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Digestion of Peptidoglycan and Analysis of Soluble Fragments
肽聚糖消化和可溶性片段分析   

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Molecular Microbiology
Dec 2016

Abstract

Peptidoglycan (murein) is a vital component of the cell wall of nearly all bacteria, composed of sugars linked by short peptides. This protocol describes the purification of macromolecular peptidoglycan from cultured bacteria and the analysis of enzyme-digested peptidoglycan fragments using high performance liquid chromatography (HPLC). Digested peptidoglycan fragments can be identified by mass spectrometry, or predicted by comparing retention times with other published chromatograms. The quantitative nature of this method allows for the measurement of changes to peptidoglycan composition between different species of bacteria, growth conditions, or mutations. This method can determine the overall architecture of peptidoglycan, such as peptide stem length, the extent of cross-linking, and modifications. Muropeptide analysis has been used to study the function of peptidoglycan-associated proteins and the mechanisms by which bacteria acquire antibiotic resistance.

Keywords: Muropeptides analysis (胞壁肽分析), Peptidoglycan (肽聚糖), PG (PG), HPLC (HPLC), Muropeptide (胞壁肽)

Background

Peptidoglycan is composed of a sugar backbone linked together by peptide stems that creates a mesh-like structure important for cell shape, and turgor pressure of bacterial cells. The macromolecular peptidoglycan is assembled from monomeric units that are synthesized in the cytoplasm and consist of an N-acetylglucosamine and N-acetylmuramic acid disaccharide with a five amino acid stem. When the monomer is flipped into the periplasm, it is added to the glycan chain by transglycosylation and a portion of the peptide stems are linked together by transpeptidation.

The amino acids comprising the peptide stem can vary by species but are generally attached to muramic acid in the order L-alanine, D-glutamic acid, meso-diaminopimelic acid, D-alanine, D-alanine, with L-lysine taking the place of diaminopimelic acid in some Gram-positives. Cross-linking occurs through the free amine of the third amino acid linking either the third or fourth amino acid directly or through linker amino acids (Schleifer and Kandler, 1972). Other common modifications include amidation of amino acids (Kato and Strominger, 1968) and O-acetylation (Clarke and Dupont, 1992) or N-deacetylation (Araki et al., 1971) of sugars.

A variety of enzymes act on peptidoglycan during growth and cell division. Classes of enzymes known as lytic transglycosylases cleave glycan chains between disaccharide units at the same position as lysozyme. The important difference is that lytic transglycosylases create a 1,6-anhydro bond, in contrast to a reducing end created by lysozyme and mutanolysin. Thus the relative abundance of 1,6-anhydro bonds can be used as an approximation of glycan chain length (Ward, 1973). Different classes of peptidases act at different bonds of the peptide stem and cross-links. For example, D,D-carboxypeptidases will cleave between the fourth and fifth amino acid, while, L,D-carboxypeptidases will cleave between the third and fourth amino acid (Holtje and Tuomanen, 1991).

Muropeptide analysis can resolve the different modifications and cross-linking to give a model of the overall structure of the macromolecular peptidoglycan. One of the first observations using HPLC-based peptidoglycan analysis was the discovery that Caulobacter crescentus lacks D,D-carboxypeptidase activity (Markiewicz et al., 1983). The first comprehensive peptidoglycan analysis was done on Escherichia coli with 80 different muropeptides species identified (Glauner et al., 1988). This method has also been used to show penicillin-resistance in Neisseria meningitidis is correlated with differences in peptidoglycan structure (Antignac et al., 2003a).

Interest in peptidoglycan has seen an increase in recent years. Continued bacterial resistance to peptidoglycan-targeting antibiotics created a need for a more complete understanding of peptidoglycan metabolism. The discovery of the human peptidoglycan-recognizing proteins, NOD1 and NOD2 have also led to increased investigations into how host cells recognize peptidoglycan and how they are able to differentiate between commensal and pathogenic bacteria (Clarke and Weiser, 2011).

The following method has a number of advantages over other types of peptidoglycan analysis, including ultra-performance liquid chromatography (UPLC)-based methods. The first advantage is that nearly all of the equipment and materials are a standard part of most laboratories, so a large investment is not needed. Second, the almost 30 year history of this protocol allows comparisons with similar chromatograms to be made, allowing for preliminary identification of peptidoglycan fragments to be made quickly, then using mass spectrometry to positively identify fragments that change or are of particular interest. The third advantage is the scale of this method, which yields enough separated material for additional analysis by mass spectrometry or enzymatic reactions. More information on the history and uses of HPLC-based peptidoglycan analysis can be found in this review (Desmarais et al., 2013)

