Abstract
Vancomycin is known to bind to Zn(II) and can induce a zinc starvation response in bacteria. Here we identify a novel polymerization of vancomycin dimers by structural analysis of vancomycin-Zn(II) crystals and fibre X-ray diffraction. Bioassays indicate that this structure is associated with an increased antibiotic activity against bacterial strains possessing high level vancomycin resistance mediated by the reprogramming of peptidoglycan biosynthesis to use precursors terminating in D-Ala-D-Lac in place of D-Ala-D-Ala. Polymerization occurs via interaction of Zn(II) with the N-terminal methylleucine group of vancomycin, and we show that the activity of other glycopeptide antibiotics with this feature can also be similarly augmented by Zn(II). Construction and analysis of a model strain predominantly using D-Ala-D-Lac precursors for peptidoglycan biosynthesis during normal growth supports the hypothesis that Zn(II) mediated vancomycin polymerization enhances the binding affinity towards these precursors.
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Introduction
Antibiotics are a diminishing resource, with the emergence and spread of resistance in bacterial populations currently outstripping the pace of discovery and approval of novel compounds. There is consequently a renewed interest in finding leads for novel antibacterial activities, and for the development of methods capable of enhancing the effectiveness of existing treatments.
Vancomycin, a natural product glycopeptide antibiotic, is a last resort treatment for serious infections caused by Gram-positive bacterial pathogens, particularly those associated with MRSA (Methicillin resistant Staphylococcus aureus) or drug-resistant enterococci. The emergence of vancomycin resistance in these pathogenic strains is however threatening to reduce its usefulness as a front-line therapy1, 2. Vancomycin acts by binding to the D-Ala-D-Ala termini of bacterial cell wall peptidoglycan (PG) and of the PG precursor Lipid II, thereby compromising the synthesis and strength of the bacterial cell wall and leading to reduced growth and cell death3. A small number of semisynthetic second-generation lipoglycopeptide antibiotics have recently been developed to improve the treatment options available for Gram-positive infections, but possess the same mode of action as the parent template glycopeptide vancomycin4, 5.
Vancomycin has recently been characterized as a zinc chelator, binding Zn(II) via the N-terminal methylleucine amino acid group and inducing a zinc starvation response in treated bacteria6, 7. Although it is not common, some antibiotics are known to require metal ions for their activity8. Bacitracin requires binding of divalent metal ions for sequestration of its molecular target undecaprenyl pyrophosphate, with Zn(II) being most effective at stabilizing complex formation through neutralization of the pyrophosphate charge9. Quinolone antibiotics also use metal ions to help bind their target molecules, DNA topoisomerase enzymes10, 11. When not required to mediate target binding, interaction of metal ions with antibiotics can provide an additional mode of antibacterial action, as seen for example with the sequestration of iron by tetracyclines12. Here we report for the first time that zinc potentiates the antibiotic activity of vancomycin, and certain other glycopeptide antibiotics, specifically against bacterial strains exhibiting inducible glycopeptide resistance. Structural analysis indicates a novel polymerized arrangement of vancomycin molecules resulting from the interaction with Zn(II), and we use data from in vitro and in vivo studies to propose a new mode of action for this vancomycin polymer. We consider the significance of the interaction with Zn(II) for patient treatment.
