Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

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Science  28 May 2010:
Vol. 328, Issue 5982, pp. 1168-1172
DOI: 10.1126/science.1185723


Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.

Plectasin is a 40–amino acid residue fungal defensin produced by the saprophytic ascomycete Pseudoplectania nigrella (1). Plectasin shares primary structural features with defensins from spiders, scorpions, dragonflies and mussels and folds into a cystine-stabilized alpha-beta structure (CSαβ). In vitro and in animal models of infection, plectasin is potently active against drug-resistant Gram-positive bacteria such as streptococci, whereas the antibacterial spectrum of an improved derivative, NZ2114 (2), also includes staphylococci such as methicillin-resistant Staphylococcus aureus (MRSA).

We set out to determine the molecular target and specific mechanism by which plectasin kills bacteria. Although many host defense peptides (HDPs) act on and disintegrate the bacterial membrane, several observations suggested that this is not the case for plectasin.

Growth kinetic measurements of the Gram-positive bacterium Bacillus subtilis exposed to plectasin clearly demonstrated that plectasin exhibited kinetic behavior similar to cell wall–interfering agents (such as vancomycin, penicillin, and bacitracin) and not to the rapidly lytic membrane-active agents (such as polymyxin and novispirin) or non-lytic antibiotics with replication (ciprofloxacin), transcription (rifampicin), or protein translation (kanamycin, tetracycline) as their primary target (Fig. 1A) (3). Consistently with this, killing kinetics indicated that over a period of approximately one generation time (0.5 hours) treated cells were unable to multiply, but remained viable (Fig. 1B inset), before the number of colony-forming units decreased (Fig. 1B). Next, the effect of plectasin on macromolecular biosynthesis pathways was investigated. The incorporation of radiolabeled isoleucine into protein and of thymidine into nucleic acids was not affected, whereas glucosamine—an essential precursor of bacterial peptidoglycan—was no longer incorporated (Fig. 1C). Lastly, treatment of B. subtilis with plectasin induced severe cell-shape deformations as visualized through phase-contrast microscopy (fig. S1). These characteristics are all typical for compounds interfering with cell-wall biosynthesis rather than for membrane disintegration (4, 5). Consistently, neither pore formation as measured by K+ efflux (Fig. 1E), nor changes in membrane potential by use of TPP+ or DiBAC4 (fig. S2, A and B), nor carboxy-fluorescein efflux from liposomes were detected (fig. S2C). Thus, despite its amphipathic nature, plectasin does not compromise membrane integrity, reducing the risk of unspecific toxicity.

Fig. 1

Effect of plectasin on intact cells. (A) Classification of antimicrobial compounds by using optical density measurements. Growth kinetic measurements of B. subtilis exposed to plectasin or various antibiotics with known cellular targets. Two to four times the minimal inhibitory concentration (MIC) of the respective compounds were used. Plectasin (black) falls into the cluster of cell-wall biosynthesis inhibiting antibiotics (red colors). (B) Killing kinetics of plectasin; Staphylococcus simulans 22 treated with plectasin at 2 × MIC (open diamonds) and 4 × MIC (squares); control is without peptide (triangles). (Insert) A similar experiment with more time points within the first 60 min demonstrating the absence of killing in the first 30 min of treatment. (C) Impact of plectasin on macromolecular biosynthesis in B. subtilis 168. Incorporation of [14C]-thymidine into nucleic acids, of L-[14C]-isoleucine into protein, and of [3H]–glucosamine in cell wall was measured in untreated controls (squares) and plectasin-treated cells (open circles); glucosamine incorporation into cell-wall material was selectively inhibited. (D) Intracellular accumulation of the ultimate soluble cell-wall precursor UDP-MurNAc-pentapeptide in vancomycin-treated (dotted line) and plectasin-treated (dashed line) cells of S. simulans 22. Cells were treated for 30 min with plectasin or vancomycin, which is known to form a complex with Lipid II. Treated cells were extracted with boiling water, and the intracellular nucleotide pool was analyzed by means of reverse HPLC. UDP-MurNAc-pentapeptide was identified by means of mass spectrometry using the negative mode and 1 mg/ml 6-aza-2-thiothymine [in 50% (v/v) ethanol/20 mM ammonium citrate] as matrix; the calculated monoisotopic mass is 1149.35; in addition to the singly charged ion, the mono- and disodium salts are detected. (E) Plectasin is unable to form pores in the cytoplasmic membrane of S. simulans 22. Potassium efflux from living cells was monitored with a potassium-sensitive electrode. Ion leakage is expressed relative to the total amount of potassium released after addition of 1 μM pore-forming lantibiotic nisin (100%, open diamonds). Plectasin was added at 0.2 μM (triangles) and 1 μM (open triangles); controls were without peptide antibiotics (squares).