Materials and Reagents

  1. Pipette tips
  2. Nalgene Oak Ridge high-speed PPCO centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0050 )
  3. 1.7 ml microcentrifuge tubes (MIDSCI, catalog number: AVSS1700 )
  4. Amicon Ultra-0.5 centrifugal filter unit with ultracel-10 membrane (EMD Millipore, catalog number: UFC501024 )
  5. Aluminum foil (Fisher Scientific, catalog number: 01-213-100 )
  6. Filter (0.22 µm)
  7. Bacterial growth medium (species specific)
  8. Micrococcus lysodeikticus ATCC No. 4698, lyophilized cells (Sigma-Aldrich, catalog number: M3770 )
  9. α-Amylase from porcine pancreas (Sigma-Aldrich, catalog number: A6255 )
  10. Pronase protease, Streptomyces griseus (EMD Millipore, catalog number: 53702 )
  11. 1 N HCl
  12. Sodium azide (NaN3) (Sigma-Aldrich, catalog number: S2002 )
  13. Mutanolysin from Streptomyces globisporus ATCC (Sigma-Aldrich, catalog number: M9901 )
  14. Sodium borohydride (Sigma-Aldrich, catalog number: 213462 )
  15. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) (Fisher Scientific, catalog number: S369 )
  16. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: S373 )
  17. Sodium dodecyl sulfate (SDS) (Fisher Scientific, catalog number: BP166-100 )
  18. Boric acid (Acros Organics, catalog number: 327132500 )
  19. Water (HPLC-grade) (Fisher Scientific, catalog number: W5SK-4 )
  20. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-1 )
  21. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358 )
  22. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366 )
  23. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P285 )
  24. o-Phosphoric acid, 85% (HPLC) (Fisher Scientific, catalog number: A260-500 )
  25. Methanol (HPLC-grade) (Fisher Scientific, catalog number: A452SK-4 )
  26. Trifluoroacetic acid (Sigma-Aldrich, catalog number: 302031 )
  27. Acetonitrile (HPLC-grade) (Fisher Scientific, catalog number: A998 )
  28. Phosphate buffer pH = 6 (PB) (see Recipes)
  29. PB with 8% (w/v) SDS (see Recipes)
  30. 0.5 M borate buffer pH = 8 (see Recipes)
  31. Phosphate buffered saline (PBS) (see Recipes)
  32. HPLC separation buffer A (see Recipes)
  33. HPLC separation buffer B (see Recipes)
  34. HPLC desalting buffer A (see Recipes)
  35. HPLC desalting buffer B (see Recipes)

Equipment

  1. Kimax baffled culture flasks (Fisher Scientific, catalog numbers: 10-140-6A and 10-140-6B)
    Manufacturer: DWK Life Sciences, Kimble, catalog numbers: 25630250 and 25630500 .
  2. Pipettes (Gilson, catalog number: F167700 )
  3. Nalgene PPCO centrifuge bottle 500 ml (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3120-0500 )
  4. SLA-3000 fixed angle rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 07149 )
  5. SS-34 fixed angle rotor (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28020 )
  6. Sorvall RC-6 plus (Thermo Fisher Scientific, Thermo ScientificTM, model: Sorvall RC 6 Plus , catalog number: 46910)
  7. HiTemp hot water bath (Fisher Scientific, catalog number: 11-481Q )
  8. Accumet AB150 pH benchtop meter (Fisher Scientific, model: Accumet AB150TM, catalog number: 13-636-AB150 )
  9. UV-Vis spectrophotometer (Thermo Fischer Scientific, Thermo ScientificTM, model: GENESYSTM 10S , catalog number: 840-208100)
  10. Vortex-Genie 2 (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  11. Astec C18 HPLC column (Sigma-Aldrich, catalog number: 55024AST )
    Note: This product has been discontinued.
  12. Beckman Coulter System Gold HPLC with 126 Solvent Module, 168 Detector, and SC100 Fraction collector (Beckman Coulter, model: System Gold )
    Note: This product has been discontinued.

Procedure

  1. Growth of bacteria cultures
    1. Grow up 200 ml or larger bacterial cultures to obtain enough peptidoglycan for analysis of Gram-negative bacteria. It may be possible to use less culture for Gram-positive bacteria. Use growth media suited to the bacteria being grown. Since the composition of peptidoglycan is growth-phase dependent (Pisabarro et al., 1985), it is important that all cultures are grown to the same optical density to limit differences due to growth phase. Harvesting cells at a final optical density corresponding to mid- or late-logarithmic growth phase will provide a high yield of cells, while minimizing the chance of harvesting dead or lysed cells.
    2. Once cells have reached the desired optical density, chill cultures in an ice bath and transfer to a 500 ml centrifuge bottle.
    3. Centrifuge cultures at 5,000 x g for 10 min at 4 °C.
    4. Decant supernatant and wash cells once with 25 ml of cold PBS.
    5. Centrifuge cultures at 5,000 x g for 10 min at 4 °C. Decant supernatant and save cell pellet at -80 °C.

  2. Isolation of macromolecular peptidoglycan from cells
    1. Suspend frozen cell pellets in 10 ml PB (25 mM sodium phosphate pH = 6, see Recipes). The slightly acidic pH protects the acetyl groups present in some bacterial species.
    2. For each cell pellet, add 10 ml of PB with 8% (w/v) SDS to a new Oak Ridge tube and place tubes in a boiling water bath.
    3. Add cell suspensions drop-wise to tubes with boiling SDS. Cover tubes with vented aluminum foil to prevent evaporation, and place in boiling water. Boil samples for 30 min.
    4. Cap tubes and allow them to equilibrate to room temperature. Do not cool samples on ice because it will cause the SDS to precipitate.
    5. Centrifuge in an SS-34 rotor at 45,000 x g for 30 min at 15 °C to pellet the insoluble peptidoglycan.
    6. Decant the supernatant containing soluble cell components. The peptidoglycan pellet will appear glassy and be about the size of a thumbnail (Figure 1).


      Figure 1. Pelleted peptidoglycan. Peptidoglycan following the first boiling SDS treatment and washes (left) and following the second boiling SDS treatment (right).

