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Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues
纯化制备及检测胎盘和胎儿组织中革兰氏阳性细菌的细胞壁   

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Abstract

Cell wall is a complex biopolymer on the surface of all Gram-positive bacteria. During infection, cell wall is recognized by the innate immune receptor Toll-like receptor 2 causing intense inflammation and tissue damage. In animal models, cell wall traffics from the blood stream to many organs in the body, including brain, heart, placenta and fetus. This protocol describes how to prepare purified cell wall from Streptococcus pneumoniae, detect its distribution in animal tissues, and study the tissue response using the placenta and fetal brain as examples.

Keywords: Cell wall(细胞壁), Peptidoglycan(肽聚糖), Bacterial inflammation(细菌炎症), Neuroproliferation(神经增殖), Fetal neurogenesis(胎儿神经发生), Placental trafficking(胎盘运输), Toll like receptor 2 ligand(Toll样受体2配体), Streptococcus pneumonae(肺炎链球菌)

Background

Host response to infection involves recognition of many bacterial components including the cell wall (CW), a complex macromolecule that forms the surface of all Gram-positive bacteria. The CW of Gram-positive bacteria is formed by the covalent network of peptidoglycan and teichoic acid. Streptococcus pneumoniae, a leading cause of pneumonia, sepsis, and meningitis, has served as an important model organism for studying the innate immune response to Gram-positive bacterial infection including CW.

When upon Streptococcus pneumoniae (pneumococcal) infection CW components are released from bacteria during growth or antibiotic-induced death, it circulates in the blood stream and crosses cellular barriers, including the placenta and blood brain barrier. CW components have inflammatory activities equal to or greater than intact bacteria (Tuomanen et al., 1985a and 1985b). The CW can be viewed as the Gram-positive equivalent of endotoxin. The vast amount of CW pieces released during infection greatly stimulates the host inflammatory response by activating the innate immune receptor, Toll like receptor 2 (TLR2) (Yoshimura et al., 1999). Responses differ depending on the organ infected: the postnatal brain undergoes apoptosis, scarring predominates in heart, and the fetal brain escapes damage, showing striking neuroproliferation (Orihuela et al., 2006; Braun et al., 1999; Fillon et al., 2006; Humann et al., 2016).

This protocol describes how to prepare purified CW from Streptococcus pneumoniae (Tuomanen et al., 1985b) and follow its distribution in mice after intravenous injection, focusing on the placenta and fetal brain as examples (Humann et al., 2016). This model yields histopathologic sections of organs for study of the tissue response to CW components. Our model’s focus on pneumococcal CW derives from its well-described role in inflammation and injury in many organs, its extensive known chemistry and its recognition as a classic TLR2 pathogen associated molecular pattern.

