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Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)
使用薄层色谱法(TLC)和高压液相色谱法(HPLC)进行细菌细胞壁前体磷酸酶测定   

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eLIFE
Jul 2017

Abstract

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.

Keywords: Lipid II (脂质II), Undecaprenyl pyrophosphate (十一异戊烯焦磷酸盐), Phosphatase activity (磷酸酶活性), Peptidoglycan (肽聚糖), HPLC (HPLC), TLC (TLC)

Background

The peptidoglycan (PG) sacculus is an essential bacterial macromolecule that protects the cell from bursting due to its turgor and maintains the shape of the cell (Vollmer and Bertsche, 2008; Typas et al., 2012). PG is composed by glycan chains connected by short peptides. The PG from different species varies in the structure of the peptides and presence of secondary modifications (Vollmer et al., 2008). PG precursors are synthesized inside the cell and equipped with a carrier lipid for transport across the membrane prior to their polymerization at the outer leaflet of the cytoplasmic membrane (Barreteau et al., 2008). The universal bacterial carrier lipid is undecaprenyl phosphate (C55-P), which is synthesized in two steps. First, UppS uses farnesyl pyrophosphate (C15-PP) and eight isopentenyl pyrophosphate (C5-PP) molecules to produce the diphosphate form of the carrier lipid (C55-PP), which is then dephosphorylated to C55-P by membrane embedded phosphatases (UppP, or PAP2-type phosphatases) (Manat et al., 2014).

The final precursor for PG synthesis is lipid II, the GlcNAc-MurNAc(pentapepide) building block linked to C55-PP. Lipid II is synthesized in two steps at the inner leaflet of the cytoplasmic membrane from UDP-MurNAc-pentapeptide, UDP-GlcNAc and C55-P by the enzymes MraY and MurG (Bouhss et al., 2008). PG glycosyltransferases (GTases) polymerize lipid II at the outer leaflet of the membrane to glycan chains. This reaction releases C55-PP which is dephosphorylated for new rounds of precursor synthesis and transport.

Peptidoglycan synthesis is a prime target for antibacterial compounds and enzymes. Bacteria and higher organisms often produce antibacterial compounds to target competing bacteria and invading pathogens, respectively (Malanovic and Lohner, 2016). Bacterial competition is particularly fierce in dense populations such as biofilms and soil communities. Whilst the group of actinomycetes are known for their capability to secrete a repertoire of small metabolites that often show antibacterial activity, many Gram-negative bacteria utilize sophisticated type VI secretion systems to target adjacent bacterial cells by antimicrobial enzymes (Russell et al., 2011; 2012 and 2014). Another type of bacterial toxins are colicins, which are secreted by certain strains of Escherichia coli (Cascales et al., 2007). Colicins use energized nutrient uptake systems to enter the periplasm of susceptible strains of E. coli. Most colicins kill the target cell by inserting into the cytoplasmic membrane to form pores (Braun and Patzer, 2013). An exception is colicin M, which has a phosphatase activity against lipid II, cleaving the essential peptidoglycan precursor to disaccharide pyrophosphate and undecaprenol (El Ghachi et al., 2006).

More recently, it was shown that some Gram-positive species use a type VII secretion system to target other bacteria (Cao et al., 2016). So far the best example is Streptococcus intermedius, which uses a type VII secretion system to deliver an antibacterial toxin, TelC, to target bacteria (Whitney et al., 2017). TelC was shown to degrade lipid II and C55-PP to release disaccharide pentapeptide and pyrophosphate, respectively, and undecaprenol. S. intermedius also produces the immunity protein TipC, which inactivates TelC by direct interaction to prevent the lysis of the toxin-producing cell (Whitney et al., 2017). In this methods paper, we provide a detailed description of the TLC and HPLC methods that established the degradation of lipid II and C55-PP by TelC (Whitney et al., 2017). These methods can be generally used to assess the activity and specificity of phosphatases against membrane-bound bacterial cell wall precursors.

Materials and Reagents

  1. Pipette tips (STARLAB, catalog numbers: S1111-3700 , S1113-1700 , S1111-6701 )
  2. 1.5 ml micro-tubes (SARSTEDT, catalog number: 72.690.001 )
  3. Aluminium HPTLC silica gel 60 plates, 20 x 20 cm, without fluorescent indicator (Merck, catalog number: 1.05547.0001 )
  4. Glass vials (Soda glass, w/o rim, round bottom, 40 x 8 x 0.8-1.0 mm) (VWR, catalog number: 212-0011 )
  5. pH indicator strips (Merck, catalog number: 1.09531.0001 )
  6. HPLC vials (Agilent Technologies, catalog number: 5182-0553 )
  7. Vial inserts, 400 μl, glass, flat bottom (Agilent Technologies, catalog number: 5181-3377 )
  8. Hypodermic needles (FINE-JECT® for single use) (VWR, catalog number: 613-2022 )
  9. 2 ml micro-tubes (SARSTEDT, catalog number: 72.695.500 )
  10. MF-Millipore membrane filter 0.22 μm, mixed cellulose esters (Merck, catalog number: GSWP04700 )
  11. Enzyme of interest/putative phosphatase
  12. Potassium chloride (KCl) (Analytical Reagent Grade) (Fisher Scientific, CAS number: 7447-40-7)
  13. Triton X-100 (Roche Diagnostics, catalog number: 10789704001 )
  14. Lipid II (Lys version) (gift from Eefjan Breukink, University of Utrecht) (Egan et al., 2015)
    Note: Lipid II can be produced and purified by previously reported methods (Brötz et al., 1995; Qiao et al., 2017).
  15. Scintillation cocktail ProFlow G+ (Meridian Biotechnologies, catalog number: ProFlow G+ )
    Note: Used together with the radioactivity flow-through detector.
  16. Calcium chloride anhydrous (CaCl2) (Melford Laboratories, catalog number: C1103 )
  17. Undecaprenyl monophosphate diammonium salt (Larodan, catalog number: 62-1055 )
  18. Magnesium chloride hexahydrate (MgCl2·6H2O) (VWR, catalog number: 25108.260 )
  19. Farnesyl pyrophosphate ammonium salt (Sigma-Aldrich, catalog number: F6892 )
  20. Isopentenyl pyrophosphate triammonium salt solution (Sigma-Aldrich, catalog number: I0503 )
  21. Undecaprenyl pyrophosphate synthase (UppS) from E. coli, purified as described in Pan et al., 2000
  22. Undecaprenol (Larodan, catalog number: 60-1055 )
  23. n-Butanol (Honeywell International, catalog number: 537993 )
  24. Pyridine, anhydrous 99.8% (Sigma-Aldrich, catalog number: 270970 )
  25. Iodine (Sigma-Aldrich, catalog number: I3380 )
  26. [14C]GlcNAc-labeled lipid II (Lys version) (gift from Eefjan Breukink, University of Utrecht) (Egan et al., 2015)
  27. Chloroform (Sigma-Aldrich, catalog number: 32211-M )
  28. Methanol (Fisher Scientific, catalog number: 10284580 )
  29. Sodium chloride (NaCl) (VWR, catalog number: 27810.295 )
  30. Methanol (CHROMASOLVTM, gradient grade, for HPLC, ≥ 99.9%) (Honeywell International, Riedel-de HaënTM, catalog number: 34885 )
  31. Peptidoglycan synthase PBP1B and its cognate activator LpoB proteins from E. coli, purified as described in Bertsche et al. (2006) and Egan et al. (2014)
  32. Sodium borohydride (Merck, catalog number: 1.06371.0100 )
  33. Milli Q quality water (ddH2O)
  34. Ammonium hydroxide (Honeywell International, catalog number: 05003 )
  35. Acetic acid > 99.8% (Sigma-Aldrich, catalog number: 33209-M )
  36. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (VWR, catalog number: 441485H )
  37. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958-500G )
  38. Sodium hydroxide (NaOH) (VWR, catalog number: 28245.298 )
  39. Sodium hydroxide for HPLC (semiconductor grade, 99.99% trace metals basis) (Sigma-Aldrich, catalog number: 306576 )
  40. ortho-Phosphoric acid, 85-90% (HPLC) (Honeywell International, FlukaTM, catalog number: 79606 )
  41. Boric acid (99.97% trace metals basis) (Sigma-Aldrich, catalog number: 339067 )
  42. Muramidase cellosyl (provided by Höchst AG, Frankfurt, Germany)
    Note: Alternatively, the muramidase mutanolysin (Sigma-Aldrich, catalog number: M9901 ) can be used.
  43. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
  44. Mobile phase (see Recipes)
  45. Undecaprenol and undecaprenyl monophosphate diammonium salt (see Recipes)
  46. 6 M pyridinium acetate (see Recipes)
  47. n-Butanol/pyridinium acetate pH 4.2 (see Recipes)
  48. HEPES/KOH stock solution (1 M, pH 7.5) (see Recipes)
  49. HEPES/NaOH stock solution (1 M, pH 7.5) (see Recipes)
  50. Sodium phosphate buffer (80 mM, pH 4.8) (see Recipes)
  51. Muramidase cellosyl (0.5 μg μl-1) (see Recipes)
  52. Sodium borate (0.5 M, pH 9.0) (see Recipes)
  53. HPLC buffer A (see Recipes)
  54. HPLC buffer B (see Recipes)