    7. Wash the pellet by adding 10 ml of PB and vortex until the pellet is completely suspended. Centrifuge in an SS-34 rotor at 45,000 x g for 30 min at 15 °C.
    8. Repeat the previous wash step to remove SDS. The residual SDS will cause foaming that will decrease with each wash. Continue washing until there are no more signs of SDS, usually 4 to 6 washes depending on the size of the initial culture.
    9. After the last wash, suspend the pellet in 1 ml PB and transfer to a microcentrifuge tube.
      Note: At this point the sacculi have been isolated from most cellular components not covalently bound. Treatments with α-amylase and pronase are used to remove trapped high-molecular weight glycogen and peptidoglycan-associated proteins, respectively. For Gram-positives, an additional treatment of 1 N HCl is also needed to remove teichoic acids (Kuhner et al., 2014) after treatment with pronase.
    10. Dilute α-amylase saline suspension to a 1 mg ml-1 working concentration with water. Add 100 µg of α-amylase to each suspended peptidoglycan pellet. Vortex briefly and incubate for 1 h at 37 °C.
    11. Dissolve necessary pronase powder to 2 mg ml-1 with water. Add 200 µg of pronase to each suspended peptidoglycan pellet. Vortex briefly and incubate for between 2 h to overnight at 37 °C with agitation.
    12. Add sample drop-wise into an Oak Ridge tube containing 10 ml PB with 4% (w/v) SDS placed into a boiling water bath and boil for 30 min.
    13. Cap tubes and allow samples to equilibrate to room temperature.
    14. Centrifuge samples in an SS-34 rotor at 45,000 x g for 30 min at 15 °C and decant the supernatant. Suspend pelleted peptidoglycan in 10 ml PB. Repeat spins and washes until no residual SDS remains.
    15. After the last wash, suspend peptidoglycan in 0.5 ml of PB, and then normalize samples to 4 mg ml-1. Peptidoglycan can be normalized using a standard curve of Micrococcus lysodeikticus and measuring absorption at either 206 nm or 254 nm. Absorbance at 206 nm is used to detect carboxyl groups, ester linkages, and peptide bonds, while absorbance at 254 nm is used to detect the sugars of the glycan backbone. Samples can be stored long-term at -20 °C or can be stored with 0.02% sodium azide at 4 °C for one month, possibly longer.

  3. Preparation of peptidoglycan fragments for HPLC
    1. Transfer 0.5 ml (2 mg) of purified peptidoglycan to a microcentrifuge tube and add 10 µl of 2 mg ml-1 mutanolysin. Incubate at 37 °C for 4 h to overnight with agitation. It is important that the concentration of peptidoglycan is sufficiently high prior to the addition of mutanolysin because insoluble fragments cannot be concentrated by centrifugation.
    2. Wash an Amicon Ultra-0.5 Centrifugal Filter Ultracel-10 twice with 0.5 ml water by centrifugation at 14,000 x g for 5 min and discard flow-through to remove trace amounts of glycerol on the membrane. Do not allow membrane to dry completely.
    3. To remove mutanolysin and insoluble peptidoglycan, add mutanolysin-digested samples to the washed filter with a new collection tube and centrifuge at 14,000 x g for 20 min.
    4. Transfer flow-through, containing soluble peptidoglycan fragments, to a new microcentrifuge tube and discard the filter.
    5. In a chemical hood, add 10 mg ml-1 sodium borohydride to 0.5 M borate buffer (see Recipes) immediately before using.
      Note: Before handling sodium borohydride read the MSDS for necessary handling precautions. Avoid contact with skin and note that it reacts violently with water. The sodium borohydride reaction rapidly creates enough hydrogen gas to pop the cap off on a microcentrifuge tube with enough force that the tube will jump and spray your sample over a meter. It is important that the microcentrifuge cap is either secured with a cap lock or left open.
    6. Add an equal volume of sodium borohydride solution (0.5 ml) to soluble peptidoglycan samples. Vortex and then uncap samples in a chemical hood and repeat after 10 min. Sodium borohydride is used to reduce sugars so that each fragment will elute as a single peak.
    7. After exactly 20 min, stop the reaction by adjusting the pH to between 2 and 4 by adding approximately 20 µl of 85% phosphoric acid to each reaction. An overview of the peptidoglycan isolation and preparation for HPLC can be seen in Figure 2.


      Figure 2. Summary and brief description of the steps necessary to prepare peptidoglycan for HPLC analysis

  4. Detection of muropeptides by HPLC
    1. Set up an HPLC method that runs separation buffer A (see Recipes) for 10 min and then goes from 0 to 100% separation buffer B (see Recipes) over 120 min with a flow rate of 0.5 ml per minute. Detect peaks using a 206 nm wavelength. An injection loop of 0.5 ml is recommended. Use a reversed-phase octadecyl silica (ODS) C18 column to separate muropeptides. A column incubator set to 30 °C will ensure a more consistent separation.
    2. Run a blank (buffer only) sample to establish a baseline and ensure that there are no contaminating residual compounds remaining on the column. Clean the column after each run with 100% buffer B for 20 min at 1 ml min-1, then equilibrate with 100% buffer A for 30 min at 1 ml min-1 before each run.
      Note: The retention time of the muropeptides is dependent on the pH of the buffers, the percentage of solvent, and temperature (Glauner, 1988). The buffer pH is near the equivalence point, making them difficult to replicate exactly even with a good pH meter. It is useful to make enough buffer to run all comparative samples. Maintaining the column at 30 °C will also help make retention times more consistent. The methanol causes a positive baseline drift, which can make analysis more difficult. One solution is to add approximately 10 µg ml-1 sodium azide (NaN3) to lower the absorbance (Glauner et al., 1988). With newer detectors the baseline drift can be fixed with the subtraction of a blank run from a sample run or the use of a reference wavelength.
    3. Inject up to 0.5 ml of sample for each HPLC run. If desired, collect separated muropeptides in 1-min (0.5 ml) fractions in a fraction collector for later analysis/confirmation by mass spectrometry. The peptidoglycan fragment peaks will separate and form peaks that can be visualized using absorbance at 206 nm (Figure 3).
    4. The amounts of each type of peptidoglycan fragment can be quantified by measuring the area under the peak using the software included with your HPLC.


      Figure 3. Chromatogram of separated peptidoglycan fragments from the rod-shaped Escherichia coli and the diplococcus Neisseria gonorrhoeae. Many of the peptidoglycan fragments are the same between species like the two prominent Tetra and Tetra-Tetra fragments. Some of the differences are due N. gonorrhoeae having O-acetylated peptidoglycan, but lacking Braun’s lipoprotein and Tri-Lys-Arg attachments. The identity of the peaks follows the naming convention used by Glauner (1988).