We obtained further support for the cell wall–interfering activity using DNA microarrays to compare the transcriptional responses of plectasin-treated cells with response patterns obtained for a range of reference antibiotics. For both B. subtilis 168 and S. aureus SG511, we found that the transcriptional profiles overlapped those of established cell-wall biosynthesis inhibitors, such as vancomycin and bacitracin (69) (fig. S3 and tables S1 and S2).

The biosynthesis of bacterial cell walls requires a number of steps (10). Initially, the N-acetylmuramic acid-pentapeptide (MurNAc-pentapeptide)—a major constituent of the cell-wall building block—is produced in the cytoplasm as an uridine diphosphate (UDP)–activated precursor before it is transferred onto a membrane carrier, bactoprenolphosphate (Fig. 2B, reaction I). The resulting membrane-anchored precursor Lipid I is then further modified to the structural cell-wall subunit, Lipid II (Fig. 2B, reaction II). In some Gram-positive bacteria, Lipid II (Fig. 2A) is further decorated by an interpeptide bridge [a pentaglycine peptide in the case of S. aureus (11)] (Fig. 2B, reaction III) before it gets translocated across the cytoplasmic membrane to the outside, where it is incorporated into the peptidoglycan polymer through the activity of transglycosylases and transpeptidases (Fig. 2B, reaction IV). We analyzed the intracellular pool of cell-wall precursors by means of reverse high-performance liquid chromatography (HPLC) and mass spectrometry and found accumulation of the soluble molecule UDP-MurNAc-pentapeptide in plectasin-treated cells (Fig. 1D), suggesting that one of the later membrane-associated or extracellular processes may be targeted by plectasin.

Fig. 2

Inhibition of membrane-associated cell-wall biosynthesis steps. (A) Structure of the cell-wall precursor Lipid II. (B) The membrane-bound steps of cell-wall precursor biosynthesis and bactoprenol (C55P) carrier cycling in staphylococci. Cell-wall biosynthesis starts in the cytoplasm with the formation of the soluble precursor UDP-MurNAc-pentapeptide (UDP-MurNAc-pp). This precursor is linked to the membrane carrier bactoprenolphosphate (C55P) by MraY yielding Lipid I (reaction I). Lipid II is formed by MurG, which adds N-acetyl-glucosamine (GlcNAc) (reaction II). When the interpeptide bridge, which only occurs in some Gram-positive bacteria, is accomplished (reaction III), the monomeric peptidoglycan unit is translocated across the cytoplasmic membrane to the outside and incorporated into the cell wall (reaction IV). (C) Inhibition of membrane-associated steps of cell-wall biosynthesis by plectasin. In all tests, plectasin was added in molar ratios of 0.1 to 1 with respect to the amount of the appropriate lipid substrate C55P, Lipid I, or Lipid II used in the individual test system. The amount of reaction products synthesized in the absence of plectasin was taken as 100%. Product analysis was done by means of TLC and subsequent scintillation counting of stained and excised product-containing bands; radiolabeling was based on [3H]-labeled C55P (for Lipid I), [14C]-GlcNAc for Lipid II, and [14C]-glycine for Lipid II-Gly1. Error bars represent ±SD, and the experiments were repeated at least three times. Technical details on the assays and the cloning and purification of the enzymes are given in (3). (D) Estimation of the stoichiometry of plectasin:Lipid II binding. Lipid II was incubated in the presence of plectasin at the molar concentration ratios indicated. The stable complex of plectasin with the Lipid II remains at the application spot, whereas both components migrate to the sites indicated. At a molar ratio of 1:1, neither free Lipid II nor free plectasin were observed.

