Uropathogenic Escherichia coli wield enterobactin-derived catabolites as siderophores

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.

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

(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.

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

(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.

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

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.

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

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.

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.

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

(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.

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

(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.