  5. Identification of peptidoglycan peaks by mass spectrometry
    1. The phosphate in the buffers used to separate peptidoglycan peaks is incompatible with many mass spectrometry methods. To desalt peptidoglycan peaks by HPLC, first pool fractions containing peaks of interest and concentrate using a vacuum concentrator.
    2. The samples can then be desalted on the same C18 HPLC column. Create a program that runs 100% desalting buffer A (see Recipes) for 10 min, then a gradient from 0% to 100% desalting buffer B (see Recipes) for 10 min, and 100% desalting buffer B for an additional 10 min at 1 ml min-1. A more gradual gradient can be used to separate peaks containing more than one type of peptidoglycan fragment.
    3. As before, run a blank sample to establish a baseline, then run samples and use a fraction collector to collect eluted peptidoglycan fragments.
    4. Vacuum concentrate collected samples containing your peptidoglycan fragment. Mass can be determined by positive ion electrospray ionization time of flight mass spectrometry (POS ES-TOF), or Matrix Assisted Laser Desorption/Ionization–Time Of Flight (MALDI-TOF MS) mass spectrometry.

Data analysis

  1. To make comparisons between bacterial strains, growth conditions, or mutations, a minimum of three peptidoglycan samples from independent cultures should be used. Use absorbance at 206 nm (A206) to identify peaks of interest. Establishing a baseline is important for analysis, especially for larger fragments with longer retention times. Software between HPLC systems will be different, but being consistent with how samples are run and how data is analyzed will ensure accurate comparison.
  2. The two common ways of expressing peak area (also known as area under the peak) are as absorbance/min and as percentage of total chromatogram. The second option offers more biological relevance and is more forgiving of samples when the amount of peptidoglycan injected is not equal. Biological replicates can be compared using a two-sample t-test assuming equal variance to compare equivalent peaks between samples.
  3. Annotated chromatograms and other information can be found in (Glauner, 1988; Glauner et al., 1988; Pisabarro et al., 1985; Antignac et al., 2003b).

Recipes

  1. PB (25 mM phosphate buffer pH = 6)
    0.2973% (w/v) NaH2PO4·H2O
    0.0926% (w/v) Na2HPO4·7H2O
    Confirm pH = 6, autoclave
  2. PB with 8% (w/v) SDS
    0.2973% (w/v) NaH2PO4·H2O
    0.0926% (w/v) Na2HPO4·7H2O
    8.0% (w/v) SDS
  3. 0.5 M borate buffer pH = 8
    1. Dissolve 6.18 g boric acid in 140 ml water
    2. Adjust pH to 8 with 10 N NaOH
    3. Adjust volume to 200 ml
    4. Filter sterilize (0.22 µm)
  4. Phosphate buffered saline (PBS)
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    1.8 mM KH2PO4
    Autoclave and store at 4 °C
  5. HPLC separation buffer A (50 mM sodium phosphate pH = 4.33)
    1. Dissolve 2.0 g NaOH in 700 ml HPLC grade water
    2. Adjust pH to 4.33 with phosphoric acid
    3. Bring up volume to 1 L with HPLC-grade water
    4. Filter sterilize (0.22 µm) and degas
  6. HPLC separation buffer B (50 mM sodium phosphate pH = 5.10 with 15% (v/v) methanol)
    1. Dissolve 1.7 g NaOH in 600 ml HPLC-grade water
    2. Adjust pH to 5.10 with phosphoric acid
    3. Bring up volume to 850 ml with HPLC-grade water
    4. Add 150 ml methanol
    5. Filter and degas. Seal container to protect from evaporation
  7. HPLC desalting buffer A
    HPLC-grade water with 0.05% (v/v) trifluoroacetic acid
  8. HPLC desalting buffer B
    50% acetonitrile (50% HPLC-grade water) with 0.05% (v/v) trifluoroacetic acid

Acknowledgments

This work was supported by NIH grant R01 AI097157. Dr. Dillard has consulted for, and received consulting fees from, Pfizer Inc. for work related to their Neisseria meningitidis vaccine patents. This protocol was adapted from (Dougherty, 1985) and (Glauner et al., 1988).

