The siderophore yersiniabactin binds copper to protect pathogens during infection - PubMed (original) (raw)

The siderophore yersiniabactin binds copper to protect pathogens during infection

Kaveri S Chaturvedi et al. Nat Chem Biol. 2012 Aug.

Abstract

Bacterial pathogens secrete chemically diverse iron chelators called siderophores, which may exert additional distinctive functions in vivo. Among these, uropathogenic Escherichia coli often coexpress the virulence-associated siderophore yersiniabactin (Ybt) with catecholate siderophores. Here we used a new MS screening approach to reveal that Ybt is also a physiologically favorable Cu(II) ligand. Direct MS detection of the resulting Cu(II)-Ybt complex in mice and humans with E. coli urinary tract infections demonstrates copper binding to be a physiologically relevant in vivo interaction during infection. Ybt expression corresponded to higher copper resistance among human urinary tract isolates, suggesting a protective role for this interaction. Chemical and genetic characterization showed that Ybt helps bacteria resist copper toxicity by sequestering host-derived Cu(II) and preventing its catechol-mediated reduction to Cu(I). Together, these studies reveal a new virulence-associated function for Ybt that is distinct from iron binding.

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Conflict of interest statement

Competing Financial Interests Statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. A yersiniabactin neutral loss screen reveals formation of a novel copper(II)-yersiniabactin complex in human urine

(a) Yersiniabactin and its metal complexes exhibit a dominant 187 mass unit neutral loss upon CID fragmentation of the positive ESI-derived ion. This neutral loss is consistent with rearrangement to lose the terminal carboxylated thiazoline. (b) A 187 mass unit neutral loss is evident in MS/MS product ion spectra of ferric-yersiniabactin (Fe(III)-Ybt) at m/z 535, aluminum-yersiniabactin (Al(III)-Ybt) at m/z 506, and gallium-yersiniabactin (Ga(III)-Ybt) at m/z 548. A constant neutral loss (CNL) scan based on this conserved fragmentation pathway was used as a metallomic screen to identify physiologic yersiniabactin binding partners. (c) Representative constant neutral loss chromatograms of urine samples in the presence and absence of purified _apo_-yersiniabactin. The combination of _apo_-yersiniabactin and urine results in formation of a prominent new peak (peak 1). Peaks corresponding to the internal standard (int. std) are indicated. These results were confirmed in three independent experiments. (d) High resolution positive ion ESI mass spectrum is consistent with the empiric formula for a singly charged Cu(II)-Ybt ion and demonstrates the prominent natural abundance M+2 ion expected from 65Cu. (d) Competitive binding experiments were conducted by titrating cupric sulfate into solutions containing a fixed concentration of 0.01 M ferric chloride and 0.01 M _apo_-yersiniabactin. Data indicate competitive binding between cupric and ferric ion for the ligand. Cu(II)-Ybt/Fe(III)-Ybt ratios were determined by comparing selected ion chromatogram peak ratios to those from a standard curve.

Figure 2

Figure 2. Cupric-yersiniabactin is produced in cystitis patients infected with yersiniabactin-producing strains

(a) A scanning constant neutral loss spectrum reveals the spectrum for Cu(II)-Ybt at its expected retention time. The expected copper isotope peaks at m/z 543 for 63Cu and m/z 545 for 65Cu peak are present at the expected ~2:1 ratio. (b) Urinary Cu(II)-Ybt was detected in 13 of 15 patients infected with a yersiniabactin-expressing pathogen and in none of the patients with yersiniabactin non-expressors. Cu(II)-Ybt levels are reported as a fraction of the corresponding 13C internal standard peak height. (c) In urine samples with detectable yersiniabactin complexes, the median Cu(II)-Ybt (m/z 543) to Fe(III)-Ybt (m/z 535) ratio is 2.941, indicating preferential in vivo copper (II) binding.