Equipment

  1. Pipettes (Gilson, catalog numbers: F167300 and F167500 )
  2. AccuTherm Microtube Shaking Incubator (Labnet International, model: AccuThermTM, catalog number: I-4002-HCS )
  3. Vacuum concentration system (Labogene, model: MaxiVac Alpha )
  4. Bench top centrifuge, for example Accuspin Micro 17 microcentrifuge (Fisher Scientific, model: accuSpinTM Micro 17 , catalog number: 75002460)
  5. Chemical fume hood
  6. TLC developing chamber (VWR, catalog number: 552-0363 )
  7. Proheat® heat gun (Sigma-Aldrich, catalog number: Z673722-1EA )
  8. Small beaker (Petri dish, Steriplan®) (VWR, catalog number: 391-2840 )
  9. Dry bath (Digital Dry Bath, Labnet International, catalog number: D1100-230V )
  10. HPLC apparatus (Agilent Technologies, model: 1200 Series ) with flow detector for radioactivity (LabLogic Systems, model: Beta-RAM 5 )
  11. ProntoSIL 120-3-C18AQ3um 250 x 4.6 mm HPLC column (Bischoff Chromatography, catalog number: 2546F184PS030 )
  12. IKA RH basic 2 magnetic stirrer (IKA, catalog number: 0003339002 )
  13. Vortex (IKA, model: Minishaker MS2 )
  14. pH meter (Cole-Parmer, Jenway, model: 3510 , catalog number: 351001)
  15. Incubator (Genlab, catalog number: INC/100/DIG )
  16. Brown glass vials (Fisher Scientific, catalog number: 11531474 )

Part I: Thin-layer chromatography assay

Procedure

  1. Enzymatic digestion of lipid II or undecaprenyl pyrophosphate
    Note: All reactions are carried out in 1.5 ml microtubes and incubated using a microtube shaking incubator at 800 rpm.
    1. Reactions are carried out in a final volume of 50 μl and set up as described below for each substrate. All enzyme substrates are dried under vacuum and subsequently solubilized in the reaction mixture.
      Note: Take into account the constituents present in the storage buffer of the assayed proteins to calculate the buffer mixture.
      1. Lys-type lipid II
        Prepare enzyme reactions with final concentrations of 30 mM HEPES/KOH pH 7.5, 150 mM KCl, 0.1% (w/v) Triton X-100, 40 µM lipid II (L-Lys). Add 2 µM phosphatase (e.g., TelCt), phosphatase-inhibitor complex (e.g., TelCt-TipC complex) or no enzyme (control) and incubate for 90 min at 37 °C.
      2. Undecaprenyl monophosphate
        Prepare enzyme reactions with final concentrations of 20 mM HEPES/KOH pH 7.5, 150 mM KCl, 1 mM CaCl2, 0.1% (w/v) Triton X-100, 100 µM undecaprenyl monophosphate. Add 2 µM phosphatase (e.g., TelCt) or no enzyme (control) and incubate for 90 min at 37 °C.
      3. Undecaprenyl pyrophosphate synthesis coupled to the degradation by TelCt
        Prepare enzyme reactions with final concentrations of 20 mM HEPES/KOH pH 7.5, 50 mM KCl, 0.5 mM MgCl2, 1 mM CaCl2, 0.1% (w/v) Triton X-100, 40 µM farnesyl pyrophosphate, 400 µM isopentenyl pyrophosphate, 10 µM UppS. Add 2 µM phosphatase (e.g., TelCt), phosphatase-inhibitor complex (e.g., TelCt-TipC complex) or no enzyme (control) and incubate for 5 h at 25 °C, followed by an additional incubation for 90 min at 37 °C.
      4. Undecaprenol
        Prepare enzyme reactions with final concentrations of 30 mM HEPES/KOH pH 7.5, 150 mM KCl, 0.1% (w/v) Triton X-100 and 100 µM undecaprenol. Incubate for 90 min at 37 °C.
    2. Terminate the reactions by adding 50 µl of n-butanol/pyridine acetate (2:1) pH 4.2.
    3. Vortex for 1 min and centrifuge for 3 min at 17,000 x g using a bench-top centrifuge to separate the organic phase (n-butanol) from the aqueous phase (pyridine-acetate, water).
      Note: This step is essential to extract hydrophobic (lipid II, C55-P, C55-OH) and amphiphilic (C55-PP and Triton X-100) substances from the mixture. These substances will be found in the upper, organic phase which will contribute to 1/3 of the total volume.

  2. Thin layer chromatography
    Note: All steps are carried out in a chemical fume hood at room temperature if not indicated otherwise. The basic procedure for thin layer chromatography is shown in the published movie (Cockburn and Koropatkin, 2015).
    1. Pour the mobile phase into the developing chamber and adjust the solvent level to 1 cm.
    2. Close the chamber with the lid and allow for saturation of the gaseous phase with solvent (60 min).
    3. Incubate the TLC plate for at least 1 h at 60 °C to remove any humidity left from storage.
    4. Use a pencil to draw a line 1.5 cm from the bottom of the plate and mark sample spots. Sample spots are separated by 2 cm, and the distance from the outer spots to the edge of the plate should be at least 4 cm.
    5. Load the complete organic phase (upper phase, see Step A3) in 10 µl aliquots on the sample spots. After the addition of each aliquot, the spot is dried with a heat gun. Alternatively, the plate is left under a fume hood for each drying step.
      Note: It is important that the lower (aqueous) phase is not transferred on the plate, as this will result in smearing of the spots. When using a heat gun, it is important to not overheat the spots, as this can lead to degradation of compounds and additional bands.
    6. Place the TLC carefully in the developing chamber such that the solvent does not reach the spots. Optimally, there should be a distance of 0.5 cm between the solvent level and the pencil line.
    7. The TLC plate is incubated in the chamber with the lid on until the solvent front reaches 4/5 of the plate length, which takes 1.5-2 h.

