Strange India All Strange Things About India and world


Phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1; DOPC) was purchased from Avanti Polar Lipids, Inc.

Sample production

Production of teixobactin

Uniformly 13C,15N-labelled teixobactin was produced by fermentation in its native host Eleftheria terrae. In brief, the isolate was grown from a freezer stock on SMSR4 agar (0.125 g casein digest, 0.1 g potato starch, 1 g casamino acids, 1 g d-glucose, 0.1 g yeast extract, 0.3 g proline, 1 g MgCl2-6H2O, 0.4 g CaCl2-2H2O, 0.02 g K2SO4, 0.56 g TES free acid (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid) per 1 l dIH2O, pH to 7 with KOH and 20 g of bacto agar autoclaved at 121 °C for 45 min) for 9 days at 28 °C. Of biomass, 1 cm2 was transferred to 20 ml of modified Celtone-RAZDAZ (10 g d-glucose U-13C6, #CLM-1396, 1.1 g Celtone Base Powder 13C;15N, #CGM-1030P-CN, 0.5 g l-isoleucine 13C6; 15N, #CNLM-561-H, 10 g MgCl2-6H2O, 4 g CaCl2-2H2O, 0.2 g K2SO4, 5.6 g TES free acid per litre, pH to 7 with KOH and autoclaved at 121 °C for 45 min) and grown at 28 °C for 4 days. All labelled material was purchased from Cambridge Isotope Laboratories. Of the grown liquid culture, 20 ml was transferred to 1 l of modified Celtone-RAZDAZ and grown at 28 °C for 6 days. Biomass was harvested by centrifugation at 4,200 r.p.m. and the pellet was extracted with 1 l of 50% aqueous acetonitrile and the suspension again centrifuged for 30 min. The acetonitrile was removed from the supernatant by rotary evaporation under reduced pressure until only water remained. The mixture was then extracted twice with 1 l of n-BuOH. The organic layer was transferred to a round bottom flask and the n-BuOH was removed by rotary evaporation under reduced pressure. The resulting yellow solid was dissolved in DMSO and subjected to preparatory HPLC (high-performance liquid chromatography) (solid phase:C18, mobile phase:H2O/MeCN/0.1% TFA). The fractions containing teixobactin were then pooled and the acetonitrile was removed by rotary evaporation under reduced pressure. The remaining aqueous mixture was then lyophilized to leave a white powder (trifluoroacetate salt). In addition, a quasi-molecular ion peak of m/z 1,315.8683 for 13C58H9615N15O15 [M+H]+ (calculated 1,315.8706 for 13C58H9615N15O15) was determined by high-resolution electrospray ionization mass spectroscopy, confirming 13C58H9515N15O15 as the molecular formula.

Synthesis and purification of lipid II

Lipid II was produced according to published methods based on enzymatic lipid reconstitution using the lipid II precursors UDP-GlcNAc, UDP-MurNAc-pentapeptide and polyisoprenolphosphate as substrates24. Lysine-form UDP-MurNAc-pentapeptide was extracted from S. simulans 22. 13C,15N-labelled UDP-GlcNAc and UDP-MurNAc-pentapeptide (lysine form) were extracted from S. simulans 22 grown in [13C/15N]-labelled rich medium (Silantes) and supplemented with [U-13C]-d-glucose and [15N]-NH4Cl19. Polyisoprenolphosphate was synthesized via phosphorylation of polyisoprenol obtained from Laurus nobilis48. The headgroup precursors were extracted from bacteria and polyisoprenol was extracted from leaves as previously described49. After synthesis, lipid II was extracted with 2:1 BuOH:(Pyr/acetate; 6 M) and then purified with a DEAE cellulose resin using a salt gradient of 0–600 mM NH4HCO3 with 2:3:1 CHCl3:MeOH:[H2O + salt]. Fractions containing pure lipid II were pooled, dried and dissolved in 2:1 chloroform/methanol. The concentration of lipid II was estimated through an inorganic phosphate determination50.

ssNMR sample preparation

Multi-lamellar vesicles of DOPC doped with 4 mol% lysine-lipid II in buffer (40 mM Na2PO4 and 25 mM NaCl, pH 7.2) were collected by centrifugation (60,000g) and loaded into ssNMR rotors. For 3.2-mm rotors, we used 800 nmol of teixobactin with unlabelled lipid II, whereas we used 400 nmol with labelled lipid II. For 1.3-mm rotors, samples contained 200 nmol of antibiotic for unlabelled lipid II.