Results
Zn(II) potentiates the activity of vancomycin against inducibly resistant bacteria
To investigate the effect of the interaction of Zn(II) with vancomycin on antibiotic activity, we performed susceptibility tests using strains known to be inducibly resistant to vancomycin, and isogenic relatives lacking this resistance. Streptomyces coelicolor M60013 and Enterococcus faecalis JH2-2::I14 both possess dedicated resistance genes which direct the synthesis of modified PG precursors ending in D-Ala-D-Lac, to which glycopeptide antibiotics exhibit very low binding affinities. Expression of resistance is induced by vancomycin via a two-component regulatory system composed of a sensor kinase (VanS) and a cognate response regulator (VanR) that acts as a transcriptional activator when phosphorylated. Strikingly, co-administration of vancomycin with increasing concentrations of zinc sulphate (0-200 μM) increased the zone of growth inhibition against vancomycin-resistant S. coelicolor M600 in a paper disc diffusion assay (Fig. 1a). This effect is specific for Zn(II). Both Ni(II) and Cu(II), the latter known to more strongly interact with vancomycin than Zn(II)7, 15, 16, had no effect on the activity of vancomycin. As expected, the presence of Zn(II) also enhanced the antibiotic activity of bacitracin9, but did not influence kanamycin (Fig. 1a). 200 μM Zn(II) also failed to increase the antibacterial activity of ramoplanin, moenomycin, novobiocin, apramycin and tunicamycin against S. coelicolor M600 (Supplementary Fig. 1), indicating that the concentration of Zn(II) used in the experiments are in the non-toxic range. Teicoplanin, a naturally occurring lipoglycopeptide antibiotic, also showed no increase in activity in the presence of Zn(II) (Fig. 1a). Importantly, the potentiation of vancomycin activity by Zn(II) was also observed when using E. faecalis JH2-2::I as the test strain indicating that this is a more general phenomenon (Fig. 1b). Minimal inhibitory concentration (MIC) measurements attribute a 4- to 8-fold increase in susceptibility towards vancomycin to Zn(II) in E. faecalis JH2-2::I and S. coelicolor M600, respectively (Fig. 1b). Notably, the use of the vancomycin-sensitive strains S. coelicolor J320117 and E. faecalis JH2-214 abolished the influence of Zn(II) on vancomycin activity, but had no abrogating effect on the enhancement of bacitracin activity (Fig. 1b). The VanRS two-component regulatory system required for inducing vancomycin resistance is deleted in S. coelicolor J3201, while E. faecalis JH2-2 lacks the transposon carrying all the vancomycin resistance genes that is present in JH2-2::I. This implies that the potentiation of vancomycin by Zn(II) is directly related to the functioning of the vancomycin resistance system.
Only glycopeptide antibiotics which are capable of activating inducible resistance are potentiated by Zn(II)
To define the relationship between the effect of Zn(II) on glycopeptide antibiotic activity and induction of glycopeptide resistance, a broader panel of natural glycopeptides were assayed for activity in different streptomycete indicator strains (Fig. 2a and Supplementary Fig. 2). Glycopeptide-strain combinations where the strength of induction via the VanS sensor kinase had previously been characterized13, 17,18,19,20,21 were selected, and assayed for any potentiation by Zn(II). In strains possessing functional inducible glycopeptide resistance systems, Zn(II)-dependent enhancement of glycopeptide antibiotic activity was closely correlated with the ability of the glycopeptide to act as an inducer of glycopeptide resistance. Thus in S. coelicolor M600, balhimycin and vancomycin are both the strongest inducers and exhibit the most marked potentiation of activity by Zn(II). In contrast, poor inducers of VanS in this strain background i.e. ristocetin, A47934, chloroeremomycin, and teicoplanin show only small or negligible changes in activity on addition of Zn(II) (Fig. 2a). This correlation is confirmed by observations in Streptomyces toyocaensis, where, in contrast to S. coelicolor M600, differences in the amino acid sequence of the VanS sensor kinase (VansSst) dictate that vancomycin is a poor inducer of resistance and A47934 the strongest inducer (Supplementary Fig. 2)19,20,21. The influence of Zn(II) on the activity of these antibiotics is also reversed between the two strains, with vancomycin activity against S. toyocaensis no longer affected by Zn(II) but the activity of A47934 being most markedly increased. Also consistent with these observations, control strains lacking any inducible glycopeptide resistance, S. coelicolor J3201 and Streptomyces avermitilis, exhibited no interaction between Zn(II) and glycopeptide activity (Fig. 2a and Supplementary Fig. 2).