  3. Staining
    1. Remove the plate from the chamber and dry it with a heat gun. The plate should be completely dried to avoid the appearance of solvent bands during staining.
    2. Place a small beaker with iodine in the chamber and put the lid on. Saturation will take 20-30 min at room temperature.
    3. Place the plate in the development chamber saturated with iodine vapor and incubate it until the spots are clearly visible (20 min-1 h).
    4. Representative examples of each reaction are shown in Figure 1.


      Figure 1. Lipid II and C55-PP, but not C55-P, are substrates of TelCt. Thin-layer chromatography analysis of the products obtained in reactions of TelCt (toxin domain of TelC) or TelCt-TipC with (A) C55-P, (B) C55-PP or (C) lipid II. D. C55-OH migrates at the solvent front. Control samples (-) contained no protein. TelCt was active against lipid II and C55-PP and was inhibited by its immunity protein TipC. The figure was adopted from Whitney et al. (2017).
      Note: Only lipid II and undecaprenyl monophosphate will appear as sharp bands. Due to its amphiphilic nature undecaprenyl pyrophosphate will appear as a crescent-shaped band between lipid II and undecaprenyl phosphate. The hydrophobic undecaprenol will always migrate at the solvent front, but will be visible after iodine staining.

Data analysis

Take a high-resolution picture and determine the retention factor (Rf) using commercially available programs (e.g., ImageJ). Bands present in control reactions serve as a standard.

Note: The distance is measured from the application line to the middle of the substance spots. For asymmetric spots (here: undecaprenyl pyrophosphate) measure the distance between the application line and lowest point of the spot. Spots in reaction mixtures should have similar Rf values, shape and color as spots derived from the standard compounds.

Part II: High performance liquid chromatography assay

Procedure

  1. Lipid II reactions for HPLC assay
    Note: A control reaction, containing the peptidoglycan synthase PBP1B and its cognate activator LpoB, is assayed to polymerize lipid II into short glycan chains with C55-PP at the terminal MurNAc residue.
    1. Dry 10,000 dpm (~1 nmol) of [14C]GlcNAc-labeled lipid II-Lys, stored in chloroform/methanol (1:1), in a glass vial using a vacuum.
    2. Resuspend the lipid II in 5 μl of 0.2% (w/v) Triton X-100 and vortex for 10 sec at 1,800 rpm.
    3. Prepare in a 1.5 ml microtube a reaction buffer mixture with final concentrations of 15 mM HEPES/NaOH, pH 7.5, 10 mM MgCl2, 150 mM NaCl, 0.023% (w/v) Triton X-100 and 0.4 mM CaCl2 (the PBP1B-LpoB control reaction did not contain CaCl2) in a total reaction volume of 100 μl.
      Note: Take into account the constituents present in the storage buffer of the assayed proteins to calculate the buffer mixture.
    4. Add 2 μM phosphatase (e.g., TelCt or Colicin M) or phosphatase-inhibitor complex (TelCt-TipC) to the reaction buffer. For a control sample add 0.75 μM PBP1B and 1.5 μM LpoB to the reaction buffer.
    5. Add the reaction mixture to the resuspended lipid II and incubate it for 60 min in a microtube shaking incubator at 37 °C with shaking (800 rpm).
    6. Spin down the condensation using a microcentrifuge.

    Reactions with phosphatases (TelCt, TelCt-TipC or Colicin M) are processed as follows:
    1. Adjust the pH of the sample to 3.5-4.0 using 20% phosphoric acid and pH indicator stripes.
      Note: Measure the pH by putting 0.3 μl sample onto the pH indicator stripe.
    2. Centrifuge the sample in a microcentrifuge for 15 min at maximum speed and room temperature. Transfer the supernatant into an HPLC vial containing a 400 µl vial insert.

    The control reaction with PBP1B-LpoB requires additional steps to digest the peptidoglycan with a muramidase and reduce the resulting unphosphorylated muropeptides. After Step A6 the reaction must be processed as follows:
    1. Incubate samples for 5 min at 100 °C using a dry bath, then spin down the condensation using a microcentrifuge.
    2. Let the samples cool down at room temperature for 2 min.
    3. Add 30 μl of cellosyl buffer (80 mM sodium phosphate, pH 4.8) and 10 µl of 0.5 µg µl-1 cellosyl (or mutanolysin) to the sample.
    4. Incubate the samples for 70 min in a microtube shaking incubator at 37 °C with shaking (800 rpm).
    5. Spin down the condensation using a microcentrifuge.
    6. Boil the reaction for 10 min at 100 °C on a dry bath and centrifuge the sample using a microcentrifuge for 15 min at maximum speed and room temperature.
    7. Punch a hole in the lid of a new 2 ml microcentrifuge tube using a needle.
      Note: The hole will allow releasing the H2 gas produced during the reduction step.
    8. Transfer the supernatant to the 2 ml microcentrifuge tube.
    9. Reduce the muropeptides by adding 100 μl of 0.5 M sodium borate, pH 9.0 and a tip of a spatula of solid sodium borohydride (ca. 1 mg).
    10. Incubate the sample for 30 min at room temperature in a microcentrifuge at 4,700 x g to prevent spillage due to gas bubbles.
    11. Adjust the pH of the sample to 3.5-4.0 using 20% phosphoric acid and pH indicator stripes.
      Note: Measure the pH by putting 0.3 μl of sample onto the pH indicator stripe.
    12. Centrifuge the sample in a microcentrifuge for 15 min at maximum speed and room temperature. Transfer the supernatant into an HPLC vial containing a 400 µl vial insert.

  2. Detection of lipid II products by HPLC
    System and set up conditions:
    HPLC connected to a radioactivity flow-through detector
    C18 reversed-phase column
    Flow rate: 0.5 ml min-1
    Column temperature: 55 °C
    1. Wash with 100% methanol for 20 min at room temperature.
    2. Increase column temperature to 55 °C.
    3. Start a linear gradient for 30 min from 100% methanol to 100% Milli Q water, holding 100% Milli Q water for further 20 min.
    4. Wash with HPLC buffer B for 20 min and equilibrate the column with HPLC buffer A for 40 min.
    5. Do a buffer run following the same method used for the samples of interest (Steps B6-B9) but without injecting any sample.
    6. Inject the sample (leave 20 µl of the total reaction volume in the vial insert) and flush the injection loop with HPLC buffer A for 2 min.
    7. Start a linear elution gradient for 60 min from 100% HPLC buffer A to 50% HPLC buffer B, holding 50% HPLC buffer B for further 10 min.
    8. Re-equilibrate the column with 100% HPLC buffer A for 30 min.
    9. Inject the next sample.
    Representative HPLC chromatograms of each sample are shown in Figure 2.