Materials and Reagents

  1. 0.22 µm bottle top filter (Corning, catalog number: 431096 )
  2. Glass tubes (Thermo Fisher Scientific, Fisher Scientific, catalog number: 14-961-32 )
  3. 1,000 ml centrifuge bottle (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3120-1000 )
  4. 30 ml centrifuge bottle (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0050 )
  5. Serological pipettes, 1 case of 5 ml, 10 ml, 25 ml
  6. Microcentrifuge tubes (Eppendorf, catalog number: 022364111 )
  7. Aluminum foil (Thermo Fisher Scientific, Fisher Scientific, catalog number: 01-213-100 )
  8. 25 gauge needles (BD, catalog number: 305122 )
  9. 50 ml polypropylene tubes (SARSTEDT, catalog number: 62.547.254 )
  10. Petri dishes (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875712 )
  11. Small electric razor to shave animals
  12. Superfrost microslides (VWR, catalog number: 48311-703 )
  13. Premium cover glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-548-5P )
  14. Magnetic stir bars
  15. Inoculation loops (Thermo Fisher Scientific, Fisher Scientific, catalog number: 22-363-595 )
  16. 1 ml syringe (BD, catalog number: 309628 )
  17. Acid washed 106 µm glass beads (Sigma-Aldrich, catalog number: G4649 )
  18. Plastic embedding molds for histology (Polysciences, catalog number: 18646D-1 )
  19. Streptococcus pneumoniae strains - CW is much easier to purify from strains that are unencapsulated such as R6 (ATCC, catalog number: BAA-255 )
  20. C57Bl6 mice, mixture of male and female (THE JACKSON LABORATORY, catalog number: 000664 )
  21. Water (Sigma-Aldrich, catalog number: W3500-1L )
  22. Ultrapure water (example: Milli-Q, EMD Millipore)
  23. Tryptic soy agar (TSA) (EMD Millipore, catalog number: 105458 )
  24. Sterile defibrinated sheep blood (i-Tek Medical Technologies, catalog number: 103-100-3 )
  25. Glycerol
  26. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4390 )
    Note: This product has been discontinued.
  27. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 71382 )
  28. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: 208094 )
  29. DNase I (Sigma-Aldrich, catalog number: DN25-10MG )
  30. RNase A (Sigma-Aldrich, catalog number: R6513-10MG )
  31. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C-3881 )
  32. Trypsin (AMRESCO, catalog number: M150 )
  33. α-amylase (Sigma-Aldrich, catalog number: A3176 )
  34. Lithium chloride (LiCl) (Sigma-Aldrich, catalog number: L9650 )
  35. Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  36. Acetone (Thermo Fisher Scientific, Fisher Scientific, catalog number: A946-4 )
  37. PierceTM LAL Chromogenic Endotoxin Quantitation Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88282 )
  38. Fluorescein isothiocyanate isomer I (FITC) (Sigma-Aldrich, catalog number: F7250 )
  39. Dulbecco’s phosphate buffered saline (DPBS) (Mediatech, catalog number: 21-030- CV )
  40. Paraformaldehyde, 16% w/v (Alfa Aesar, catalog number: 43368 )
  41. Sucrose (Sigma-Aldrich, catalog number: S9378 )
  42. Tissue Tek OCT compound (SAKURA FINETEK USA, catalog number: 4583 )
  43. Prolong Gold Antifade with DAPI (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P36931 )
  44. Trizma HCl (Sigma-Aldrich, catalog number: T5941 )
  45. Tris-base (Sigma-Aldrich, catalog number: 1070897600 )
  46. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331-500ml )
  47. Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: 451614 )
  48. Sodium bicarbonate (NaHCO3) (Sigma-Aldrich, catalog number: S-6014 )
  49. Magnesium chloride hexahydrate (MgCl2) (Sigma-Aldrich, catalog number: M0250 )
  50. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C1016 )
  51. MnSO4 monohydrate (Sigma-Aldrich, catalog number: M7634 )
  52. Glucose (Sigma-Aldrich, catalog number: G7528 )
  53. Adenosine (Oakwood Products, catalog number: 093333 )
  54. Uridine (EMD Millipore, catalog number: 6680 )
  55. Glutamine (Sigma-Aldrich, catalog number: G8540 )
  56. Nicotinic acid (Sigma-Aldrich, catalog number: N4126 )
  57. (B6) Pyridoxine HCl (Sigma-Aldrich, catalog number: P9755 )
  58. Ca-pantothenate (D-pantothenic acid) (Sigma-Aldrich, catalog number: P3161 )
    Note: This product has been discontinued.
  59. Thiamine HCl (Sigma-Aldrich, catalog number: 5871-100GM )
  60. Riboflavin (Sigma-Aldrich, catalog number: R4500 )
  61. Biotin (Sigma-Aldrich, catalog number: B4639 )
  62. 10 N NaOH solution (Thermo Fisher Scientific, Fisher Scientific, catalog number: SS255-1 )
  63. Ferrous sulfate heptahydrate (FeSO4·7H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: I146-500 )
  64. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C8027 )
  65. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z4750 )
  66. Manganese chloride tetrahydrate (MnCl2·4H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M87-500 )
  67. Pyruvic acid (Sigma-Aldrich, catalog number: 107360-25g )
  68. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  69. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  70. Yeast extract (BD, BactoTM, catalog number: 212750 )
  71. Sodium acetate anhydrous (Sigma-Aldrich, catalog number: S8625 )
  72. Casamino acids technical (BD, BactoTM, catalog number: 223120 )
  73. L-tryptophan (Sigma-Aldrich, catalog number: T8941 )
  74. L-cysteine HCl (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP376-100 )
  75. Asparagine (Sigma-Aldrich, catalog number: A4284 )
  76. Choline chloride (Sigma-Aldrich, catalog number: C7527 )
  77. Dry ice
  78. Solutions used to treat CW during purification
    1. 50 mM Tris-HCl, pH 7.0 (see Recipes)
    2. 5% (v/v) SDS (see Recipes)
    3. 1 M NaCl (see Recipes)
    4. 100 mM Tris, pH 7.5 (see Recipes)
    5. 1 M MgSO4 (see Recipes)
    6. 1 M CaCl2 (see Recipes)
    7. 1% (v/v) SDS (see Recipes)
    8. 8 M LiCl (see Recipes)
    9. 100 mM EDTA (see Recipes)
    10. Carbonate buffer, pH 9.2 (see Recipes)
  79. Preparation of C+Y components
    1. ‘3 in 1’ salts (see Recipes)
    2. 20% glucose (see Recipes)
    3. 50% sucrose (see Recipes)
    4. Adenosine (2 mg/ml) (see Recipes)
    5. Uridine (2 mg/ml) (see Recipes)
    6. Glutamine (1 mg/ml) (see Recipes)
    7. Adams I (see Recipes)
    8. Adams II (see Recipes)
    9. 2% pyruvate (see Recipes)
    10. 1 M KH2PO4 (see Recipes)
    11. 1 M K2HPO4 (see Recipes)
    12. 5% yeast extract (see Recipes)
  80. Media preparation
    1. PreC media (see Recipes)
    2. Supplement (see Recipes)
    3. Adams III (see Recipes)
    4. 1 M potassium phosphate buffer (see Recipes)
    5. C+Y medium (see Recipes)