We then analyzed the effect of plectasin on the membrane-bound steps of cell-wall biosynthesis in vitro. Cytoplasmic membranes with associated cell-wall biosynthesis apparatus were isolated and incubated with plectasin and radiolabeled substrates that are necessary for Lipid II formation. Using thin-layer chromatography and subsequent scintillation counting, we found the overall synthesis reaction to be strongly inhibited (Fig. 2C). For a more detailed analysis, we cloned the individual cell-wall biosynthesis genes from S. aureus, expressed them in Escherichia coli, and analyzed the activity of the purified enzymes in the presence of plectasin by measuring the amount of product formed. These enzymes included MraY (Fig. 2B, reaction I), MurG (Fig. 2B, reaction II), FemXAB (Fig. 2B, reaction III), and PBP2 (Fig. 2B, reaction IV). Whereas the MraY reaction was not affected by plectasin, we found the MurG, FemX, and PBP2 reactions to be inhibited in a dose-dependent fashion (Fig. 2C). For these three enzymes, Lipid I (MurG) or Lipid II (FemX and PBP2) are substrates, and significant inhibition of the reactions was only observed when plectasin was added in equimolar concentrations with respect to Lipid I or Lipid II (Fig. 2C). Thus, plectasin—similarly to glycopeptide antibiotics [such as vancomycin (12, 13)] and lantibiotics (14, 15)—may form a stoichiometric complex with the substrate rather than inhibiting the enzyme. To further validate this, we incubated either Lipid I or II with plectasin in various molar ratios and used thin-layer chromatography (TLC) to analyze the migration behavior. Free Lipid I and II as well as free peptide were found to migrate to defined positions in the chromatogram, whereas the Lipid I/II–plectasin complex remained at the start point (Fig. 2D). Free Lipid I/II and free peptide were not detectable only at an equimolar ratio, indicating the formation of a 1:1 stoichiometric complex.

We further analyzed the interaction of both Lipid I and II with plectasin using a liposome system with membranes composed of phosphatidylcholine and Lipid II [0.2 or 0.5 mole percent (mol %)] and 14C-labeled plectasin. We found the maximum number of plectasin molecules that bound to liposomes to approximately match the number of Lipid II molecules available on the liposome surface (fig. S4). Using Scatchard plot analysis, we determined an equilibrium-binding constant of 1.8 × 10−7 mol for Lipid II and 1.1 × 10−6 mol for Lipid I, suggesting that the second sugar in Lipid II, the N-acetyl glucosamine, contributes to the stability of the complex.

To gain further insight into the structural nature of the plectasin/Lipid II interaction at the membrane interface, we measured chemical shift changes for 15N-labeled plectasin. Heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra were measured either in solution or on binding membrane-mimicking dodecylphosphocholine (DPC) micelles (figs. S5 and S6). Fitting the binding data to a Langmuir isotherm yielded a free enthalpy of binding ΔG = –27 ± 1 kJ/mol (fig. S7). Backbone HN and N atoms of 10 residues [G6, W8, D9, A31, K32, G33, G34, F35, V36, and C37 (16)], which in the tertiary structure all locate to one end of plectasin, exhibited marked changes in chemical shifts [Δδobs > 0.15 parts per million (ppm)] (Fig. 3A, residues labeled yellow), suggesting an orientation in which one end of plectasin specifically is located in the membrane interface.

Fig. 3

NMR-based model of the plectasin/Lipid II-complex. (A) Surface representation of plectasin with the residues showing substantial chemical shift perturbations upon binding to DPC micelles, which are indicated in yellow. (B) Surface representation of plectasin with the residues showing substantial chemical shift perturbations upon Lipid II titration, which is shown in magenta. (C) Detailed view of the pyrophosphate-binding pocket. In this proposed HADDOCK-generated model, the pyrophosphate moiety forms hydrogen bonds to F2, G3, C4, and C27, and the d-γ-glutamate of Lipid II forms a salt bridge with the N terminus of plectasin and the side-chain of His18.

To identify the residues on plectasin that bind Lipid II, we then titrated plectasin bound to DPC with Lipid II. With increasing concentrations of Lipid II, another set of NMR signals appeared and became stronger, whereas the NMR signals of apo-plectasin bound to DPC micelles became weaker until they disappeared at equimolar concentrations of plectasin and Lipid II, supporting the 1:1 binding stoichiometry found by means of TLC. Addition of extra plectasin to the mixture brought the signals of apo-plectasin forward again, and further addition of Lipid II to equimolarity led to the disappearance of the signals again. From a three-dimensional (3D)–HNCA spectrum, we could assign backbone HN, N, and Cα signals of the plectasin:Lipid II:DPC complex. The strongest changes in chemical shift (Δδobs > 0.22 ppm) were obtained for amino acids F2, C4, D12, Y29, A31, G33, C37, and K38 (figs. S5 and S6). Most of these residues localize in a coherent patch in close proximity to the residues affected by binding to DPC (Fig. 3B, residues labeled magenta). A31, G33, and C37 exert chemical shift-changes both upon addition of DPC and Lipid II. To further verify this, site-saturated mutagenesis (in which a given amino acid is changed to each of the other 19 natural amino acids) was carried out at all positions in plectasin except the six cysteines. The mutant libraries were expressed in S. cerevisiae, and 400 to 600 transformants of each position tested for activity against S. aureus in a plate overlay assay. No amino acid substitutions at positions D12, Y29, or G33 resulted in activity against S. aureus, whereas only the very conservative mutations of A31 to G and K38 to R resulted in activity against S. aureus. At other amino acid positions not involved in DPC or Lipid II binding, a wide range of non-homologous amino acid substitutions gave rise to plectasin variants retaining antimicrobial activity.