    Figure 2. TelCt cleaves lipid II between undecaprenyl and pentapeptide-pyrophosphate. A. HPLC chromatograms of the radiolabeled products resultant from reactions containing Lys-Lipid II and the indicated proteins. PBP1B + LpoB reaction was further digested with cellosyl and reduced with sodium borohydride. B. Proposed structures of the main products (peaks 1-3 in panel A) of each reaction. GlcNAc, N-acetylglucosamine; MurNAc-PP, N-acetylmuramic acid pyrophosphate; MurNAc-P, N-acetylmuramic acid phosphate; MurNAc(r), N-acetylmuramitol; L-Ala, L-alanine; L-Lys, L-lysine; D-iGlu, D-isoglutamic acid; D-Ala, D-alanine. The figure was adopted from Whitney et al. (2017).

Data analysis

  1. Each reaction should be assayed in triplicate.
  2. The produced muropeptides are identified based on their retention time, using the software provided with the HPLC system (e.g., LauraTM, LabLogic Systems Ltd).
  3. If needed, the phosphatase product can be verified by mass spectrometry. For this, use 16 nmol of non-radioactive lipid II (Lys version) as substrate to perform the reaction as described above, using a UV detector set at 205 nm, and collect the product fraction. Dry the collected fraction in a SpeedVac and store it at -20 °C until mass spectrometry analysis as reported (Bui et al., 2009).
    Note: The products can be collected either manually or using the HPLC collector module.

Recipes

Note: Unless otherwise indicated, all stock solutions are prepared using Milli Q water.

  1. Mobile phase (according to Rick et al., 1998)
    Mix components in the following order under gentle stirring:
    10 ml water
    1 ml 36% ammonium hydroxide
    48 ml methanol
    88 ml chloroform
    Store in a brown glass bottle
    Note: Chloroform should be added step-wise and slowly to prevent phase separation.
  2. Undecaprenol and undecaprenyl monophosphate diammonium salt
    Prepare 1 mM solutions in chloroform-methanol (2:1, v:v)
    Store in brown glass vials at -20 °C
  3. 6 M pyridinium acetate
    Mix 51.5 ml glacial acetic acid with 48.5 ml of pyridine
  4. n-Butanol/pyridinium acetate pH 4.2 (according to van Heijenoort et al., 1992)
    Mix 50 ml n-butanol with 25 ml 6 M pyridinium acetate
  5. HEPES/KOH stock solution (1 M, pH 7.5)
    Dissolve 23.83 g N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) in 90 ml of Milli Q water
    Adjust pH to 7.5 with potassium hydroxide (1 M)
    Adjust to a final volume of 100 ml
  6. HEPES/NaOH stock solution (1 M, pH 7.5)
    Dissolve 23.83 g N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) in 90 ml of Milli Q water
    Adjust pH to 7.5 with sodium hydroxide (1 M)
    Adjust to a final volume of 100 ml
  7. Sodium phosphate buffer (80 mM, pH 4.8)
    Dissolve 0.64 g sodium hydroxide (for HPLC) in 150 ml Milli Q water
    Adjust pH to 4.8 with phosphoric acid (85% and 20%)
    Adjust to a final volume of 200 ml
  8. Muramidase cellosyl (0.5 μg μl-1)
    Dissolve 5 mg of freeze-dried muramidase cellosyl stock in 10 ml of 20 mM sodium phosphate, pH 4.8
    Aliquot in microtubes and store at -20 °C
  9. Sodium borate (0.5 M, pH 9.0)
    Dissolve 3.09 g boric acid in 75 ml Milli Q water
    Adjust pH to 9.0 with sodium hydroxide (10 M)
    Adjust to a final volume of 100 ml
  10. HPLC buffer A (50 mM sodium phosphate, pH 4.31 with 10 μl of 10% sodium azide per liter of buffer)
    Dissolve 4 g sodium hydroxide (for HPLC) in 1,900 ml Milli Q water
    Adjust pH to pH 4.31 with phosphoric acid (85% and 20%)
    Adjust to a final volume of 2 L
    Filter the buffer using a 0.22 μm filter
    Add 20 μl of 10% sodium azide
  11. HPLC buffer B (75 mM sodium phosphate, pH 4.95, 15% v/v methanol)
    Dissolve 6 g sodium hydroxide (for HPLC) in 1,500 ml Milli Q water
    Adjust pH to pH 4.95 with phosphoric acid (85% and 20%)
    Adjust to a final volume of 1.7 L
    Filter the buffer through a 0.22 μm filter
    Add 300 ml of methanol for HPLC

Acknowledgments

This work reports in detail the methods previously used to demonstrate the cleavage site of TelC in peptidoglycan precursors (Whitney et al., 2017). This work was funded by the UK Medical Research Council (MRC) within the Joint Programming Initiative on Antimicrobial Resistance ANR-14-JAMR-0003 (NAPCLI) and the AMR Cross-council initiative Collaborative Grant MR/N002679/1. The authors declare that they have no conflict of interest or competing interest.