Equipment

  1. 37 °C CO2 incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: 3110 )
  2. Benchtop microcentrifuge (Eppendorf, model: 5417C )
  3. 1,000 ml Erlenmeyer flasks
  4. 4,000 ml Erlenmeyer flasks
  5. Sorvall centrifuge RC 5C Plus and appropriate rotor for the centrifuge tubes or bottles
  6. 500 ml beaker
  7. Stirring hotplate
  8. Vortex mixer
  9. Speed-vac (Savant, model: SC110A )
  10. Water-bath sonicator (Thermo Fisher Scientific, Fisher Scientific, model: FS20 )
  11. Heating pad
  12. Cryostat microtome (Microme, model: HM505E )
  13. Zeiss LSM 510 NLO Meta confocal microscope
  14. Spectrophotometer (Turner, Model: 340 )
  15. Spectra MAX340 plate reader to measure absorbance at 405 nm (Molecular Device, model: Spectra MAX340 )

Software

  1. Zen 2008 software package (Carl Zeiss MicroImaging, Inc.)
  2. ImageJ (imagej.net/Particle_Analysis)
  3. Graphpad Prism (Graphpad)

Procedure

  1. Preparation of pneumococcal CW: peptidoglycan-teichoic acid complex
    Notes:
    1. Prepare all the solutions and do all washes with distilled water (use cell culture distilled water from Sigma [#W3500] with low endotoxin concentration).
    2. Filter all the solutions before use (with filter 0.22 µm).
    3. Preferably use new clean glassware and equipment to avoid any contamination with endotoxin (LPS).
    4. Prior to large-scale growth, check pneumococcal stock on blood plate without antibiotics to confirm the absence of contamination.
    5. The protocol described here uses C+Y medium (see Recipes) as a partially defined growth medium. Other commercially available media are also suitable but have more diverse contaminating components such as endotoxin that needs to be removed for achieving a pure preparation.
    1. Preparation of stock of Streptococcus pneumoniae
      CW can be purified from any pneumococcal strain. However, the procedure is most successful with unencapsulated strains such as R6 (Tuomanen et al., 1985b).
      1. Streak strain on TSA agar (20 g TSA per 500 ml water, autoclave) containing 3% (v/v) sterile defibrinated sheep blood and incubate the plate at 37 °C, 5% CO2 for 16-18 h (Figure 1). The growth on blood agar allows for the visualization of hemolysis, which is characteristic for S. pneumonia.


