The frontline antibiotic vancomycin induces a zinc starvation response in bacteria by binding to Zn(II)

Abstract

Vancomycin is a front-line antibiotic used for the treatment of nosocomial infections, particularly those caused by methicillin-resistant Staphylococcus aureus. Despite its clinical importance the global effects of vancomycin exposure on bacterial physiology are poorly understood. In a previous transcriptomic analysis we identified a number of Zur regulon genes which were highly but transiently up-regulated by vancomycin in Streptomyces coelicolor. Here, we show that vancomycin also induces similar zinc homeostasis systems in a range of other bacteria and demonstrate that vancomycin binds to Zn(II) in vitro. This implies that vancomycin treatment sequesters zinc from bacterial cells thereby triggering a Zur-dependent zinc starvation response. The Kd value of the binding between vancomycin and Zn(II) was calculated using a novel fluorometric assay, and NMR was used to identify the binding site. These findings highlight a new biologically relevant aspect of the chemical property of vancomycin as a zinc chelator.

Figure 1. Evidence for the activation of Zur-dependent zinc homeostasis systems by vancomycin.

(a) Zur-regulated genes specifically induced by 10 μg/ml vancomycin (6.9 μM) in the transcriptome of S. coelicolor. Fold change corresponds to the change in expression of each gene 30 min after vancomycin treatment relative to time 0. (b) Regulation of gene expression by Zur in response to Zn(II). In high Zn(II) concentrations, zinc-bound Zur represses transcription of its target genes by binding to a Zur binding site in the promoter region. When Zn(II) is low, zinc-free Zur is released from the promoter allowing transcription.

Figure 2. Vancomycin induces Zur-dependent zinc responsive mechanisms in different bacterial species.

qRT-PCR analysis of the transcriptional response to vancomycin of (a) the six Zur-regulated genes SCO0475, SCO2505, SCO7676, SCO7677, SCO7681 and SCO7682 in S. coelicolor (b) the putative Zur regulated genes SGR5019 (SCO2505 orthologue), SGR0545 (SCO3429 orthologue) and SGR0546 (SCO3428 orthologue) in S. griseus (c) the putative Zur regulated genes SSGG01683 (SCO2505 orthologue), SSGG00253 (SCO0476 orthologue) and SSGG06641 (SCO3428 orthologue) in S. roseosporus (d) znuA, znuB and znuC from the zinc transport system in E. coli and (e) B. subtilis znuABC. “+Zn” indicates a sample from a culture grown in the presence of ~3 μM of zinc sulphate, and “+Zn +Van” or “+Zn +EDTA” indicates a sample from similar cultures treated with 2 mM of either vancomycin or EDTA for 30 min. The y axis indicates transcript abundance normalized to a reference gene presented in a log scale. Data are presented as means ± SD.

Figure 3. In vitro analysis of the interaction between vancomycin and Zn(II).

(a) Affinity chromatography demonstrating that vancomycin binds to Zn(II) and Cu(II) but not to Ni(II). Vancomycin was applied to an uncharged HiTrap column (control) and to columns charged with Ni(II), Zn(II) or Cu(II). Elution of vancomycin from the columns was quantified by the analysis of 0.5 ml fractions using UV absorbance at 282 nm. (b) Analysis of the interaction of vancomycin with metal ions using fluorometry (λexcitation 280 nm, Slit: 5 nm, Tris-Cl pH 7.3). Cu(II) and Zn(II) alter the fluorescence emission of vancomycin but Ni(II) does not. (c) Analysis of the changes in fluorescence of vancomycin (50 μM) in the presence of increasing molar equivalent ratios of Zn(II) using optimized fluorescence conditions (λexcitation 280 nm, Slit: 5 nm, Tris-Cl pH 7.3). Fluorescence is most enhanced with a 1:1 ratio then progressively quenched with higher Zn(II) concentrations. Fluorescence titration data were analysed using DynaFit 4 to determine the equilibrium dissociation constant (Kd) for the vancomycin-Zn(II) binding (right panel). (d) Analysis of the changes in fluorescence of vancomycin (50 μM) in the presence of increasing molar equivalent ratios of Cu(II) using the same conditions as in (c). The wavelengths corresponding to the maximum fluorescence intensities in (c) and (d) are indicated by dotted lines.

Figure 4. ¹H NMR analysis of Zn(II) binding to the vancomycin structure in deuterium oxide (D₂O) at pD7.1.

(a) The chemical shift (ppm) of certain proton resonances in the ¹H NMR spectrum of vancomycin are affected by increasing concentrations of Zn(II) ions. The arrows indicate the resonances changing in the 2.5 to 4 ppm spectral region as the concentration of Zn(II) present is increased from 0 mM (bottom spectrum, 1 mM vancomycin) to 5 mM (top spectrum, Van: Zn(II) = 1:5). (b) Complete shift analysis summarizing the difference in proton resonance shifts between each zinc condition and the control. The differences between the control (0 mM Zn(II)) and 0.5 mM (Van:Zn(II) = 2:1), 1 mM (Van:Zn(II) = 1:1), 2 mM (Van:Zn(II) = 1:2) and 5 mM (Van:Zn(II) = 1:5) zinc chloride are named Δδ(1–2), Δδ(1–3), Δδ(1–4) and Δδ(1–5) respectively. The largest changes are mapped onto the vancomycin structure defining the Zn(II) binding site.