References

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  7. Cao, Z., Casabona, M. G., Kneuper, H., Chalmers, J. D. and Palmer, T. (2016). The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria. Nat Microbiol 2: 16183.
  8. Cascales, E., Buchanan, S. K., Duche, D., Kleanthous, C., Lloubes, R., Postle, K., Riley, M., Slatin, S. and Cavard, D. (2007). Colicin biology. Microbiol Mol Biol Rev 71(1): 158-229.
  9. Cockburn, D. and Koropatkin, N. (2015). Product analysis of starch active enzymes by TLC. Bio-protocol 5(20): e1621.
  10. Egan, A. J. F., Biboy, J., van’t Veer, I., Breukink, E. and Vollmer, W. (2015). Activities and regulation of peptidoglycan synthases. Phil Trans R Soc B 370: 20150031.
  11. Egan, A. J., Jean, N. L., Koumoutsi, A., Bougault, C. M., Biboy, J., Sassine, J., Solovyova, A. S., Breukink, E., Typas, A., Vollmer, W. and Simorre, J. P. (2014). Outer-membrane lipoprotein LpoB spans the periplasm to stimulate the peptidoglycan synthase PBP1B. PNAS 111(22): 8197-8202.
  12. El Ghachi, M., Bouhss, A., Barreteau, H., Touze, T., Auger, G., Blanot, D. and Mengin-Lecreulx, D. (2006). Colicin M exerts its bacteriolytic effect via enzymatic degradation of undecaprenyl phosphate-linked peptidoglycan precursors. J Biol Chem 281(32): 22761-22772.
  13. Malanovic, N. and Lohner, K. (2016). Antimicrobial peptides targeting gram-positive bacteria. Pharmaceuticals (Basel) 9(3).
  14. Manat, G., Roure, S., Auger, R., Bouhss, A., Barreteau, H., Mengin-Lecreulx, D. and Touze, T. (2014). Deciphering the metabolism of undecaprenyl-phosphate: the bacterial cell-wall unit carrier at the membrane frontier. Microb Drug Resist 20(3): 199-214.
  15. Pan, J. J., Chiou, S. T. and Liang, P. H. (2000). Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction. Biochemistry 39(35): 10936-10942.
  16. Qiao, Y., Srisuknimit, V., Rubino, F., Schaefer, K., Ruiz, N., Walker, S. and Kahne, D. (2017). Lipid II overproduction allows direct assay of transpeptidase inhibition by β-lactams. Nat Chem Biol 13(7): 793-798.
  17. Rick, P. D., Hubbard, G. L., Kitaoka, M., Nagaki, H., Kinoshita, T., Dowd, S., Simplaceanu, V. and Ho, C. (1998). Characterization of the lipid-carrier involved in the synthesis of enterobacterial common antigen (ECA) and identification of a novel phosphoglyceride in a mutant of Salmonella typhimurium defective in ECA synthesis. Glycobiology 8(6): 557-567.
  18. Russell, A. B., Hood, R. D., Bui, N. K., LeRoux, M., Vollmer, W. and Mougous, J. D. (2011). Type VI secretion delivers bacteriolytic effectors to target cells. Nature 475(7356): 343-347.
  19. Russell, A. B., Peterson, S. B. and Mougous, J. D. (2014). Type VI secretion system effectors: poisons with a purpose. Nat Rev Microbiol 12(2): 137-148.
  20. Russell, A. B., Singh, P., Brittnacher, M., Bui, N. K., Hood, R. D., Carl, M. A., Agnello, D. M., Schwarz, S., Goodlett, D. R., Vollmer, W. and Mougous, J. D. (2012). A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11(5): 538-549.
  21. Typas, A., Banzhaf, M., Gross, C. A. and Vollmer, W. (2012). From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat Rev Microbiol 10(2): 123-136.
  22. van Heijenoort, Y., Gomez, M., Derrien, M., Ayala, J. and van Heijenoort, J. (1992). Membrane intermediates in the peptidoglycan metabolism of Escherichia coli: possible roles of PBP 1b and PBP 3. J Bacteriol 174(11): 3549-3557.
  23. Vollmer, W. and Bertsche, U. (2008). Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim Biophys Acta 1778(9): 1714-1734.
  24. Vollmer, W., Blanot, D. and de Pedro, M. A. (2008). Peptidoglycan structure and architecture. FEMS Microbiol Rev 32(2): 149-167.
  25. Whitney, J. C., Peterson, S. B., Kim, J., Pazos, M., Verster, A. J., Radey, M. C., Kulasekara, H. D., Ching, M. Q., Bullen, N. P., Bryant, D., Goo, Y. A., Surette, M. G., Borenstein, E., Vollmer, W. and Mougous, J. D. (2017). A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria. Elife 6: e26938.

简介

肽聚糖包裹细菌细胞质膜以保护细胞免于因膨胀而导致的溶解。 肽聚糖合成的最后步骤需要称为脂质II的膜锚定底物,其中肽聚糖亚基通过焦磷酸部分连接至载体脂质十一碳烯醇。 脂质II是糖肽抗生素和几种抗微生物肽的靶标,并且通过参与细菌竞争的“攻击”酶来降解以诱导裂解。 在这里,我们分别描述了两种使用薄层色谱法(TLC)和高压液相色谱法(HPLC)的方案来测定磷脂酶如Colicin M或来自中间链球菌的LXG毒素蛋白TelC对脂质II的消化,的。 TLC方法也可以监测十一异戊二烯基(pyro)磷酸盐的消化,而HPLC方法允许分离脂质II的二 - ,单 - 或非磷酸化二糖五肽产物。

【背景】肽聚糖(PG)球囊是一种必需的细菌大分子,它可以保护细胞免受由于其膨胀引起的破裂并保持细胞的形状(Vollmer和Bertsche,2008; Typas等人,2012)。 PG由通过短肽连接的聚糖链组成。来自不同物种的PG在肽的结构和二级修饰的存在方面有所不同(Vollmer等人,2008)。 PG前体在细胞内合成,并装备有载体脂质,以在膜在细胞质膜的外部小叶处聚合之前穿过膜转运(Barreteau等人,2008)。通用的细菌载体脂质是十一烯丙基磷酸酯(C155-p-em),其分两步合成。首先,UppS使用焦磷酸法尼酯(C15:PP)和八种异戊烯基焦磷酸(C15:PP)分子产生载体脂质的二磷酸酯形式(C55-PPM),然后将其去磷酸化为C55S-P (UppP或PAP2型磷酸酶)(Manat等人,2014)。

PG合成的最终前体是脂质II,连接到C55 - / - PP的GlcNAc-MurNAc(五pepepide)结构单元。通过酶MraY和MurG,在来自UDP-MurNAc-五肽,UDP-GlcNAc和C- 55 -p的细胞质膜的内部小叶上分两步合成脂质II (Bouhss et al。,2008)。 PG糖基转移酶(GTases)使膜外部小叶中的脂质II聚合成聚糖链。该反应释放出去磷酸化的C55新鲜前体合成和运输的新一轮的PP 。

肽聚糖合成是抗菌化合物和酶的主要目标。细菌和高等生物通常分别产生抗菌化合物以针对竞争细菌和侵入病原体(Malanovic and Lohner,2016)。生物膜和土壤群落等密集种群的细菌竞争尤其激烈。虽然放线菌群以其能够分泌通常显示抗菌活性的小代谢物的能力而闻名,但许多革兰氏阴性菌利用复杂的VI型分泌系统通过抗微生物酶靶向相邻的细菌细胞(Russell等人, ,2011; 2012和2014)。另一种类型的细菌毒素是大肠杆菌素,其由大肠杆菌的某些菌株分泌(Cascales等人,2007)。大肠杆菌素使用活化的营养吸收系统进入E易感菌株的周质。大肠杆菌。大多数大肠杆菌素通过插入细胞质膜形成孔隙来杀死靶细胞(Braun and Patzer,2013)。一个例外是大肠菌素M,其具有针对脂质II的磷酸酶活性,将基本肽聚糖前体切割成焦磷酸二糖和十一碳烯醇(El Ghachi et al。,2006)。

最近,研究表明,一些革兰氏阳性菌种使用VII型分泌系统来靶向其他细菌(Cao等人,2016年)。到目前为止,最好的例子是中间链球菌(Streptococcus intermedius),它使用VII型分泌系统将抗菌毒素TelC递送到靶细菌(Whitney等人,2017)。已显示TelC分别降解脂质II和C 55以分别释放二糖五肽和焦磷酸以及十一碳烯醇。 S上。 intermedius 也产生免疫蛋白TipC,它通过直接相互作用灭活TelC以防止产毒细胞的裂解(Whitney等人,2017)。在这篇方法论文中,我们提供了TLC和HPLC方法的详细描述,这些方法建立了TelC(Whitney等人)的降解脂质II和C55 - / - ,2017)。这些方法通常可用于评估磷酸酶对膜结合细菌细胞壁前体的活性和特异性。