To visualize the complex between Lipid II and plectasin, docking studies using the GOLD and HADDOCK programs were performed (17, 18). In accordance with the NMR data, evidence in favor of a primary binding site involving the interaction of the pyrophosphate moiety of Lipid II with the amide protons F2, G3, C4, and C37 of plectasin via hydrogen bonding was obtained (Fig. 3C). Several of the other large chemical shift changes are present in residues involved in secondary structure interactions (such as the formation of beta-sheets), which most likely undergo structural changes upon binding to the target. Taken together, these data strongly support a model in which plectasin gains affinity and specificity through binding to the solvent-exposed part of Lipid II, whereas the hydrophobic part of plectasin is located in the membrane interface. Thus, plectasin shares functional features with the lantibiotic nisin in that for both peptides the pyrophosphate moiety is most relevant for binding of Lipid II, although nisin inserts deeply into the membrane bilayer, forming pores and causing major delocalization of Lipid II (19, 20).

To test whether inhibition of cell-wall biosynthesis is restricted to plectasin or represents a general feature, we tested a series of defensin peptides from other fungi, mollusks, and arthropods for Lipid II binding and inhibition of the overall Lipid II synthesis and FemX reaction (fig. S8A). Two fungal defensins, oryzeasin (from Aspergillus oryzea) and eurocin (from Eurotium amstelodami), did inhibit the enzymatic reactions and bind to Lipid II in stoichiometric numbers, as did the two defensins from invertebrates, lucifensin from maggots of the blowfly Lucilia sericata and gallicin from the mussel Mytilus galloprovinciali (fig. S8, B to D). In contrast, heliomicin from the tobacco budworm Heliothis virescens, which shares the conserved cysteine pattern, did not show affinity for Lipid II and had no activity in these assays. These data clearly demonstrate that among the host defense peptides of eukaryotic organisms, specific inhibitors of cell-wall biosynthesis can be found that directly target Lipid II, “the bacterial Achilles’ heel” for antibiotic attack (21).

Vancomycin, one of the very few remaining drugs for the treatment of multi-resistant Gram-positive infections, has been shown to predominantly bind the D-alanyl-D-alanine (D-ala-D-ala) part of the pentapeptide in Lipid II (Fig. 2A) (12). However, high-level vancomycin resistance has been observed in both enterococci (VRE) and staphylococci (VRSA). There is no cross-resistance between vancomycin and plectasin, and in contrast to vancomycin, plectasin is not competitively inhibited by the presence of the D-ala-D-ala ligand (fig. S9). This further demonstrates that the primary interactions to Lipid II differ between plectasin and vancomycin, and taken together, these results suggest that future development of true cross-resistance between vancomycin and plectasin is unlikely.

Plectasin and its improved derivatives such as NZ2114 possess a range of features—such as potent activity in vitro under physiological conditions and in animal models of infection, low potential for unwanted toxicities, extended serum stability and in vivo half-life, and cost-effective large-scale manufacturing—which combined with a validated microbial target make it a promising lead for further drug development.

Supporting Online Material

Materials and Methods

Figs. S1 to S9

Tables S1 and S2


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  3. We thank M. Josten, A. Hansen, and M. R. Markvardsen for expert technical assistance and acknowledge the Carlsberg Research Center for use of the 800-MHz NMR spectrometer and the Obel Foundation for supporting the NMR laboratory at Aalborg University. H.G.S. acknowledges financial support by the European Union (PIAP-GA-2008-218191), the German Research Foundation (SA 292/10-2 and SA 292/13-1), the Bundesministerium für Bildung und Forschung (SkinStaph), and by the BONFOR program of the Medical Faculty, University of Bonn. A.M.J.J.B. acknowledges financial support from the Netherlands Organization for Scientific Research (VICI grant 700.56.442). A.S.A. acknowledges financial support from the Danish Research Council for Technology and Production (274-05-0435). Novozymes AS holds a patent on plectasin (patent number WO2003/044049/044049) and has filed patent applications on improved variants of plectasin. L.D.M., A.K.N. and D.S.R. hold stock options in Novozymes AS. DNA microarray data can be accessed through ArrayExpress, accession number E-MTAB-60. NMR assignment of 1H, 15N, and 13C atoms of plectasin have been deposited in the BioMagResBank (accession number 16739).
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