        Figure 1. Overnight growth of unencapsulated strain R6 on TSA blood agar. Pure culture of pneumococcus is the source of bacteria for seed stocks made in step A1b.

      2. Scrape all the colonies off the agar plate with an inoculation loop, and inoculate into 10 ml C+Y medium in a test tube. Incubate the culture at 37 °C, 5% CO2. When the optical density of the culture at 620 nm (OD620) reaches 0.4, harvest the culture by centrifugation at 1,500 x g, 4 °C for 10 min in a microcentrifuge.
      3. Remove the culture supernatant carefully. Resuspend the bacterial pellet in 5 ml of freezing medium (2.5 ml C+Y medium and 2.5 ml 80% [v/v] glycerol), and make 1 ml aliquots. Store the stocks in cryovials at -80 °C. These stocks contain approximately 1 x 107 cfu/ml.
    2. Large scale cultivation of pneumococcus
      1. Prepare 10 L of C+Y medium (see Recipes) and aliquot in three 4,000 ml Erlenmeyer flasks (3,300 ml per flask). Incubate the medium at 37 °C, 5% CO2 overnight to confirm absence of contamination.
      2. Inoculate 1 ml/flask of pneumococcal stock (107 cfu/ml) into the medium. Incubate the culture at 37 °C, 5% CO2 without shaking.
        Notes:
        1. To estimate bacterial growth in flasks, transfer one 10 ml aliquot (using sterile serological pipet) from flask into a sterile glass tube when the culture begins to appear turbid. Read the culture turbidity at 620 nm (OD620) of this transferred culture. Keep the bacterial culture in the glass tube, and incubate it in the incubator with the flask culture. Monitor the OD620 of the glass tube culture over time.
        2. OD620 in flask will be approximately 0.1 higher than OD620 in glass tube.
      3. When the OD620 in the flask reaches 0.7-0.8, remove the flask from the incubator and immerse it into ice for 15 min. Swirl the flask every 2 min to cool culture rapidly.
      4. Pour the cooled culture into pre-chilled 1,000 ml centrifuge bottles, and harvest the culture by centrifugation at 4 °C, 4,000 x g for 10 min in the Sorvall centrifuge.
      5. Decant supernatant carefully by pouring. Pellets can be stored at -80 °C until use
    3. Preliminary harvest and mechanical breakage of the bacterial cells
      Note: The operational definition of crude CW is material that is precipitable by boiling in SDS.
      1. Resuspend each pellet with 10 ml/bottle ice cold 50 mM Tris-HCl, pH 7.0 (see Recipes). 
        Note: Perform this step as quickly as possible to avoid bacterial autolysis.
      2. Boil 200 ml 5% (v/v) SDS (see Recipes) in a 500 ml beaker on a hotplate. Carefully monitor temperature control to prevent boiling over the beaker’s edge.
      3. Combine the resuspended bacterial pellets and add the bacterial suspension into the boiling SDS slowly.
      4. Boil the suspension for 15 min, and cool it to room temperature.
      5. Aliquot the boiled suspension into 30 ml centrifuge tubes. Centrifuge the suspension at room temperature, 12,000 x g for 10 min in the Sorvall centrifuge. Discard the supernatant carefully to avoid dislodging the pellet.
      6. Resuspend the pellet in 20 ml/tube 1 M NaCl (see Recipes). Centrifuge the suspension at room temperature, 12,000 x g for 10 min in the Sorvall centrifuge. Discard the supernatant gently and carefully. Repeat this step for another 2 times.
      7. Resuspend the pellet in 20 ml/tube water. Centrifuge the suspension at room temperature, 12,000 x g for 10 min in the Sorvall centrifuge. Discard the supernatant gently and carefully. Repeat this step for another 7 times to remove detergent.
      8. Resuspend the pellet in 2 ml water, and add equal volume of glass beads.
      9. Vortex the pellet-glass beads suspension at 4 °C, maximum speed for 16-18 h.
    4. Elimination of DNA, RNA, and proteins 
      1. Harvest the broken CWs by centrifugation at room temperature, 5,000 x g for 10 min in the Sorvall centrifuge.