Bioassay results indicate that in the S. coelicolor M600 background the activities of vancomycin and balhimycin are each capable of being enhanced by Zn(II) (Fig. 2a). Vancomycin is known to bind Zn(II)7. To determine whether balhimycin can participate in a similar physical interaction, affinity column chromatography, fluorometry and Calf Intestinal Alkaline Phosphatase (CIAP) inhibition assays were performed as previously described for vancomycin7 (Fig. 2b and Supplementary Figs 3–5). All three methods indicated an interaction with Zn(II) that is notably stronger than that observed for teicoplanin whose antibiotic activity is influenced only marginally by Zn(II). Zn(II)-dependent enhancement of glycopeptide activity therefore appears to require both the ability of the antibiotic to induce glycopeptide resistance via VanS, and physical interaction with Zn(II).
The potentiation of vancomycin activity by Zn(II) requires remodelling of PG termini to D-Ala-D-Lac
Three different hypotheses could explain the effect of Zn(II) on vancomycin (and other glycopeptide) antibiotic activity in inducibly resistant bacterial strains, and these were tested experimentally. An inhibitory effect on the Zn(II)-dependent D-Ala-D-Ala dipeptidase activity of the VanX vancomycin resistance enzyme was ruled out by analysis of the S. coelicolor ∆vanX mutant J322513 which was found to respond to Zn(II)-vancomycin in the same way as the M600 parent strain (Supplementary Fig. 6). The hypothesis that interaction of vancomycin with Zn(II) increased sensitivity by inhibiting activation of the VanS sensor kinase by the glycopeptide was also eliminated through the analysis of mutant strains (Supplementary Fig. 7). vanS mutants of S. coelicolor (J3200)17 and E. faecalis (JH2-2::C1)14 which exhibit constitutively active glycopeptide resistance, with no requirement for the presence of antibiotic inducer, again show a normal enhancement of vancomycin activity in response to Zn(II) (Supplementary Fig. 7). The remaining possibility is that interaction of vancomycin with Zn(II) increases the inhibition of PG biosynthesis that takes place using the remodelled vancomycin-resistant PG precursors possessing D-Ala-D-Lac termini. To investigate this hypothesis we exploited the activities of the homologous enzymes VanA and DdlA in S. coelicolor. VanA is a non-essential bifunctional D-Ala-D-Ala and D-Ala-D-Lac ligase enzyme which preferentially synthesizes D-Ala-D-Lac in the presence of D-Lactate (D-Lac), and supplies the substrate required for the remodelling of PG biosynthesis in response to vancomycin13, 22. In contrast, DdlA encodes the essential D-Ala-D-Ala ligase activity responsible for cell wall formation during normal growth using PG precursors with D-Ala-D-Ala termini22, 23. To generate a strain (H7400) constitutively using PG terminating in D-Ala-D-Lac during normal growth, we introduced the vanA gene under the control of the strong and constitutive ermE promoter (ermEp) into the S. coelicolor ∆vanRS mutant J3201, then successfully deleted the normally essential ddlA gene. An analogous strain (H7300) expressing ddlA from the ermEp was also generated as a control. Use of the ∆vanRS mutant background ensures that there is no contribution from the native vancomycin-inducible vanA gene. Vancomycin antibiotic activity was potentiated by Zn(II) only in strain H7400 where PG precursors terminating in D-Ala-D-Lac are used for cell wall biosynthesis, not in H7300 which uses PG precursors terminating in D-Ala-D-Ala (Fig. 3a). This effect was further enhanced when the growth medium was supplemented with D-Lactate to promote the D-Ala-D-Lac ligase activity of VanA (Fig. 3a). This implies that the interaction with Zn(II) can specifically increase the binding affinity of vancomycin for PG molecules terminating in D-Ala-D-Lac. This interpretation is supported by in vitro fluorometric analysis which shows a marked quenching of vancomycin fluorescence on addition of Zn(II) to a solution containing vancomycin and D-Ala-D-Lac, but not vancomycin in the presence of D-Ala-D-Ala (Fig. 3b). In contrast, addition of Zn(II) to vancomycin enhances fluorescence in the control indicating different binding (Fig. 3b).
