Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin

Final purification of corynebactin by C8 reversed-phase HPLC.

<p>Corynebactin was recovered as a single activity peak coincident with an A₂₁₀ absorbance peak at approximately 9.4% acetonitrile during elution with a 0% to 80% linear gradient of acetonitrile in 0.6% formic acid. A₂₁₀ is shown as a dashed line; the linear acetonitrile gradient is shown as a solid line; and CAS activity for each fraction is shown as a dotted line.</p

Quantitative bioassay for corynebactin.

<p>A. Varying concentrations of corynebactin (expressed as EDDA equivalents) were added to wells containing 25 µM FeCl₃. Growth of <i>C. diphtheriae</i> C7(β) Δ<i>ciuE</i> was stimulated by corynebactin, and the growing bacteria reduced the triphenyltetrazolium chloride indicator dye and turned red. No visible bacterial growth was present around the control well without corynebactin. The figure shows the results of a representative bioassay. B. Within the range from 6.75 µM to 108 µM EDDA equivalents, the average diameter of the growth stimulation zone for <i>C. diphtheriae</i> C7(β) Δ<i>ciuE</i>, based on triplicate samples, was directly proportional to the log₂ of the concentration of the corynebactin sample in the well.</p

Analysis of corynebactin by electrospray ionization mass spectra (negative ion mode).

<p>A. A representative mass spectrum for corynebactin. The average <i>m/z</i> value for corynebactin was 493.138 based on the average of five separate mass spectra. Additional peaks at 475.28, 500.31, 501. 28, and 546.31 correspond to the following ions: [M-H-H₂O]⁻, [M-H+Fe²⁺-2H-HCOOH]⁻, [M-H+Fe³⁺-3H-HCOOH]⁻, and [M-H+Fe³⁺-3H]⁻, respectively.</p

Comparison of structures of corynebactin, staphyloferrin A, and rhizoferrin.

<p>A. Structure of corynebactin determined by NMR spectroscopy. B. The published structure of staphyloferrin. C. The published structure of rhizoferrin.</p

Analysis of corynebactin structure by NMR spectroscopy.

<p>A. ¹H-¹H DQF-COSY spectrum of corynebactin dissolved in 90% H₂O at pH 4.0. Dashed lines illustrate the correlations between the protons in the central lysine core moiety B. Overlay of the ¹H-¹³C HSQC Spectrum of corynebactin dissolved in D₂O at pH 6.0 (black) with the ¹H-¹³C HMBC (red). The inset shows an expansion of the region indicated by the dashed box.</p

Effects of purified corynebactin on uptake of ⁵⁵Fe by wild-type and mutant strains of <i>C. diphtheriae</i>.

<p>The strains of <i>C. diphtheriae</i> tested are as follows: wild type C7(β) (▪, □); corynebactin production deficient C7(−) Δ<i>ciuE</i> (•, ○); and corynebactin utilization deficient C7(β) Δ<i>ciuA</i> (▴, ▵). Filled symbols show the results of assays performed in medium without added corynebactin, and open symbols show the results of assays performed in medium with addition of purified corynebactin at 4 µM.</p

List of Chemical Shifts for Corynebactin.

<p>The group assignment is as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034591#pone-0034591-g005" target="_blank">Fig. 5</a>-C. All shifts are referenced to internal DSS.</p