ssNMR spectroscopy

1H-detected ssNMR experiments were performed at 60 kHz magic angle spinning (MAS) using magnetic fields of 700 and 950 MHz (1H frequency). 3D CαNH and CONH experiments51 for the sequential assignment of teixobactin were performed with dipolar transfer steps using low-power PISSARRO52 decoupling in all dimensions. 1H-detected 15N T1rho relaxation experiments18,51 were acquired with a 15N spin lock-field of 18 kHz and spin-lock durations of 0, 10, 20, 40, 70 and 100 ms. T1rho trajectories were fit to single exponentials. 2D CC experiments were acquired with PARISxy33,53 recoupling (m = 1) at 950 MHz magnetic field and 18 kHz MAS. A 2D CαN experiment was acquired at 700 MHz, 12 kHz MAS and 5 ms N to C cross-polarization transfer time. To characterize lipid II-bound teixobactin, we used CC magnetization transfer times of 50 and 600 ms. To probe interfacial contacts between 13C,15N-teixobactin and 13C,15N-lipid II, we used CC magnetization transfer times of 50, 150 and 300 ms. The scalar TOBSY35 experiment was acquired at 700 MHz using 8 kHz MAS with 6 ms CC mixing time. The mobility edited H(H)C experiment25 was measured at 700 MHz with 16.5 kHz MAS at a temperature of 300 K using a T2 relaxation filter of 2.5 ms. 1D MAS 31P experiments were acquired at 500 MHz magnetic field and 12 kHz MAS. 2D HP experiments were acquired at 800 MHz and 60 kHz MAS using 1 and 2 ms 1H to 31P cross-polarization contact time. Static 31P ssNMR experiments were acquired at 500 MHz magnetic field without sample spinning. Note that the phosphorus nuclei of lipids give rise to an anisotropic powder pattern signal, whose shape depends on the orientation of lipid headgroups25. Further experimental details of ssNMR experiments are given in the Supplementary Information.

Fluorescence microscopy

GUVs preparation

We used a self-assembled GUV cell, aligned with two titanium electrodes in a closed Teflon chamber (volume = 500 μl). Of 0.5 mM DOPC doped with Atto 550-labelled lipid II (0.1 mol%), 1 μl was brushed on the titanium electrodes. The GUV cell was dried under vacuum. Next, the chamber was filled with 350 μl 0.1 M sucrose solution, the electrodes dipped in and connected to a power supply of a sine wave (2.5 V; 10 Hz; 90 min). Each microscopy slide (m-slide 8 well, Ibidi) was incubated with 350 μl BSA solution (1 mg ml−1) for 1 h. To detach the GUVs, the power supply was changed to square wave (2 V; 2 Hz; 15 min). The slides were washed once with water and 0.1 M glucose solution. The slides were immersed in 300 μl of 0.1 M glucose solution to which 50 μl of GUVs was added. These were incubated for 3 h with 1 μM teixobactin and later observed under a Zeiss LSM 880 confocal microscope. GUVs were imaged using Zeiss LSM 880 with ×63/1.2 NA glycerol and ×100/1.2 NA oil objective lenses. The Atto 550 label appeared red upon excitation by the 560-nm laser. The brightfield was used for detection and location of the GUVs and to observe their shape. Zeiss Zen Black software was used for the analysis of the images.

Bacterial imaging

B. megaterium was grown overnight at 37 °C in LB media. Secondary culture was grown for 3 h until the OD600 = 0.3 was reached. Of cells, 500 μl were centrifuged at 3,000g for 5 min. The supernatant was discarded, and the cells were resuspended in 200 μl solution from a 1 μg ml−1 stock of the fluorescent analogue26 Lys(Bodipy FL)10-teixobactin. The cells were allowed to incubate for the desired timepoints (1 min, 15 min and 45 min) at 37 °C. After incubation, they were centrifuged and washed with buffer (100 mM Na2HPO4 and 18 mM KH2PO4, pH 7.4) three times. For fixing the cells, they were resuspended in a 4% formalin and allowed to incubate at 37 °C for 10 min. They were washed once again with the buffer and resuspended in 200 μl of buffer. Of the stained and fixed cells, 50 μl were then pipetted onto the agarose beds and covered with coverslip. The bacterial coverslips were imaged using Zeiss LSM 700 with a ×100/1.2 NA oil objective lens. Lys(Bodipy FL)10-teixobactin was excited using a 488-nm laser. A z-stack containing 15 planes at a 0.56-μm interval was acquired with 0.1-μm pixel size, and maximum intensity projections were made for analysis and display. Icy software’s Spot detector was used to analyse the images and calculate the average intensity of the clusters in all images54.