References

  1. Antignac, A., Boneca, I. G., Rousselle, J. C., Namane, A., Carlier, J. P., Vazquez, J. A., Fox, A., Alonso, J. M. and Taha, M. K. (2003a). Correlation between alterations of the penicillin-binding protein 2 and modifications of the peptidoglycan structure in Neisseria meningitidis with reduced susceptibility to penicillin G. J Biol Chem 278(34): 31529-31535.
  2. Antignac, A., Rousselle, J. C., Namane, A., Labigne, A., Taha, M. K. and Boneca, I. G. (2003b). Detailed structural analysis of the peptidoglycan of the human pathogen Neisseria meningitidis. J Biol Chem 278(34): 31521-31528.
  3. Araki, Y., Nakatani, T., Hayashi, H. and Ito, E. (1971). Occurrence of non-N-substituted glucosamine residues in lysozyme-resistant peptidoglycan from Bacillus cereus cell walls. Biochem Biophys Res Commun 42(4): 691-697.
  4. Clarke, A. J. and Dupont, C. (1992). O-acetylated peptidoglycan: its occurrence, pathobiological significance, and biosynthesis. Can J Microbiol 38(2): 85-91.
  5. Clarke, T. B. and Weiser, J. N. (2011). Intracellular sensors of extracellular bacteria. Immunol Rev 243(1): 9-25.
  6. Desmarais, S. M., De Pedro, M. A., Cava, F. and Huang, K. C. (2013). Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell wall structure and assembly. Mol Microbiol 89(1): 1-13.
  7. Dougherty, T. J. (1985). Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography. J Bacteriol 163(1): 69-74.
  8. Glauner, B. (1988). Separation and quantification of muropeptides with high-performance liquid chromatography. Anal Biochem 172(2): 451-464.
  9. Glauner, B., Holtje, J. V. and Schwarz, U. (1988). The composition of the murein of Escherichia coli. J Biol Chem 263(21): 10088-10095.
  10. Holtje, J. V. and Tuomanen, E. I. (1991). The murein hydrolases of Escherichia coli: properties, functions and impact on the course of infections in vivo. J Gen Microbiol 137(3): 441-454.
  11. Kato, K. and Strominger, J. L. (1968). Structure of the cell wall of Staphylococcus aureaus. IX. Mechanism of hydrolysis by the L11 enzyme. Biochemistry 7(8): 2745-2761.
  12. Kuhner, D., Stahl, M., Demircioglu, D. D. and Bertsche, U. (2014). From cells to muropeptide structures in 24 h: peptidoglycan mapping by UPLC-MS. Sci Rep 4: 7494.
  13. Markiewicz, Z., Glauner, B. and Schwarz, U. (1983). Murein structure and lack of DD- and LD-carboxypeptidase activities in Caulobacter crescentus. J Bacteriol 156(2): 649-655.
  14. Pisabarro, A. G., de Pedro, M. A. and Vazquez, D. (1985). Structural modifications in the peptidoglycan of Escherichia coli associated with changes in the state of growth of the culture. J Bacteriol 161(1): 238-242.
  15. Schleifer, K. H. and Kandler, O. (1972). Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36(4): 407-477.
  16. Ward, J. B. (1973). The chain length of the glycans in bacterial cell walls. Biochem J 133(2): 395-398.

简介

肽聚糖(murein)是由短肽连接的糖组成的几乎所有细菌的细胞壁的重要组成部分。 该方案描述了从培养细菌中纯化大分子肽聚糖和使用高效液相色谱(HPLC)分析酶消化的肽聚糖片段。 消化的肽聚糖片段可以通过质谱鉴定,或通过比较保留时间与其他公开的色谱图预测。 该方法的定量性质允许测量不同种类的细菌,生长条件或突变之间肽聚糖组成的变化。 该方法可以确定肽聚糖的总体结构,如肽长度,交联程度和修饰。 已经使用神经肽分析来研究肽聚糖相关蛋白的功能和细菌获得抗生素抗性的机制。
【背景】肽聚糖由通过肽干连接在一起的糖骨架组成,其产生对细胞形状重要的网状结构和细菌细胞的膨胀压力。大分子肽聚糖从在细胞质中合成的单体单元组装,并由具有5个氨基酸的茎组成的具有5个氨基酸的葡萄糖胺和N-乙酰氨基葡萄糖二糖组成。当单体翻转到周质中时,通过反式糖基化将其加入到聚糖链中,并且通过转肽酶将一部分肽茎连接在一起。
 包含肽干的氨基酸可以根据物种变化,但通常以L-丙氨酸,D-谷氨酸,内消旋二氨基庚二酸,D-丙氨酸,D-丙氨酸,丙氨酸,在一些革兰氏阳性中,L-赖氨酸代替二氨基庚二酸。通过直接或通过连接氨基酸连接第三或第四氨基酸的第三个氨基酸的游离胺进行交联(Schleifer和Kandler,1972)。其他常见的修饰包括氨基酸的酰胺化(Kato和Strominger,1968)和O-乙酰化(Clarke和Dupont,1992)或者N-脱乙酰化(Araki et al。 al。,1971)糖。
各种酶在生长和细胞分裂过程中作用于肽聚糖。已知为裂解转糖基酶的酶类在与溶菌酶相同的位置处在双糖单位之间切割聚糖链。重要的区别是裂解转糖基酶产生1,6-脱水键,与由溶菌酶和变溶菌素形成的还原末端相反。因此,1,6-脱水键的相对丰度可以用作聚糖链长度的近似值(Ward,1973)。不同类型的肽酶在肽干和交联的不同键上起作用。例如,D,D-羧基肽酶将在第四和第五个氨基酸之间切割,而L,D-羧基肽酶将在第三和第四个氨基酸之间切割(Holtje和Tuomanen,1991)。
神经肽分析可以解决不同的修饰和交联,给出大分子肽聚糖的总体结构模型。使用基于HPLC的肽聚糖分析的第一次观察之一是发现新月牙菌素缺乏D,D-羧肽酶活性(Markiewicz等人,1983)。第一次全面的肽聚糖分析在大肠杆菌中进行,其中鉴定了80种不同的神经肽物种(Glauner等人,1988)。这种方法也用于显示脑膜炎奈瑟氏球菌中的青霉素抗性与肽聚糖结构的差异相关(Antignac等人,2003a)。
肽聚糖的兴趣近年来有所增加。对肽聚糖靶向抗生素的持续细菌耐药性需要更全面地了解肽聚糖代谢。人类肽聚糖识别蛋白NOD1和NOD2的发现也导致对宿主细胞如何识别肽聚糖以及它们如何能够区分共生和致病细菌的研究有了更多的研究(Clarke和Weiser,2011)。
以下方法比其他类型的肽聚糖分析具有许多优点,包括基于超高效液相色谱(UPLC)的方法。第一个优点是几乎所有的设备和材料都是大多数实验室的标准组成部分,因此不需要大量的投资。其次,该方案近30年的历史可以进行与相似色谱图的比较,从而可以快速进行肽聚糖片段的初步鉴定,然后使用质谱法来确定变化或特别感兴趣的片段。第三个优点是该方法的规模,其产生足够的分离材料用于通过质谱法或酶反应进行额外分析。关于基于HPLC的肽聚糖分析的历史和用途的更多信息可参见本综述(Desmarais等人,2013)