Figure 3

Figure 3. Yersiniabactin promotes E. coli growth in copper-toxic conditions

Urinary and non-urinary E. coli isolates from a UTI patient population were cultured in the presence of 10 μM copper (II) sulfate for 18 hours. Growth was determined and expressed as total CFU/mL. (a) Urinary strains demonstrate greater resistance to copper toxicity than coexisting rectal strains. For each patient, CFU/mL from the non-urinary strain was subtracted from CFU/mL from the coincident urinary strain to yield a difference. In the four patients from whom multiple coincident urinary and non-urinary strains were recovered, the mean difference in colony forming units is reported. The median value of these differences was 2.11 x 107 CFU/mL, with a range of −5.4 x 103 to 1.66 x 108. (b) Yersiniabactin-expressors were more resistant to copper toxicity than non-expressors (p<0.0013). These results were confirmed in three independent experiments. (c) Yersiniabactin-expressor (UTI89) and non-expressor (UTI89Δ_ybtS_) cultures treated with 0–25 μM copper (II) sulfate revealed an average of ten-fold survival advantage for the yersiniabactin expressor (_p_-value = 0.012, 0.0004, 0.009, 0.002 and 0.023, respectively, _t_-test). (d) Purified _apo_-yersiniabactin or Cu(II)-Ybt was added in 1.5-fold molar excess over 10 μM copper (II) sulfate to yersiniabactin-deficient (UTI89Δ_ybtS_) culture. Samples containing copper alone demonstrated a >3 log CFU/mL decrease in viability. _Apo_-yersiniabactin (_apo_-Ybt) addition restores growth to untreated wild type levels (p = ns). This cytoprotective effect is unique to _apo_-yersiniabactin, and is not observed upon addition of pre-formed Cu(II)-Ybt. These results were confirmed in three independent experiments.

Figure 4

Figure 4. Catecholate siderophores and yersiniabactin exert opposing effects on copper cytotoxicity

(a) Growth of wild type (UTI89), yersiniabactin (Δ_ybtS_), catecholate siderophore (Δ_entB_), or total siderophore (Δ_entB_Δ_ybtS_) expression mutants in the presence of copper was determined. Results were consistent with copper-dependent cytoprotective effect for yersiniabactin and cytotoxic effect for catecholate siderophores. (b) Exogenous addition of 20 μM of the siderophore enterobactin, or its catecholate moiety 2,3-dihydroxybenzoate (DHB) enhances copper (II) sulfate toxicity in UTI89. (c) _Apo-_yersiniabactin prevents catechol-dependent reduction of copper (II) sulfate to copper (I) in an order-of-addition dependent manner. The complete reaction system consisted of 17.5 μM copper(II) sulfate, either 20 μM enterobactin (ent) or its catecholate moiety 2,3-dihydroxybenzoic acid (DHB), 25 μM _apo_-yersiniabactin (Ybt), and 25 μM of the copper(I) indicator bathocuproine sulfonate. Reagents were added in the order indicated and Cu(I)-bathocuproine absorbance was determined 30 min after addition of the last reagent. Results were confirmed in three independent experiments.

References

    1. Miethke M, Marahiel MA. Siderophore-based iron acquisition and pathogen control. Microbiol Mol Biol Rev. 2007;71:413–451. doi: 10.1128/MMBR.00012-07. -DOI -PMC -PubMed
    1. Devireddy LR, Hart DO, Goetz DH, Green MR. A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell. 2010;141:1006–1017. doi: 10.1016/j.cell.2010.04.040. -DOI -PMC -PubMed
    1. Kim HJ, Galeva N, Larive CK, Alterman M, Graham DW. Purification and physical-chemical properties of methanobactin: a chalkophore from Methylosinus trichosporium OB3b. Biochemistry. 2005;44:5140–5148. doi: 10.1021/bi047367r. -DOI -PubMed
    1. Kim HJ, et al. Methanobactin, a copper-acquisition compound from methane-oxidizing bacteria. Science. 2004;305:1612–1615. doi: 10.1126/science.1098322. -DOI -PubMed
    1. Clarke SE, Stuart J, Sanders-Loehr J. Induction of siderophore activity in Anabaena spp. and its moderation of copper toxicity. Appl Environ Microbiol. 1987;53:917–922. -PMC -PubMed

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