations of triscatechol siderophores framed on serine and threonine macrolactone scaffolds. Metallomics 9, 824–839 [DOI] [PubMed] [Google Scholar]
  • 56.Noinaj N., Guillier M., Barnard T. J., and Buchanan S. K. (2010) TonB-dependent transporters: regulation, structure, and function. Annu Rev Microbiol 64, 43–60 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Rudra S., Dasmandal S., Patra C., Kundu A., and Mahapatra A. (2016) Binding affinities of Schiff base Fe(II) complex with BSA and calf-thymus DNA: Spectroscopic investigations and molecular docking analysis. Spectrochim Acta A Mol Biomol Spectrosc 166, 84–94 [DOI] [PubMed] [Google Scholar]
  • 58.Makarska-Bialokoz M. (2018) Interactions of hemin with bovine serum albumin and human hemoglobin: A fluorescence quenching study. Spectrochim Acta A Mol Biomol Spectrosc 193, 23–32 [DOI] [PubMed] [Google Scholar]
  • 59.Lemaitre C., Bidet P., Benoist J. F., Schlemmer D., Sobral E., d'Humieres C., and Bonacorsi S. (2014) The ssbL gene harbored by the ColV plasmid of an Escherichia coli neonatal meningitis strain is an auxiliary virulence factor boosting the production of siderophores through the shikimate pathway. J Bacteriol 196, 1343–1349 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Scarrow Robert C., D. J. E., Ng Chiu, Liu Sylvia, and Raymond Kenneth N.. (1990) Iron(III) coordination chemistry of linear dihydroxyserine compounds derived from enterobactin. Inorg. Chem. 30, 900–906 [Google Scholar]
  • 61.Raines D. J., Moroz O. V., Blagova E. V., Turkenburg J. P., Wilson K. S., and Duhme-Klair A. K. (2016) Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product. Proc Natl Acad Sci U S A 113, 5850–5855 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Cobessi D., Celia H., and Pattus F. (2005) Crystal structure at high resolution of ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa. J Mol Biol 352, 893–904 [DOI] [PubMed] [Google Scholar]
  • 63.Braud A., Hannauer M., Mislin G. L., and Schalk I. J. (2009) The Pseudomonas aeruginosa pyochelin-iron uptake pathway and its metal specificity. J Bacteriol 191, 3517–3525 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Knosp O, M. v. T., Page W J. (1984) Siderophore-mediated uptake of iron in Azotobacter vinelandii. J Bacteriol 159, 341–347 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.H Nikaido E. Y. R. (1990) Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups. J Bacteriol 172, 1361–1367 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Grinter R., and Lithgow T. (2019) The structure of the bacterial iron-catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism. J Biol Chem 294, 19523–19534 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Chekabab S. M., Rehman M. A., Yin X., Carrillo C., Mondor M., and Diarra M. S. (2019) Growth of Salmonella enterica Serovars Typhimurium and Enteritidis in Iron-Poor Media and in Meat: Role of Catecholate and Hydroxamate Siderophore Transporters. J Food Prot 82, 548–560 [DOI] [PubMed] [Google Scholar]
  • 68.Klebba P. E., Newton S. M. C., Six D. A., Kumar A., Yang T., Nairn B. L., Munger C., and Chakravorty S. (2021) Iron Acquisition Systems of Gram-negative Bacterial Pathogens Define TonB-Dependent Pathways to Novel Antibiotics. Chem Rev 121, 5193–5239 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Ito A., Sato T., Ota M., Takemura M., Nishikawa T., Toba S., Kohira N., Miyagawa S., Ishibashi N., Matsumoto S., Nakamura R., Tsuji M., and Yamano Y. (2018) In Vitro Antibacterial Properties of Cefiderocol, a Novel Siderophore Cephalosporin, against Gram-Negative Bacteria. Antimicrob Agents Chemother 62 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Klein S., Boutin S., Kocer K., Fiedler M. O., Storzinger D., Weigand M. A., Tan B., Richter D., Rupp C., Mieth M., Mehrabi A., Hackert T., Zimmermann S., Heeg K., and Nurjadi D. (2022) Rapid Development of Cefiderocol Resistance in Carbapenem-resistant Enterobacter cloacae During Therapy Is Associated With Heterogeneous Mutations in the Catecholate Siderophore Receptor cirA. Clin Infect Dis 74, 905–908 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Padovani M., Bertelli A., Corbellini S., Piccinelli