关键字:胞壁肽分析, 肽聚糖, PG, HPLC, 胞壁肽

材料和试剂

  1. 移液器提示
  2. Nalgene Oak Ridge高速PPCO离心管(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3119-0050)
  3. 1.7ml微量离心管(MIDSCI,目录号:AVSS1700)
  4. 带超薄膜的Amicon Ultra-0.5离心过滤器(EMD Millipore,目录号:UFC501024)
  5. 铝箔(Fisher Scientific,目录号:01-213-100)
  6. 过滤器(0.22μm)
  7. 细菌生长培养基(物种特异性)
  8. 粟粒微球菌ATCC No.4698,冻干细胞(Sigma-Aldrich,目录号:M3770)
  9. 来自猪胰腺的α-淀粉酶(Sigma-Aldrich,目录号:A6255)
  10. 苍耳酶蛋白酶,灰色链霉菌(EMD Millipore,目录号:53702)
  11. 1 N HCl
  12. 叠氮化钠(NaN 3 3)(Sigma-Aldrich,目录号:S2002)
  13. 来自全球链球菌(ATCC)的Mutanolysin(Sigma-Aldrich,目录号:M9901)
  14. 硼氢化钠(Sigma-Aldrich,目录号:213462)
  15. 磷酸二氢钠一水合物(NaH 2 PO 4·H 2 O)(Fisher Scientific,目录号:S369)
  16. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(Fisher Scientific,目录号:S373)
  17. 十二烷基硫酸钠(SDS)(Fisher Scientific,目录号:BP166-100)
  18. 硼酸(Acros Organics,目录号:327132500)
  19. 水(HPLC级)(Fisher Scientific,目录号:W5SK-4)
  20. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318-1)
  21. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358)
  22. 氯化钾(KCl)(Fisher Scientific,目录号:BP366)
  23. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P285)
  24. O-磷酸,85%(HPLC)(Fisher Scientific,目录号:A260-500)
  25. 甲醇(HPLC级)(Fisher Scientific,目录号:A452SK-4)
  26. 三氟乙酸(Sigma-Aldrich,目录号:302031)
  27. 乙腈(HPLC级)(Fisher Scientific,目录号:A998)
  28. 磷酸盐缓冲液pH = 6(PB)(参见食谱)
  29. 具有8%(w / v)SDS的PB(参见食谱)
  30. 0.5 M硼酸盐缓冲液pH = 8(见配方)
  31. 磷酸盐缓冲盐水(PBS)(见食谱)
  32. HPLC分离缓冲液A(参见食谱)
  33. HPLC分离缓冲液B(参见食谱)
  34. HPLC脱盐缓冲液A(参见食谱)
  35. HPLC脱盐缓冲液B(参见食谱)

设备

  1. Kimax挡板培养瓶(Fisher Scientific,目录号:10-140-6A和10-140-6B)
    制造商:DWK Life Sciences,Kimble,目录号:25630250和25630500。
  2. 移液器(Gilson,目录号:F167700)
  3. Nalgene PPCO离心机瓶500毫升(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3120-0500)
  4. SLA-3000固定角转子(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:07149)
  5. SS-34固定角度转子(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:28020)
  6. Sorvall RC-6 plus(Thermo Fisher Scientific,Thermo Scientific TM,型号:Sorvall RC 6 Plus,目录号:46910)
  7. HiTemp热水浴(Fisher Scientific,目录号:11-481Q)
  8. Accumet AB150 pH台式仪表(Fisher Scientific,型号:Accumet AB150 TM,目录号:13-636-AB150)
  9. UV-Vis分光光度计(Thermo Fischer Scientific,Thermo Scientific TM,型号:GENESYS TM,目录号:840-208100)
  10. Vortex-Genie 2(Scientific Industries,型号:Vortex-Genie 2,目录号:SI-0236)
  11. Astec C18 HPLC柱(Sigma-Aldrich,目录号:55024AST)
    注意:本产品已停产。
  12. 贝克曼库尔特系统黄金HPLC与126溶剂模块,168检测器和SC100馏分收集器(Beckman Coulter,型号:System Gold)
    注意:本产品已停产。

程序

  1. 细菌培养物的生长
    1. 长达200毫升或更大的细菌培养物以获得足够的肽聚糖用于分析革兰氏阴性细菌。革兰氏阳性菌可能使用较少的培养物。使用适合生长细菌的生长培养基。由于肽聚糖的组成与生长相关(Pisabarro等人,1985),重要的是所有培养物生长至相同的光密度以限制由于生长期的差异。以对应于中期或后期对数生长期的最终光密度收集细胞将提供高产量的细胞,同时最小化收获死亡或裂解细胞的机会。
    2. 一旦细胞达到所需的光密度,在冰浴中冷却培养物并转移到500ml离心瓶中
    3. 在4℃下以5,000×g离心培养物10分钟。
    4. 用25ml冷PBS将上清液和上清液洗涤一次
    5. 在4℃下以5,000×g离心培养物10分钟。去除上清液,并在-80℃保存细胞沉淀。

  2. 从细胞中分离大分子肽聚糖
    1. 将冷冻细胞沉淀悬浮于10ml PB(25mM磷酸钠pH = 6,参见食谱)中。微酸性pH保护一些细菌物种中存在的乙酰基。
    2. 对于每个细胞沉淀物,加入10 ml具有8%(w / v)SDS的PB至新的Oak Ridge管,并将管置于沸水浴中。
    3. 将细胞悬浮液逐滴加入含沸点SDS的管中。盖上带有铝箔的管子,以防止蒸发,并置于沸水中。煮沸样品30分钟。
    4. 盖管并允许它们平衡至室温。不要在冰上冷却样品,因为会导致SDS沉淀。
    5. 在15℃下以45,000×g离心机在SS-34转子中离心30分钟,以沉淀不溶性肽聚糖。
    6. 滗析含有可溶性细胞成分的上清液。肽聚糖颗粒将出现玻璃状,约为缩略图的大小(图1)。