关键字:脂质II, 十一异戊烯焦磷酸盐, 磷酸酶活性, 肽聚糖, HPLC, TLC

材料和试剂

  1. 移液器吸头(STARLAB,产品目录号:S1111-3700,S1113-1700,S1111-6701)
  2. 1.5毫升微型管(SARSTEDT,目录号:72.690.001)
  3. 铝HPTLC硅胶60板,20×20厘米,无荧光指示剂(Merck,产品目录号:1.05547.0001)
  4. 玻璃瓶(苏打玻璃,无边缘,圆底,40×8×0.8-1.0毫米)(VWR,目录号:212-0011)
  5. pH指示条(Merck,目录号:1.09531.0001)
  6. HPLC小瓶(Agilent Technologies,目录号:5182-0553)
  7. 小瓶插入物,400微升,平底玻璃(安捷伦科技,产品目录号:5181-3377)
  8. 皮下注射针(单次使用FINE-JECT )(VWR,目录号:613-2022)
  9. 2毫升微型管(SARSTEDT,目录号:72.695.500)
  10. MF-Millipore膜过滤器0.22μm,混合纤维素酯(Merck,目录号:GSWP04700)
  11. 感兴趣的酶/推定的磷酸酶
  12. 氯化钾(KCl)(分析试剂级)(Fisher Scientific,CAS号:7447-40-7)
  13. Triton X-100(Roche Diagnostics,目录号:10789704001)
  14. Lipid II(Lys版本)(Utrecht大学Eefjan Breukink赠送)(Egan et al。,2015)
    注:脂质II可以通过先前报道的方法生产和纯化(Brötz等,1995; Qiao等,2017)。
  15. 闪烁鸡尾酒ProFlow G +(Meridian Biotechnologies,目录号:ProFlow G +)
    注:与放射性流通检测器一起使用。
  16. 无水氯化钙(CaCl 2)(Melford Laboratories,目录号:C1103)

  17. 十一烯丙基单磷酸二铵盐(Larodan,目录号:62-1055)
  18. 氯化镁六水合物(MgCl 2·6H 2 O)(VWR,目录号:25108.260)
  19. 法尼基焦磷酸铵盐(Sigma-Aldrich,目录号:F6892)
  20. 异戊烯基焦磷酸三铵盐溶液(Sigma-Aldrich,目录号:I0503)
  21. 来自大肠杆菌的脱癸烯基焦磷酸合酶(UppS),按照Pan等人2000年所述进行纯化
  22. Undecaprenol(Larodan,目录号:60-1055)
  23. 正丁醇(Honeywell International,目录号:537993)
  24. 吡啶,无水99.8%(Sigma-Aldrich,目录号:270970)
  25. 碘(Sigma-Aldrich,目录号:I3380)
  26. (Egan< et al。,2015)
    [14C] GlcNAc-标记的脂质II(Lys版本)
  27. 氯仿(Sigma-Aldrich,目录号:32211-M)
  28. 甲醇(Fisher Scientific,目录号:10284580)
  29. 氯化钠(NaCl)(VWR,目录号:27810.295)
  30. 甲醇(CHROMASOL TM TM,梯度级,用于HPLC,≥99.9%)(Honeywell International,Riedel-deHaënTM,目录号:34885)
  31. 来自E的肽聚糖合酶PBP1B及其同源激活蛋白LpoB蛋白。如Bertsche等人(2006)和Egan等人所述进行纯化。 (2014)
  32. 硼氢化钠(Merck,目录号:1.06371.0100)
  33. Milli Q优质水(ddH 2 O)
  34. 氢氧化铵(Honeywell International,目录号:05003)
  35. 乙酸> 99.8%(Sigma-Aldrich,目录号:33209-M)
  36. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(VWR,目录号:441485H)
  37. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958-500G)
  38. 氢氧化钠(NaOH)(VWR,目录号:28245.298)
  39. 用于HPLC的氢氧化钠(半导体级,基于99.99%痕量金属)(Sigma-Aldrich,目录号:306576)
  40. 正磷酸,85-90%(HPLC)(Honeywell International,Fluka,目录号:79606)
  41. 硼酸(基于99.97%痕量金属)(Sigma-Aldrich,目录号:339067)
  42. Muramidase cellosyl(由德国法兰克福HöchstAG提供)
    注意:或者,可以使用溶菌酶变溶菌素(Sigma-Aldrich,目录号:M9901)。
  43. 叠氮化钠(NaN3)(Sigma-Aldrich,目录号:S2002)
  44. 流动阶段(见食谱)
  45. Undecaprenol和undecaprenyl monophosphate二铵盐(见食谱)
  46. 6 M乙酸吡啶(见食谱)
  47. 正丁醇/乙酸吡啶鎓pH值4.2(见食谱)
  48. HEPES / KOH原液(1 M,pH 7.5)(见食谱)
  49. HEPES / NaOH储备溶液(1M,pH7.5)(见食谱)
  50. 磷酸钠缓冲液(80 mM,pH 4.8)(见食谱)
  51. 溶菌酶(0.5微克/μl)(见食谱)
  52. 硼酸钠(0.5 M,pH 9.0)(见食谱)
  53. HPLC缓冲液A(见食谱)
  54. HPLC缓冲液B(见食谱)

设备

  1. 移液器(Gilson,产品目录号:F167300和F167500)
  2. AccuTherm Microtube摇床培养箱(Labnet International,型号:AccuTherm TM,产品目录号:I-4002-HCS)
  3. 真空浓缩系统(Labogene,型号:MaxiVac Alpha)
  4. 台式离心机,例如Accuspin Micro 17微量离心机(Fisher Scientific,型号:accuSpin TM Micro 17,目录号:75002460)
  5. 化学通风橱
  6. TLC显影室(VWR,目录号:552-0363)
  7. Proheat®热风枪(Sigma-Aldrich,目录号:Z673722-1EA)
  8. 小烧杯(培养皿,Steriplan®)(VWR,目录号:391-2840)
  9. 干浴(Digital Dry Bath,Labnet International,目录号:D1100-230V)
  10. 具有放射性流量检测器的HPLC装置(Agilent Technologies,型号:1200系列)(LabLogic Systems,型号:Beta-RAM 5)
  11. ProntoSIL 120-3-C18AQ3um 250×4.6mm HPLC柱(Bischoff Chromatography,目录号:2546F184PS030)
  12. IKA RH basic 2磁力搅拌器(IKA,目录号:0003339002)
  13. 漩涡(IKA,型号:Minishaker MS2)
  14. pH计(Cole-Parmer,Jenway,型号:3510,目录号:351001)
  15. 培养箱(Genlab,目录号:INC / 100 / DIG)
  16. 棕色玻璃瓶(Fisher Scientific,目录号:11531474)