G., Gurrieri F., and De Francesco M. A. (2023) In Vitro Activity of Cefiderocol on Multiresistant Bacterial Strains and Genomic Analysis of Two Cefiderocol Resistant Strains. Antibiotics 12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 72.Scholz R. L., and Greenberg E. P. (2015) Sociality in Escherichia coli: Enterochelin Is a Private Good at Low Cell Density and Can Be Shared at High Cell Density. J Bacteriol 197, 2122–2128 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Kenan C Murphy K. G. C. (2003) Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol Biol 4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Robinson J. I., Weir W. H., Crowley J. R., Hink T., Reske K. A., Kwon J. H., Burnham C. D., Dubberke E. R., Mucha P. J., and Henderson J. P. (2019) Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections. J Clin Invest 129, 3792–3806 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Pedregosa Fabian, G. V., Gramfort Alexandre, Michel Vincent, Thirion Bertrand, Grisel Olivier, Blondel Mathieu, Müller Andreas, Nothman Joel, Louppe Gilles, Prettenhofer Peter, Weiss Ron, Dubourg Vincent, Vanderplas Jake, Passos Alexandre, Cournapeau David, Brucher Matthieu, Perrot Matthieu, Duchesnay Édouard. (2011) Scikit-learn Machine Learning in Python. Journal of Machine Learning Research 12, 2825–2830 [Google Scholar]
  • 76.Rohart F., Gautier B., Singh A., and Le Cao K. A. (2017) mixOmics: An R package for 'omics feature selection and multiple data integration. PLoS Comput Biol 13, e1005752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Le Cao K. A., Rossouw D., Robert-Granie C., and Besse P. (2008) A sparse PLS for variable selection when integrating omics data. Stat Appl Genet Mol Biol 7, Article 35 [DOI] [PubMed] [Google Scholar]
  • 78.Kampa K., Mehta S., Chou C. A., Chaovalitwongse W. A., and Grabowski T. J. (2014) Sparse optimization in feature selection: application in neuroimaging. Journal of Global Optimization 59, 439–457 [Google Scholar]
  • 79.Feng C. M., Xu Y., Liu J. X., Gao Y. L., and Zheng C. H. (2019) Supervised Discriminative Sparse PCA for Com-Characteristic Gene Selection and Tumor Classification on Multiview Biological Data. IEEE Trans Neural Netw Learn Syst 30, 2926–2937 [DOI] [PubMed] [Google Scholar]
  • 80.Yao M., Ma L., Duchoslav E., and Zhu M. (2009) Rapid screening and characterization of drug metabolites using multiple ion monitoring dependent product ion scan and postacquisition data mining on a hybrid triple quadrupole-linear ion trap mass spectrometer. Rapid Commun Mass Spectrom 23, 1683–1693 [DOI] [PubMed] [Google Scholar]
  • 81.Zhou Y., Guan J., Gao W., Lv S., and Ge M. (2018) Quantification and Confirmation of Fifteen Carbamate Pesticide Residues by Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes via LC-MS/MS QTRAP System. Molecules 23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82.Luo Minkui, H. L., Fischbach Michael A, Liu David R, Walsh Christopher T, Groves John T. (2006) Enzymatic Tailoring of Enterobactin Alters Membrane Partitioning and Iron Acquisition. ACS Chem Biol 1, 29–32 [DOI] [PubMed] [Google Scholar]
  • 83.Egbers P. H., Harder T., Koch B. P., and Tebben J. (2020) Siderophore purification with titanium dioxide nanoparticle solid phase extraction. Analyst 145, 7303–7311 [DOI] [PubMed] [Google Scholar]
  • 84.Zajdowicz S., Haller J. C., Krafft A. E., Hunsucker S. W., Mant C. T., Duncan M. W., Hodges R. S., Jones D. N., and Holmes R. K. (2012) Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae. PLoS One 7, e34591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Crosa J. H., and Walsh C. T. (2002) Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66, 223–249 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
media-1.docx (2.8MB, docx)

Data Availability Statement

The computer codes for the analyses in this study are available in Github (https://github.com/QL5001/EntMetabolome_script; branch name: main; commit ID, 82317f0). All other data generated and analyzed in this study are included in the published article and supplementary materials.

Articles from bioRxiv are provided here courtesy of Cold Spring Harbor Laboratory Preprints

RESOURCES