      图1.造粒肽聚糖。第一次沸腾SDS处理后的肽聚糖洗涤(左)和第二次煮沸的SDS处理(右)。

    7. 通过加入10ml PB洗涤沉淀,并旋转直到颗粒完全悬浮。在15℃下以45,000×g离心机在SS-34转子中30分钟。
    8. 重复上一次洗涤步骤去除SDS。残留的SDS会引起每次洗涤会降低的泡沫。继续洗涤,直到没有更多的SDS的迹象,通常根据初始培养物的大小4至6次洗涤。
    9. 最后一次洗涤后,将沉淀悬浮于1 ml PB中,并转移到微量离心管中 注意:在这一点上,神经与大多数细胞组分没有共价结合。用α-淀粉酶和链霉蛋白酶处理分别去除被捕获的高分子量糖原和肽聚糖相关蛋白。对于革兰氏阳性,在用链霉蛋白酶处理后,还需要额外处理1N HCl以除去磷酸(Kuhner等人,2014)。
    10. 将α-淀粉酶盐水悬浮液稀释至1毫克/升的工作浓度。向每个悬浮的肽聚糖颗粒中加入100μg的α-淀粉酶。短暂旋涡,37℃孵育1小时
    11. 用必需的链霉菌粉末溶解至2毫克/升。向每个悬浮的肽聚糖颗粒中加入200μg的链霉蛋白酶。短暂旋涡并在37℃下搅拌孵育2小时至过夜。
    12. 将样品滴加到含有10 ml PB的橡胶岭管中,4%(w / v)SDS放入沸水浴中煮沸30分钟。
    13. 盖管,允许样品平衡至室温。
    14. 在15℃下以45,000×g离心在SS-34转子中的样品30分钟并倾析上清液。将沉淀的肽聚糖悬浮于10ml PB中。重复旋转和洗涤,直到不残留SDS。
    15. 最后一次洗涤后,将肽聚糖悬浮于0.5ml PB中,然后将样品标准化至4mg ml -1。肽聚糖可以使用微球菌溶菌酶的标准曲线进行标准化,并测量206nm或254nm处的吸收。 206 nm处的吸光度用于检测羧基,酯键和肽键,而254 nm处的吸光度用于检测聚糖骨架的糖。样品可以长期保存在-20°C,或者可以在4°C下与0.02%叠氮化钠一起储存一个月,可能更长时间。

  3. HPLC的肽聚糖片段的制备
    1. 将0.5ml(2mg)纯化的肽聚糖转移到微量离心管中,并加入10μl2mg ml -1的溶血素。在37℃下孵育4小时,搅拌过夜。重要的是,在添加溶血素之前,肽聚糖的浓度足够高,因为不溶性片段不能通过离心浓缩。
    2. 通过以14,000xg离心5分钟,用0.5ml水洗涤Amicon Ultra-0.5离心过滤器Ultracel-10两次,并丢弃流过以除去膜上痕量的甘油。不要让膜完全干燥。
    3. 为了除去溶菌解蛋白酶和不溶性肽聚糖,用新的收集管将经溶解解菌素消化的样品加入洗涤的过滤器中,并以14,000xg离心20分钟。
    4. 将含有可溶性肽聚糖片段的流通转移到新的微量离心管中并丢弃过滤器。
    5. 在化学品罩中,在使用前立即加入10毫克/升的硼氢化钠至0.5M硼酸盐缓冲液(参见食谱)。
      注意:在处理硼氢化钠之前,请阅读MSDS的必要处理注意事项。避免与皮肤接触,并注意与水剧烈反应。硼氢化钠反应迅速产生足够的氢气,在微量离心管上弹出盖子,足够的力使管子跳动并将样品喷在一米上。重要的是,微型离心机盖可以用盖锁固定或打开。
    6. 向可溶性肽聚糖样品中加入等体积的硼氢化钠溶液(0.5ml)。涡旋,然后在化学品罩中取样,10分钟后重复。硼氢化钠用于还原糖,以使每个片段作为单个峰值洗脱。
    7. 完全20分钟后,通过在每个反应中加入约20μl的85%磷酸将pH调节至2至4停止反应。肽聚糖分离和HPLC制备的概述可以在图2中看到。


      图2.准备肽聚糖进行HPLC分析所需步骤的总结和简要说明

  4. 通过HPLC检测神经肽
    1. 设置运行分离缓冲液A(参见食谱)10分钟的HPLC方法,然后以0.5ml /分钟的流速在120分钟内从0分钟缓冲液B到达100%的分离缓冲液B(参见食谱)。使用206 nm波长检测峰。建议注射回路为0.5毫升。使用反相十八烷基二氧化硅(ODS)C18柱分离神经肽。设置为30°C的色谱柱培养箱将确保更一致的分离。
    2. 运行一个空白(仅缓冲液)样品建立基线,并确保残留在色谱柱上的污染残留化合物。在每次运行后用100%缓冲液B以1ml / min的速率清洗柱子,然后用100%缓冲液A以1ml / min的速度平衡30分钟。 >在每次运行之前。
      注意:神经肽的保留时间取决于缓冲液的pH,溶剂的百分比和温度(Glauner,1988)。缓冲液pH值接近等值点,即使使用良好的pH计也难以准确地复制。制作足够的缓冲区以运行所有比较样品是有用的。将色谱柱维持在30°C也有助于使保留时间更加一致。甲醇引起基线漂移,可以使分析更加困难。一种解决方案是加入约10μg/ ml的叠氮化钠(NaN 3 N 3)以降低吸光度(Glauner等,1988)。使用较新的检测器,可以通过从样品运行中减去空白运行或使用参考波长来固定基线漂移。
    3. 每个HPLC运行注射高达0.5毫升的样品。如果需要,在馏分收集器中收集1分钟(0.5ml)级分的分离的神经肽,以便以后通过质谱法进行分析/确认。肽聚糖片段峰将分离并形成可以使用206 nm处的吸光度可视化的峰(图3)。
    4. 可以通过使用HPLC附带的软件测量峰值下的面积来量化每种类型的肽聚糖片段的量。