Isothermal titration calorimetry

For isothermal titration calorimetry (ITC) measurements large unilamellar vesicles (LUVs) containing lysine-lipid II were prepared by incorporating 2 mol% of lysine-lipid II in DOPC from the stock solution. The lipids were dried under a nitrogen stream and hydrated with buffer (20 mM HEPES and 50 mM NaCl, pH 7) to a lipid-phosphate concentration of 20 mM. Finally, unilamellar vesicles were obtained after ten rounds of extrusion through 200-nm membrane filters (Whatman Nuclepore, Track-Etch Membranes). ITC experiments were performed with the Affinity ITC (TA Instruments-Waters LLC) to determine interaction between LUVs and teixobactin. Teixobactin was diluted in the buffer, to a final concentration of 30 μM. The samples were degassed before use. The chamber was filled with 177 μl of teixobactin, and the LUVs were titrated into the chamber at a rate of 1.96 ml per 150 s with a constant syringe stirring rate of 125 r.p.m. The number of injections was 23. Experiments were performed at 37 °C and analysed using the Nano Analyze Software (TA instruments-Water LLC). All experiments were performed in triplicates. Control experiments were performed with lipid II-free DOPC LUVs. The independent model was used to determine the interaction between teixobactin and lipid II. ITC data of R4L10-teixobactin were previously published18.

Fluorescence spectroscopy

For fluorescence spectroscopy, DOPC LUVs containing 0.5 mol% of pyrene-labelled lipid II in buffer (10 mM Tris-Cl and 100 mM NaCl, pH 8.0) were prepared as described above. All fluorescence experiments were performed with a Cary Eclipse (FL0904M005) fluorometer. All samples (1.0 ml) were continuously stirred in a 10 × 4-mm quartz cuvette and kept at 20 °C. Teixobactin was titrated to the LUVs. Pyrene fluorescence was followed with spectral recordings between 360 and 550 nm (λex350 nm, bandwidth 5 nm). The emission at 380 and 495 nm was recorded and averaged over 50 s, to obtain the values for the monomer and excimer intensity, respectively, to determine the excimer to monomer ratio for all conditions.

HS-AFM imaging

The HS-AFM images were acquired in amplitude modulation tapping mode in liquid using a high-speed atomic force microscope (RIBM). Short cantilevers (approximately 7 μm) with a nominal spring constant of 0.15 N m−1 were used (USC-F1.2-k0.15, NanoWorld). A minimal imaging force was applied by using a small set-point amplitude of 0.8 nm (for a 1 nm free amplitude). The HS-AFM results showing the assembly of teixobactin filaments and membrane deformation were obtained from imaging of supported lipid bilayers on mica. The lipid bilayer was obtained by incubating LUVs containing DOPC and lipid II (prepared as mentioned above) on top of a freshly cleaved mica for 20–30 min. After the incubation period, the mica was cleaned gently using recording buffer (10 mM Tris-Cl and 100 mM NaCl, pH 8.0). Imaging was started on the lipid bilayer surface in recording buffer. Next, a concentrated teixobactin solution was added to reach the desired final teixobactin concentration in the AFM liquid chamber of 40 µl. Images were primarily processed using built-in scripts (RIBM) in Igor Pro (Wavemetrics) and analysed using ImageJ software. The images or videos were corrected minimally for tilt, drift and contrast. Unless otherwise mentioned, the times reported in AFM images are relative to the addition of teixobactin into the imaging chamber. Image acquisition rate varies from 0.5 frames per second to 2 frames per seconds (see Fig. 2, Extended Data Figs. 5, 6,  or legends of Supplementary Videos 1 and 2), and the line rate varies from 150 lines per second to 400 lines per second. Control experiments with conventional AFM (JPK Nanowizard) supported the HS-AFM measurements as a similar height of the individual fibrils and their sheets on the membrane was observed. Stated errors are standard deviation.

Permeabilization assay

The bacterial cultures were grown overnight at 30 °C in TSB media for S. simulans and at 37 °C in LB medium for Bacillus subtilis. Secondary cultures were grown for 3 h until OD600 = 0.5 was reached. The bacterial cells were then centrifuged at 1,500g for 10 min at 4 °C and washed twice with 10 ml of buffer (10 mM Tris, 100 mM NaCl, 1 mM MgCl2 and 0.5% glucose, pH 7.2). The bacterial cells were resuspended to an OD600 = 10 in the buffer and used for the experiment. All permeability experiments were performed with a Cary Eclipse (FL0904M005) fluorometer. All samples (1.0 ml) were continuously stirred in a 10 × 4-mm quartz cuvette and kept at 20 °C. For the assay, 1 μl of the bacterial suspension was added to 1 ml of buffer. For the ion leakage assays, 1 μl of the DiSC-2 probe from a 1 mM stock was added to the cuvette and the fluorescence was measured between a wavelength of 650 nm and 670 nm (bandwidth of 5 mm) for 2 min before the addition of the antibiotic and 6 min after. For the Sytox green leakage assays, 1 μl of the Sytox green probe from a 0.25 mM stock was added to the cuvette and the fluorescence was measured between a wavelength of 500 nm and 520 nm (bandwidth of 5 mm) for 2 min before the addition of the antibiotic and 6 min after. All experiments were performed in triplicates. The concentrations of antibiotics used are 10 nM nisin (1× MIC), 10 μM vancomycin (10× MIC) and 0.5 μM plectasin/teixobactin for S. simulans (1× MIC) and 0.2 μM plectasin/teixobactin for B. subtilis (10× MIC).