Zn(II) mediates polymerisation of vancomycin
The interaction of vancomycin with Zn(II) is associated with a novel D-Ala-D-Lac binding activity. To investigate the structural basis for this activity we have crystallized the vancomycin-Zn(II) complex. Our first attempt generated a crystal structure for a vancomycin-Zn(II)-citrate complex containing two independent vancomycin molecules in an asymmetric unit with space group C222, plus a separate complex of Zn(II) coordinated by two citrate molecules and a polyethylene glycol molecule (Supplementary Fig. 8a and Supplementary Table 2). This dimeric arrangement of vancomycin is identical to that previously reported by Schäfer and colleagues (space group P 43212) (Supplementary Fig. 8c)24. Subsequent crystallization in the absence of citrate successfully generated a clean vancomycin-Zn(II) crystal structure (Fig. 4a and Supplementary Fig. 8b). The structure revealed two independent vancomycin molecules in space group H3, and a Zn(II) ion coordinated by two water molecules and two atoms derived from the N-terminal methylleucine group of vancomycin, including one amino nitrogen and one carboxyl oxygen (Fig. 4a, Supplementary Fig. 8b and Supplementary Table 2). This binding site for Zn(II) to the vancomycin molecule is entirely consistent with that predicted from our previous NMR solution structural studies, and is distinct from the binding site for Cu(II) (Fig. 4b)7, 15. The presence of Zn(II) allows vancomycin to adopt a novel conformation not previously observed in reported vancomycin crystal structures (Supplementary Fig. 8b), and is distinct from the conformation of the vancomycin-Cu(II) complex in which two independent vancomycin molecules are arranged in space group C2 (Supplementary Fig. 8d)16. This novel conformation allows the assembly of repeated vancomycin dimers held together by coordination with Zn(II) to generate a vancomycin polymer (Fig. 4a). Fibre X-ray diffraction, a technique to study the molecular structure of long assemblies of identical subunits25, confirmed the polymerization of vancomycin by Zn(II) (Supplementary Fig. 9). Analysis of a sample containing both vancomycin and Zn(II) generated a diffraction pattern consisting of three concentric reflection rings at 25 Å, 11 Å and 8 Å of Braggs’ interplanar distances, consistent with the presence of repetitive polymer fibres with a low degree of alignment26, 27. These were completely absent in similar analysis of a control sample of vancomycin lacking Zn(II) (Supplementary Fig. 9).
Discussion
High-level glycopeptide resistance in Gram-positive bacteria is due to the replacement of the D-Ala-D-Ala terminus of PG precursors with D-Ala-D-Lac, to which the antibiotics exhibit lower binding affinities4. Replacement with D-Ala-D-Ser can also occur but generates markedly lower resistance due to production of a smaller defect in antibiotic binding. The identification of a lead glycopeptide structure with increased activity against cell wall biosynthesis proceeding via PG precursors with D-Ala-D-Lac termini is therefore of significant interest. The novel polymerization of vancomycin mediated by interaction with Zn(II) is associated with increased antibiotic activity against model D-Ala-D-Lac-based glycopeptide resistant strains, including an E. faecalis VRE strain. Interestingly, a synthetic polymer of vancomycin monomers with enhanced potency against VRE has previously been reported, and surface plasmon resonance studies also indicated an increased binding affinity between the polymer and a VRE cell wall mimic containing D-Ala-D-Lac28. In this study, Zn(II) causes polymerization of vancomycin dimers, with the metal ion holding repeated dimeric units in place via interaction with nitrogen and oxygen atoms in the N-terminal methylleucine moiety (Fig. 4).