      图3.来自棒状大肠杆菌和淋球菌双球菌的分离的肽聚糖片段的色谱图。 许多肽聚糖片段在物种之间是相同的,如两个显着的Tetra和Tetra-Tetra片段。一些差异是由于。具有O-乙酰化肽聚糖的Gonorrhoeae,但缺乏Braun的脂蛋白和Tri-Lys-Arg附着物。峰值的身份遵循Glauner(1988)使用的命名约定。

  5. 通过质谱鉴定肽聚糖峰
    1. 用于分离肽聚糖峰的缓冲液中的磷酸盐与许多质谱法不相容。通过HPLC对肽聚糖峰进行脱盐,使用真空浓缩器将含有目标峰和浓缩物的第一池级分。
    2. 然后将样品在相同的C18 HPLC柱上脱盐。创建一个运行100%脱盐缓冲液A(参见食谱)10分钟,然后从0%至100%脱盐缓冲液B(参见食谱)10分钟的梯度和100%脱盐缓冲液B另外10分钟的程序1 ml min -1 。可以使用更渐进的梯度来分离含有多于一种类肽聚糖片段的峰。
    3. 如前所述,运行空白样品建立基线,然后运行样品并使用级分收集器收集洗脱的肽聚糖片段。
    4. 收集含有您的肽聚糖片段的真空浓缩物。质谱可以通过正离子电喷雾离子化飞行时间质谱(POS ES-TOF)或基质辅助激光解吸/电离飞行时间(MALDI-TOF MS)质谱法测定。

数据分析

  1. 为了比较细菌株,生长条件或突变,应使用至少三个来自独立培养物的肽聚糖样品。使用206nm处的吸光度(A <206)来鉴定感兴趣的峰。建立基线对分析很重要,特别是对于具有较长保留时间的较大片段。 HPLC系统之间的软件将不同,但与样品运行方式和数据分析方式一致将确保准确的比较。
  2. 表达峰面积(也称为峰面积)的两种常见方式如吸光度/分钟和总色谱图百分比。第二个选项提供更多的生物相关性,并且当注射的肽聚糖的量不相等时,更容易获得样品。可以使用假定相等方差的双样本t检验比较生物学重复,以比较样品之间的等效峰。
  3. 注释色谱图和其他信息可以在(Glauner,1988; Glauner等人,1988; Pisabarro等人,1985; Antignac等人, / em>,2003b)。

食谱

  1. PB(25mM磷酸缓冲液pH = 6)
    0.2973%(w / v)NaH 2 PO 4·2H 2 O / / 0.0926%(w / v)Na 2 HPO 4·7H 2 O
    确认pH = 6,高压釜
  2. PB含8%(w / v)SDS 0.2973%(w / v)NaH 2 PO 4·2H 2 O / / 0.0926%(w / v)Na 2 HPO 4·7H 2 O
    8.0%(w / v)SDS
  3. 0.5M硼酸盐缓冲液pH = 8
    1. 将6.18克硼酸溶于140毫升水中
    2. 用10N NaOH调节pH至8
    3. 调节体积至200 ml
    4. 过滤灭菌(0.22μm)
  4. 磷酸盐缓冲盐水(PBS)
    137 mM NaCl
    2.7 mM KCl
    10mM Na 2 HPO 4
    1.8mM KH PO 4
    高压灭菌并储存在4°C
  5. HPLC分离缓冲液A(50mM磷酸钠pH = 4.33)
    1. 将2.0 g NaOH溶于700 ml HPLC级水中
    2. 用磷酸调节pH至4.33
    3. 用HPLC级水将体积增加至1升
    4. 过滤灭菌(0.22μm)和脱气
  6. HPLC分离缓冲液B(50mM磷酸钠pH = 5.10,15%(v / v)甲醇)
    1. 将1.7 g NaOH溶于600 ml HPLC级水中
    2. 用磷酸调节pH至5.10
    3. 使用HPLC级水将体积增至850毫升
    4. 加入150 ml甲醇
    5. 过滤和脱气密封容器以防止蒸发
  7. HPLC脱盐缓冲液A
    具有0.05%(v / v)三氟乙酸的HPLC级水分
  8. HPLC脱盐缓冲液B
    含有0.05%(v / v)三氟乙酸的50%乙腈(50%HPLC级水)

致谢

这项工作得到NIH授权R01 AI097157的支持。 Dillard博士就辉瑞公司就他们的脑膜炎奈瑟氏球菌疫苗专利工作提供咨询,并收到咨询费用。该方案从(Dougherty,1985)和(Glauner等人,1988)改编而来。

参考

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  9. Glauner,B.,Holtje,JV和Schwarz,U.(1988)。&nbsp; 大肠杆菌的murein的组成。生物化学263(21):10088-10095。
  10. Holtje,JV和Tuomanen,EI(1991)。大肠杆菌的murein水解酶:对体内感染过程的性质,功能和影响。 J. Gen Microbiol 137(3 ):441-454。
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引用:Schaub, R. E. and Dillard, J. P. (2017). Digestion of Peptidoglycan and Analysis of Soluble Fragments. Bio-protocol 7(15): e2438. DOI: 10.21769/BioProtoc.2438.
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