Structure calculations

Parametrization of teixobactin

Parameters and topology were based on our work on R4L10-teixobactin18, substituted with d-glutamine at position 4 and lallo-enduracididine at position 10. Parameters for lallo-enduracididine were based on l-arginine, in which the guanidinium group was cyclized with ring geometry as in 2-keto-enduracididine in Protein Data Bank (PDB) 4JME55. A monomeric teixobactin starting model for HADDOCK structure calculation was then generated in CNS56, using only chemical-shift-derived restraints57. Parameters for lipid II were taken from ref. 58.

Structure calculation protocol

We used HADDOCK version 2.4 (ref. 39) for the structure calculations. An eight-body docking (four lipid II and four teixobactin molecules) was performed using ssNMR-derived distance and dihedral restraints. Seven thousand models were generated in the rigid-body docking stage of HADDOCK, of which the best-scoring 500 were subjected to the flexible refinement protocol of HADDOCK. The resulting models were energy minimized. Default HADDOCK settings were used except for doubling the weight of the distance restraints during all stages of the structure calculation. The final models were further filtered based on the topological requirements (that is, the lipid tails of all lipid II molecules must point in the same direction as the membrane-anchoring residues Ile2, Ile5 and Ile6). This resulted in a final ensemble of 25 structures.

Analysis of calculated structures

Structural and violation statistics of the final 25 structures are discussed in detail in the Supplementary Information. The average backbone RMSD (from the average structure) of the 25 teixobactin molecules in the complex was 2.3 ± 0.6 Å.

Molecular dynamics simulations

Molecular dynamics calculations were performed with GROMACS, version 4.6.3 using the g54a7 forcefield59. We simulated the ssNMR structure of four teixobactin molecules in complex with four lipid II molecules in a hydrated DOPC membrane. The truncated lipid II tail used for the ssNMR structure was manually elongated to C55 tails by transferring coordinates from ref. 60. The topologies for natural teixobactin and lipid II were generated using ATB61. The charges on the PPi group were adapted to those in ref. 58. For the starting system, the complex was placed approximately 0.5 nm (in reference to the teixobactin molecules) above a pre-equilibrated DOPC bilayer62 (extended to 512 lipids) and ten lipids were removed to accommodate the long lipid II tails. The box (dimensions 12.81 × 12.81 × 10 nm) was then rehydrated and the system electrostatically neutralized (total atom number of 120,295). After minimization, the system was equilibrated at 300 K for 1 ns in an NVT ensemble (fixed number of atoms, N, a fixed volume, V, and a fixed temperature, T) using a V-rescale thermostat with a coupling constant of 0.1 ps and a 2-fs time step with strong position restrains (force constant of 10,000 kJ mol−1 nm−2) on the complex. Next, the system was equilibrated for 100 ns in an NPT ensemble (fixed number of atoms, N, a fixed pressure, P, and a fixed temperature, T) with semi-isotropic pressure coupling at 1 bar using a Parrinello–Rahman barostat63. During this equilibration step, position restraints were gradually reduced from 1,000 to 25 kJ mol−1 nm−2. For lipid II tails, position restraints were removed to facilitate their integration into the membrane. Afterwards, the system was freely evolved in two independent simulations for 287 and 267 ns without applying ssNMR distance restraints. In one of the two simulations, chemical-shift-derived dihedral restraints57 were applied to residues 2–6 of the teixobactin molecules with a constant of force of 100 kJ mol−1 nm−2.

Average atom–atom distances in the ensemble (see Supplementary Tables 5–7) were computed with the GROMACS tool g_dist. The membrane thickness discussed in Fig. 4 was computed with g_lomepro64, considering the phosphorus atoms of DOPC to specify the representative lipid atoms and using a 100 × 100 grid. An additional simulation of 250 ns without teixobactin was performed to get the average thickness of the unperturbed membrane. To back-calculate distances between teixobactin and water or lipid tails, we counted contacts over the free molecular dynamics simulation (from 100 ns to the end of the simulation). Contacts were counted using the GROMACS tool g_mindist for a water or lipid tail atom within a distance of 0.5 nm.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Source link

Leave a Reply

Your email address will not be published.