Other glycopeptide or lipoglycopeptide antibiotics which possess the methylleucine interaction site would also be expected to bind Zn(II) and may also be susceptible to similar polymerization and enhancement of antibiotic activity. Consistent with this, Zn(II) was able to increase the activity of two antibiotics with the methylleucine group, balhimycin and A47934, but had no effect on either teicoplanin or ristocetin which lack this feature (Supplementary Table 3). In vitro assays also demonstrated markedly stronger binding of Zn(II) to balhimycin compared to teicoplanin. Chloroeremomycin does possess a potential binding site but Zn(II) did not produce an effect in a bioassay against S. coelicolor M600 since the antibiotic is not a good inducer of the D-Ala-D-Lac resistance system. Interestingly, of the three new semi-synthetic lipoglycopeptide antibiotics being brought into clinical use, both oritavancin and telavancin retain the methylleucine present in vancomycin and therefore potentially also retain the ability to interact with Zn(II). Dalbavancin has a structure related to teicoplanin and would not be predicted to interact.
Due to the well-known effect of Zn(II) on bacitracin antibiotic activity, topical treatments for application to wound infections come pre-formulated as bacitracin-zinc. The results of this study suggest that a similar approach may also be beneficial for vancomycin to augment activity against vancomycin resistant pathogens. This could be evaluated for clinical significance using a suitable infection model. Some of the known side effects of vancomycin in long term treatments also coincidently overlap with aspects of zinc deficiency syndrome, such as nephrotoxicity or muscle spasm29, 30. Careful co-administration of vancomycin with a Zn(II) additive could therefore be helpful for both antibiotic activity and the alleviation of toxic side effects. Further studies to determine the structure of the tertiary vancomycin-Zn(II)-D-Ala-D-Lac complex will be necessary to fully understand the nature of the binding to D-Ala-D-Lac PG precursors, and to be able to further exploit the enhanced affinity of polymerized vancomycin and other glycopeptide antibiotics toward D-Ala-D-Lac.
Methods
Bacterial strains, plasmids, oligos, antibiotics and culture conditions
The bacterial stains, plasmids and oligos used in this study are described and listed in Supplementary Table 4. A47934 was prepared from the producer strain S. toyocaensis as described previously31. Balhimycin and chloroeremomycin were kind gifts from Andy Truman. All other antibiotics used for this study were purchased from Sigma-Aldrich. Except where detailed below, media and culture conditions were as previously described32.
Antibiotic susceptibility tests
All susceptibility tests, including the antibiotic disc diffusion assays and MIC determinations, were performed on MMCGT agar medium33 supplemented with the required concentration of metal sulphate. Results were scored after 2–4 days of incubation at 30 °C. For the antibiotic disc diffusion assay, for S. coelicolor, 0, 50, 100, 150 and 200 μM of metal sulphate was added to the media, and for S. avermitilis 0, 100 and 200 μM of metal sulphate was used. For S. toyocaensis, the assay was performed using 0, 50 and 100 μM of metal sulphate. E. faecalis strains were exposed to 0, 100, 200, 300 and 400 μM metal sulphate. Concentrations beyond these maximum levels can cause toxicity and were avoided. For the bioassay of all Streptomyces strains, approximately 107 spores were used to seed the test plates. For the E. faecalis strains, approximately 107 cfu from an overnight LB liquid culture were spread on the MMCGT medium. The concentrations of antibiotics used in the bioassays are listed in Supplementary Table 1. For the determination of the vancomycin MICs, Etest strips purchased from Biomerieux were used. All bioassays were performed at least three times and representative results are presented. The degree of relative variation for the inhibitory halo size in plate diffusion tests was less than 10%.
Construction of S. coelicolor H7300 and H7400
S. coelicolor H7300 (∆vanRS ∆ddlA + ermEp-ddlA) and H7400 (∆vanRS ΔddlA + ermEp-vanA), were constructed from S. coelicolor ∆vanRS J320117 as follows. Initial transformation with pGN8 (pIJ10257 ermEp-ddlA)22 or pGN17 (pIJ10257 ermEp-vanA)22 generated strains H7100 and H7200, respectively. Deletion of the ddlA gene in H7100 and H7200 was then accomplished by PCR-directed mutagenesis as described previously22, creating H7300 and H7400, respectively.