第一部分:薄层色谱分析

程序

  1. 脂肪II或十一异戊烯基焦磷酸酶的酶消化
    注意:所有反应均在1.5ml微管中进行,并使用微管振荡培养箱以800rpm进行培养。
    1. 以50μl的终体积进行反应,并如下对每种底物所述进行设置。所有酶底物在真空下干燥,随后溶解在反应混合物中。
      注意:考虑存在于分析蛋白质储存缓冲液中的组分,以计算缓冲液混合物。
      1. Lys型脂质II
        用最终浓度为30mM HEPES / KOH pH 7.5,150mM KCl,0.1%(w / v)Triton X-100,40μM脂质II(L-Lys)制备酶反应。加入2μM磷酸酶(例如TelCt),磷酸酶 - 抑制剂复合物(例如,TelCt-TipC复合物)或不加酶(对照)并在37℃孵育90分钟C.
      2. Undecaprenyl monophosphate
        用最终浓度为20mM HEPES / KOH pH 7.5,150mM KCl,1mM CaCl 2,0.1%(w / v)Triton X-100,100μM十一异戊二烯基单磷酸酯制备酶反应。加入2μM磷酸酶(,例如,TelCt)或不加酶(对照),并在37℃孵育90分钟。
      3. 伴随着TelCt降解的Undecaprenyl焦磷酸盐合成
        制备具有终浓度为20mM HEPES / KOH pH 7.5,50mM KCl,0.5mM MgCl 2,1mM CaCl 2,0.1%(w / v) Triton X-100,40μM法呢基焦磷酸酯,400μM异戊烯基焦磷酸盐,10μMUppS。加入2μM磷酸酶(例如TelCt),磷酸酶 - 抑制剂复合物(例如,TelCt-TipC复合物)或不加酶(对照)并在25°孵育5小时C,然后在37°C孵育90分钟。
      4. Undecaprenol
        用最终浓度为30mM HEPES / KOH pH 7.5,150mM KCl,0.1%(w / v)Triton X-100和100μM非肽素醇制备酶反应。
        在37°C孵育90分钟

    2. 加入50μl正丁醇/吡啶乙酸盐(2:1)pH 4.2终止反应。
    3. 涡旋1分钟并使用台式离心机以17,000×gg离心3分钟以将有机相(正丁醇)从水相(吡啶 - 乙酸盐,水)中分离出来。 > 注意:此步骤对于提取疏水性(脂质II,C -P,C 55)-OH)和两亲(C-55)-PP和Triton X- 100)物质。这些物质会在上层有机相中找到,有机相中的有机相占总体积的1/3。

  2. 薄层色谱
    注:如果没有其他说明,所有的步骤都是在室温下的化学通风橱中进行的。已发表的电影(Cockburn和Koropatkin,2015)显示了薄层色谱的基本步骤。
    1. 将流动相倒入显影室,调整溶剂水平至1厘米。
    2. 用盖子关闭室,并用溶剂(60分钟)使气相饱和。

    3. 在60°C孵育薄层板至少1小时,以去除储存中的湿气。
    4. 使用铅笔从平板底部划出1.5厘米的线并标记样品点。样点间隔2厘米,从外点到平板边缘的距离至少应为4厘米。
    5. 在样品点上以10μl等分试样加载完整的有机相(上层相,见步骤A3)。加入各等分试样后,用加热枪干燥。或者,每块干燥步骤都留在通风橱下。
      注意:重要的是下层(水相)不会转移到板上,因为这会导致斑点变得模糊。使用热风枪时,不要过热斑点很重要,因为这可能会导致化合物和附加条带的降解。
    6. 小心地将TLC放在显影室中,使溶剂不能到达斑点。

      最好在溶剂水平和铅笔线之间应有0.5厘米的距离
    7. TLC板在带盖的室中孵育直至溶剂前沿达到板长度的4/5,这需要1.5-2小时。

  3. 染色
    1. 从培养箱中取出培养皿,并用加热枪进行干燥。

      该板应完全干燥,以避免在染色过程中出现溶剂带
    2. 放入一个带有碘的小烧杯并放上盖子。
      在室温下,饱和度需要20-30分钟
    3. 将平板置于充满碘蒸气的显影室中,并孵育直至斑点清晰可见(20分钟至1小时)。

    4. 每种反应的代表性例子如图1所示。


      图1.脂质II和C55 - / - ,而不是C55 - / - ,是在TelCt(TelC的毒素结构域)或TelCt-TipC与(A)C55反应中获得的产物的薄层层析分析 (B)C55-PP或(C)脂质II。 D.C55 -OH在溶剂前沿迁移。对照样品( - )不含蛋白质。 TelCt对脂质II和C55 - / - 动物具有活性,并且受到其免疫蛋白TipC的抑制。该数据来自惠特尼等人(2017)。
      注:只有脂质II和十一烯丙基单磷酸酯会出现尖锐的条带。由于其两亲性,十一异戊烯基焦磷酸将在脂质II和十一异戊烯基磷酸酯之间呈月牙形带。疏水性十一碳烯醇总是在溶剂前沿迁移,但在碘染色后将可见。

数据分析

拍摄高分辨率图片,并使用市售程序(例如,ImageJ)确定保留因子(R )。控制反应中存在的条带用作标准。

注:距离是从应用线到物质点中间的距离。对于不对称斑点(此处:十一异戊烯基焦磷酸盐)测量施用线与斑点的最低点之间的距离。反应混合物中的斑点应该具有与从标准化合物衍生的斑点相似的R em / f em / em em值,形状和颜色。

第二部分:高效液相色谱分析

程序

  1. 用于HPLC分析的脂质II反应
    注意:测定含有肽聚糖合酶PBP1B及其同源激活剂LpoB的对照反应,以用C 55/55亚单位将脂质II聚合成短聚糖链> -PP在末端MurNAc残基
    1. 使用真空在玻璃小瓶中干燥10,000dpm(〜1nmol)贮存在氯仿/甲醇(1:1)中的[14 C] GlcNAc-标记的脂质II-Lys。 >
    2. 在5μl0.2%(w / v)Triton X-100中重悬脂质II,并以1,800rpm涡旋10秒。
    3. 在1.5ml微管中制备终浓度为15mM HEPES / NaOH,pH 7.5,10mM MgCl 2,150mM NaCl,0.023%(w / v)Triton X-100的反应缓冲液混合物和0.4mM CaCl 2(PBP1B-LpoB对照反应不含有CaCl 2),总反应体积为100μl。
      注意:考虑存在于检测蛋白质储存缓冲液中的成分,以计算缓冲液混合物。
    4. 向反应缓冲液中加入2μM磷酸酶(例如,,TelCt或Colicin M)或磷酸酶 - 抑制剂复合物(TelCt-TipC)。对于对照样品,向反应缓冲液中加入0.75μMPBP1B和1.5μMLpoB。
    5. 将反应混合物加入到重悬的脂质II中,并在37℃摇动(800rpm)的微管孵育器中孵育60分钟。

    6. 使用微型离心机旋转冷凝。

    与磷酸酶(TelCt,TelCt-TipC或Colicin M)的反应如下处理:
    1. 使用20%磷酸和pH指示条将样品的pH调节至3.5-4.0。
      注意:将0.3μl样品放入pH指示剂条中测量pH。
    2. 在最大速度和室温下将样品在微型离心机中离心15分钟。将上清液转移到含有400μl小瓶插入物的HPLC小瓶中。

    与PBP1B-LpoB的对照反应需要额外的步骤以用胞壁酰胺酶消化肽聚糖并减少所得的未磷酸化的鼠肽。在步骤A6之后,反应必须如下处理:
    1. 使用干浴在100°C孵育样品5分钟,然后使用微型离心机旋转浓缩。
    2. 让样品在室温下冷却2分钟。
    3. 向样品中加入30μl纤维素缓冲液(80 mM磷酸钠,pH 4.8)和10μl0.5μgμl-1纤维素(或变溶菌素)。