Affinity column chromatography
Metal affinity column chromatography experiments were performed as described previously7. HiTrap Chelating HP (GE healthcare) columns were initially washed with 5 ml of MilliQ water with a flow rate of 1 ml min−1 then loaded with 500 μl of 100 mM copper, nickel or zinc sulphate solutions. A column loaded with buffer solution was used as an uncharged negative control. Columns were washed again with 5 ml of MilliQ water then equilibrated with 10 ml of binding buffer (20 mM NaH2PO4/Na2HPO4 (pH 7.4), 200 mM NaCl, 10% Glycerol, 0.1% Triton-X-100). 150 μl of 10 mM balhimycin or teicoplanin was applied to each column and elution performed using 5 ml of binding buffer followed by 5 ml of binding buffer supplemented with 50 mM imidazole. Sixteen 0.5 ml fractions were collected (after discarding the dead volume of the column) and the relative abundance of antibiotic in each fraction was measured at OD282 after 5x dilution in water. Aliquots of the same diluted fractions were also analysed in a bioassay. For this, 20 μl was applied to freshly prepared MMCGT bioassay plates seeded with ~107 spores of a vancomycin-sensitive S. coelicolor ∆vanRS strain (J3201)17. The results were scored after incubation at 30 °C for 2–4 days.
Fluorometry
Fluorometric analysis was performed in 100 mM Tris-Cl (pH7.3) according to the method described previously7 using a PerkinElmer LS55 fluorescence spectrometer (Waltham, MA, USA) set to a 280 nm excitation wavelength; 5 nm slit width; 300–450 nm emission scan range; and 50 nm min−1 scan speed. Vancomycin solutions were supplemented with 50 µM of Ac-Lys(Ac)-D-Ala-D-Ala-OH or Ac-Lys(Ac)-D-Ala-D-Lac-OH (Bachem) where indicated. Zn(II) was added to the required concentration from a 50 mM stock solution of zinc sulphate heptahydrate in deionized water.
Crystallization, data collection and refinement
Vancomycin hydrochloride (Sigma, V2002) and zinc sulphate heptahydrate (Sigma, Z4750) were purchased from Sigma and used without further purification. Vancomycin was used at a concentration of 50 mg ml−1 in 10 mM HEPES buffer at pH 7.022, and Zn(II) added to a 10-fold molar excess, adjusting the pH to 7.0 as required. Any insoluble material was removed by centrifugation, and the supernatant was used to set up MRC 2-drop crystallisation trial plates containing conditions from the JCSG + (Molecular Dimensions) or PEGs I (Qiagen) crystallisation suites. Plates were loaded with two drops per reservoir, each containing 0.2 μl sample + 0.2 μl of crystallisation suite mix, using a Mosquito® crystallisation robot (TTP Labtech). The plates were then incubated at 19 °C in a Rock Imager Hotel/Imager (Formulatrix) and imaged (visual and UV) over a period of 34 days. Any crystals formed were harvested using 0.2–0.3 ml cryoloops (Hampton) and cryoprotectant solution containing 26% ethylene glycol and reservoir solution. Harvested crystals were plunged into liquid nitrogen and either stored or used for diffraction experiments straight away. Well A3 of the JCSG + plate, the reservoir of which contains 0.2 M di-ammonium hydrogen citrate salt [(NH4)2H Cit (C6H5O7)] + 20% (w/v) PEG 3350, produced the crystal found to be a tertiary vancomycin-Zn(II)-citrate complex. The desired vancomycin-Zn(II) crystal was obtained from well F8 of the PEGs I plate, from a reservoir comprising 0.2 M magnesium formate salt + 20% (w/v) PEG 3350. Diffraction data were collected at the Diamond Synchrotron Facility (Oxford, UK). A summary of the conditions for the data collection and the structure refinement parameters are given in Supplementary Table 2. The crystal structures were solved either by direct methods or using the single wavelength anomalous dispersion (SAD) method, with the chlorine atoms as the anomalous scatters (Cl-SAD). Model coordinates have been deposited in the Protein Data Bank (PDB) under accession number 5M2H for the vancomycin-Zn(II)-citrate complex and under accession number 5M2K for the vancomycin-Zn(II) complex.