    4. 在37°C振荡(800 rpm)的微管振荡培养箱中孵育样品70分钟。

    5. 使用微型离心机旋转冷凝。
    6. 将反应在100℃的干燥浴中煮沸10分钟,并使用微型离心机在最高速度和室温下离心15分钟。

    7. 在一个新的2毫升微量离心管盖上打一个孔 注:孔将允许释放在还原步骤中产生的H 2气体。
    8. 将上清液转移到2 ml微量离心管中。
    9. 通过加入100μl的0.5M硼酸钠,pH9.0和尖端的固体硼氢化钠(1mg)来减少鼠肽。
    10. 在微量离心机中,以4,700×g的克数在室温下将样品孵育30分钟以防止由于气泡而溢出。

    11. 使用20%磷酸和pH指示条纹将样品的pH调节至3.5-4.0 注意:将0.3μl样品放入pH指示剂条中测量pH。
    12. 在最大速度和室温下将样品在微型离心机中离心15分钟。将上清液转移到含有400μl小瓶插入物的HPLC小瓶中。

  2. HPLC检测脂质II产品
    系统和设置条件:
    HPLC连接到放射性流通检测器
    C18反相柱
    流速:0.5ml min -1 -1>
    柱温:55℃

    1. 在室温下用100%甲醇清洗20分钟。
    2. 将色谱柱温度升至55°C。
    3. 从100%甲醇到100%Milli Q水开始线性梯度30分钟,保持100%Milli Q水20分钟。
    4. 用HPLC缓冲液B清洗20分钟,并用HPLC缓冲液A平衡柱子40分钟。
    5. 按照与目标样本相同的方法(步骤B6-B9)进行缓冲区运行,但不注入任何样本。
    6. 注入样品(将20μl总反应体积留在小瓶插入物中)并用HPLC缓冲液A冲洗注射环2分钟。
    7. 从100%HPLC缓冲液A至50%HPLC缓冲液B开始线性洗脱梯度60分钟,保持50%HPLC缓冲液B再保持10分钟。
    8. 用100%HPLC缓冲液A重新平衡柱子30分钟。
    9. 注入下一个样本。

    每个样品的代表性HPLC色谱图如图2所示。


    图2.TelCt切割十一烯丙基和五肽 - 焦磷酸之间的脂质II。 :一种。由含有Lys-脂质II和所示蛋白质的反应产生的放射性标记产物的HPLC色谱图。 PBP1B + LpoB反应用纤维素进一步消化并用硼氢化钠还原。 B.每种反应主要产物的建议结构(图A中的峰1-3)。 GlcNAc,N-乙酰葡糖胺; MurNAc- PP,N-乙酰胞壁酸焦磷酸盐; MurNAc-P em,N-乙酰胞壁酸磷酸盐; MurNAc(r),N-乙酰muramitol; L-Ala,L-丙氨酸; L-Lys,L-赖氨酸; D-iGlu,D-异谷氨酸; D-Ala,D-丙氨酸。这个数字被惠特尼等人采用。 (2017)。

数据分析


  1. 每个反应应重复测定三次
  2. 使用与HPLC系统一起提供的软件(例如,Laura TM,LabLogic Systems Ltd),基于它们的保留时间来鉴定产生的鼠肽。
  3. 如果需要,可以通过质谱法验证磷酸酶产物。为此,使用16nmol的非放射性脂质II(Lys型)作为底物以使用设定在205nm的UV检测器进行如上所述的反应,并收集产物级分。将所收集的级分在SpeedVac中干燥并将其储存在-20℃下直到报道的质谱分析(Bui等人,2009)。
    注意:可以手动或使用HPLC收集器模块收集产品。

食谱

注:除非另有说明,否则所有储备溶液均使用Milli Q水制备。

  1. 流动相(根据瑞克等人,1998年)

    在温和搅拌下按以下顺序混合组分:

    10毫升水 1毫升36%氢氧化铵
    48毫升甲醇
    88毫升氯仿
    存放在棕色玻璃瓶中
    注意:氯仿应逐步缓慢加入,以防止相分离。
  2. Undecaprenol和undecaprenyl monophosphate二铵盐
    在氯仿 - 甲醇(2:1,v:v)中制备1 mM溶液
    存放在-20°C的棕色玻璃瓶中
  3. 6 M乙酸吡啶

    将51.5 ml冰醋酸与48.5 ml吡啶混合
  4. 正丁醇/乙酸吡啶鎓pH 4.2(根据van Heijenoort等人,1992)
    将50ml正丁醇与25ml 6M乙酸吡啶Mix混合。
  5. HEPES / KOH储液(1M,pH 7.5)
    将23.83克N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)溶于90毫升Milli Q水中。
    用氢氧化钾(1M)将pH调节至7.5。
    调整到100毫升的最终体积
  6. HEPES / NaOH储备溶液(1 M,pH 7.5)
    将23.83克N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)溶于90毫升Milli Q水中。
    用氢氧化钠(1M)将pH调节至7.5。
    调整到100毫升的最终体积
  7. 磷酸钠缓冲液(80 mM,pH 4.8)

    溶解150毫升Milli Q水中的0.64克氢氧化钠(用于HPLC)
    用磷酸(85%和20%)调节pH值至4.8 调整到200毫升的最终体积
  8. 米力霉素纤维素(0.5μgμl-1)
    将5mg冻干的胞壁酰肌醇贮存液溶于10ml 20mM磷酸钠,pH4.8中 分装在微管中并储存在-20°C。
  9. 硼酸钠(0.5 M,pH 9.0)

    溶解3.09克硼酸在75毫升Milli Q水中 用氢氧化钠(10M)调节pH至9.0。
    调整到100毫升的最终体积
  10. HPLC缓冲液A(50mM磷酸钠,pH 4.31,每升缓冲液含10μl10%叠氮钠)

    4克氢氧化钠(用于HPLC)溶于1,900毫升Milli Q水中
    用磷酸(85%和20%)将pH调节至pH 4.31 调整到2 L的最终音量
    使用0.22μm过滤器过滤缓冲液。
    加20μl10%叠氮钠
  11. HPLC缓冲液B(75mM磷酸钠,pH 4.95,15%v / v甲醇)

    将6克氢氧化钠(用于HPLC)溶于1,500毫升Milli Q水中
    用磷酸(85%和20%)将pH调节至pH 4.95 调整到1.7 L的最终音量。

    通过0.22μm过滤器过滤缓冲液 加入300毫升甲醇用于HPLC

致谢

这项工作详细报道了以前用于证明肽聚糖前体中TelC切割位点的方法(Whitney等人,2017)。这项工作由英国医学研究委员会(MRC)在抗菌药物耐药性联合规划倡议ANR-14-JAMR-0003(NAPCLI)和AMR跨委员会倡议合作赠款MR / N002679 / 1中资助。作者声明他们没有利益冲突或利益冲突。

参考

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Pazos, M., Otten, C. and Vollmer, W. (2018). Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC). Bio-protocol 8(6): e2761. DOI: 10.21769/BioProtoc.2761.
  2. Whitney, J. C., Peterson, S. B., Kim, J., Pazos, M., Verster, A. J., Radey, M. C., Kulasekara, H. D., Ching, M. Q., Bullen, N. P., Bryant, D., Goo, Y. A., Surette, M. G., Borenstein, E., Vollmer, W. and Mougous, J. D. (2017). A broadly distributed toxin family mediates contact-dependent antagonism between Gram-positive bacteria. Elife 6: e26938.
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