Fibre X-ray diffraction
X-ray diffraction studies were performed at the Crystallographic X-ray facility, Department of Biochemistry, University of Cambridge using an X8 Proteum (Bruker AXS). Fibre samples were placed in the X-ray beam and the diffraction data were collected with a sample-to-detector distance of 120 mm and an exposure time of 120 sec. Fibre samples for analysis were generated from solutions of vancomycin (5 mg ml−1 solution of vancomycin hydrochloride in 10 mM HEPES buffer at pH 7.0) or vancomycin-Zn(II) (vancomycin solution plus a 10-fold molar excess of zinc sulphate heptahydrate (Sigma, Z4750) in 10 mM HEPES buffer at pH 7.0) as described in the Supplementary Methods.
References
Howden, B. P. et al. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clin. Microbiol. Rev. 23, 99–139 (2010).
Uttley, A. H. C., Collins, C. H., Naidoo, J. & George, R. C. Vancomycin-resistant enterococci. Lancet i, 57–58 (1988).
Barna, J. C. & Williams, D. H. The structure and mode of action of glycopeptide antibiotics of the vancomycin group. Annu. Rev. Microbiol. 38, 339–357 (1984).
Binda, E., Marinelli, F. & Marcone, G. L. Old and new glycopeptide antibiotics: Action and Resistance. Antibiotics (Basel) 3, 572–594 (2014).
Klinker, K. P. & Borgert, S. J. Beyond vancomycin: The tail of the lipoglycopeptides. Clin. Ther. 37, 2619–2636 (2015).
Hesketh, A. et al. Genome-wide dynamics of the bacterial response to antibiotics that target the cell envelope. BMC genomics 12, 226 (2011).
Zarkan, A. et al. The frontline antibiotic vancomycin induces a zinc starvation response in bacteria by binding to Zn(II). Sci. Rep. 6, 19602 (2016).
Ming, L. J. Structure and function of “metalloantibiotics”. Med. Res. Rev. 23, 697–762 (2003).
Economou, N. J., Cocklin, S. & Loll, P. J. High-resolution crystal structure reveals molecular details of target recognition by bacitracin. Proc. Natl. Acad. Sci. USA 110, 697-14207-14212 (2013).
Grenier, D. et al. Iron-chelating activity of tetracyclines and its impact on the susceptibility of Actinobacillus actinomycetemcomitans to these antibiotics. Antimicrob. Agents Chemother. 44, 763–766 (2000).
Chohan, Z. H. et al. Metal binding and antibacterial activity of ciprofloxacin complexes. J. Enzyme Inhib. Med. Chem. 20, 303–307 (2005).
Uivarosi, V. Metal complexes of quinolone antibiotics and their applications: an update. Molecules 18, 11153–11197 (2013).
Hong, H. J. et al. Characterization of an inducible vancomycin resistant system in Streptomyces coelicolor reveals a novel gene (vanK) required for drug resistance. Mol. Microbiol. 52, 1107–1121 (2004).
Foucault, M. L., Depardieu, F., Courvalin, P. & Grillot-Courvalin, C. Inducible expression eliminates the fitness cost of vancomycin resistance in enterococci. Proc. Natl. Acad. Sci. USA 107, 16964–16969 (2010).
Nair, U. B. et al. Elucidation of vancomycin’s enantioselective binding site using its copper complex. Chirality 8, 590–595 (1996).
Kucharczyk, M. et al. Structural features of the Cu(2+)-vancomycin complex. J. Inorg. Biochem. 102, 936–942 (2008).
Hutchings, M. I., Hong, H. J. & Buttner, M. J. The vancomycin resistance VanRS signal transduction system of Streptomyces coelicolor. Mol. Microbiol. 59, 923–935 (2006).
Kwun, M. J. & Hong, H. J. The activity of glycopeptide antibiotics against resistant bacteria correlates with their ability to induce the resistance system. Antimicrob. Agents Chemother. 58, 6306–6310 (2014).
Koteva, K. et al. A vancomycin photoprobe identifies the histidine kinase VanSsc as a vancomycin receptor. Nat. Chem. Biol. 6, 327–329 (2010).
Novotna, G. et al. A novel membrane protein, VanJ, conferring resistance to teicoplanin. Antimicrob. Agents Chemother. 56, 1784–1796 (2012).
Novotna, G., Kwun, M. J. & Hong, H.-J. In vivo characterization of the activation and interaction of the VanR-VanS two-component regulatory system controlling glycopeptide antibiotic resistance in two related Streptomyces species. Antimicrob. Agents Chemother. 60, 1627–1637 (2015).
Kwun, M. J. et al. In vivo studies suggest that induction of VanS-dependent vancomycin resistance requires binding of the drug to D-Ala-D-Ala termini in the peptidoglycan cell wall. Antimicrob. Agents Chemother. 57, 4470–4480 (2013).
Zawadzke, L. E., Bugg, T. D. & Walsh, C. T. Existence of two D-alanine:D-alanine ligases in Escherichia coli: cloning and sequencing of the ddlA gene and purification and characterization of the DdlA and DdlB enzymes. Biochemistry 30, 1678–1682 (1991).
Schäfer, M., Schneider, T. R. & Sheldrick, G. M. Crystal structure of vancomycin. Structure 4, 1509–1515 (1996).
Marvin, D. A. & Nave, C. X-Ray Fibre Diffraction. Structural Molecular Biology: Methods and Applications. New York, USA: Plenum Press (1982).
Sikorski, P., Atkins, E. D. & Serpell, L. C. Structure and texture of fibrous crystals formed by Alzheimer’s abeta(11-25) peptide fragment. Structure 11, 915–926 (2003).
Morris, K. L. & Serpell, L. C. Chapter 9 X-Ray Fibre Diffraction of Amyloid Fibrils. Amyloid Proteins – Methods and Protocols. New York, USA: Springer (2012).
Arimoto, H. et al. Multi-valent polymer of vancomycin: enhanced antibacterial activity against VRE. Chem. Commun. 15, 1361–1362 (1999).
Levine, D. P. Vancomycin: a history. Clin. Infect. Dis. 42, S5–12 (2006).
Maret, W. & Sandstead, H. H. Zinc requirements and the risks and benefits of zinc supplementation. J. Trace Elem. Med. Biol. 20, 3–18 (2006).
Truman, A. W. et al. Antibiotic resistance mechanisms inform discovery: Identification and characterization of a novel Amycolatopsis strain producing Ristocetin. Antimicrob. Agents Chemother. 58, 5687–5695 (2014).
Kieser, T. et al. Practical Streptomyces genetics. The John Innes Foundation, Norwich (2000).
Hong, H.-J., Paget, M. S. & Buttner, M. J. A signal transduction system in Streptomyces coelicolor that activates the expression of a putative cell wall glycan operon in response to vancomycin and other cell wall-specific antibiotics. Mol. Microbiol. 44, 1199–1211 (2002).
Acknowledgements
This work was supported by funding from the Royal Society, UK (516002.K5877/ROG) and the Medical Research Council, UK (G0700141). A.Z. was supported from the Said foundation, the Cambridge Trust and the Cambridge Philosophical Society. We thank Andrew Truman and Patrice Courvalin for kindly supplying some of the glycopeptide antibiotics and Enterococcal strains used in this study.
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A.Z., A.R.H., and H.J.H. designed the study and wrote the manuscript. H.R.M. assisted with some of the antibiotic activity assays. A.Z., A.D.B. and D.Y.C. performed the structural crystallization studies. A.Z. performed all other experiments.
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Zarkan, A., Macklyne, HR., Chirgadze, D.Y. et al. Zn(II) mediates vancomycin polymerization and potentiates its antibiotic activity against resistant bacteria. Sci Rep 7, 4893 (2017). https://doi.org/10.1038/s41598-017-04868-2
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DOI: https://doi.org/10.1038/s41598-017-04868-2
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