      2. Carefully transfer the supernatant (contains CW) into a fresh 30 ml centrifuge tube.
      3. Add 10 ml water to the pellet and vortex vigorously. Incubate the suspension at room temperature for 20 min to separate supernatant by sedimentation. Then, remove the supernatant carefully with a sterile serological pipette. Transfer the supernatant into the centrifuge tube in step A4b. Repeat this step at least 5 times until the supernatant appears clear.
      4. Centrifuge the CW containing supernatant at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge.
      5. Discard the supernatant gently and carefully resuspend the pellet in 10 ml 100 mM Tris, pH 7.5 (see Recipes).
      6. Add 0.2 ml of 1 M MgSO4 (see Recipes) into the suspension to make a final concentration of 20 mM.
      7. Add 10 μl DNase I and 50 μl RNase A into the suspension at final concentration of 10 and 50 µg/ml respectively. Incubate the suspension at 37 °C for 2 h.
      8. Add 0.1 ml of 1 M CaCl2 (see Recipes) into the suspension to make a final concentration of 10 mM.
      9. Add 0.1 ml of trypsin (10 mg/ml) to make a final concentration of 100 µg/ml and 0.3 U of α-amylase.
      10. Incubate the suspension at 37 °C for 12-15 h.
    5. Removal of non-covalent adducts and contaminating endotoxin
      1. Resuspend the pellet in 10 ml 8 M LiCl (see Recipes), incubate at 37 °C for 15 min.
      2. Centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant.
      3. Resuspend the pellet with 10 ml 100 mM EDTA (see Recipes), and incubate at 37 °C, for 15 min.
      4. Centrifuge the suspension at room temperature, 27,000 x g for 15 min. Discard the supernatant.
      5. Resuspend the pellet in 10 ml water, centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant.
      6. Resuspend the pellet in 10 ml acetone, centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant very carefully because the pellet is very loose.
      7. Resuspend the pellet in 10 ml water, centrifuge the suspension at room temperature, 27,000 x g for 15 min in the Sorvall centrifuge. Discard the supernatant. Repeat this step for another 5 times.
      8. Resuspend the pellet in 2 ml water.
      9. Test the CW suspension for endotoxin by PierceTM LAL Chromogenic Endotoxin Quantitation Kit according to the manufacturer’s instruction.
      10. Record the weight of the microcentrifuge tubes, and aliquot the CW suspension into the tubes.
      11. Lyophilize the CW suspension in a Speed-vac.
      12. Record the weight of the tube containing lyophilized CW. Subtract the weight of the empty tube to obtain mass of CW. Reconstitute the dried CW by adding 348 μl H2O per 46 mg CW material, which results in CW stock equivalent to 106 cfu/µl. Store the CW stocks at 4 °C.
      13. The composition of this CW material has been described (Holtje et al., 1975).
        Notes: The CW stock will have a ‘milky’ appearance (Figure 2).


        Figure 2. Final appearance of purified pneumococcal CW

    6. FITC labeling of CW
      1. Pipet the desired amount (100-200 μl depending on the number of mice) of CW from the CW stock into a fresh microcentrifuge tube.
      2. Sonicate the CW in a water-bath sonicator for 30 min.
      3. Resuspend the sonicated CW in 500 μl FITC solution (1 mg/ml, dissolved in sterile carbonate buffer; see Recipes). Protect the preparation from light by wrapping in aluminum foil. Incubate the suspension at room temperature for 1 h.
      4. Centrifuge the FITC-labeled CW at room temperature, 17,000 x g for 2 min in a microcentrifuge. Discard the supernatant, and resuspend in 1 ml DPBS. Repeat this step 2 times.
      5. Resuspend the FITC-labeled CW pellet in DPBS to a concentration of 1 x 106 bacterial equivalents/μl.


        Figure 3. Final appearance of FITC labeled pneumococcal CW

  2. Detection of CW distribution in placenta and fetal tissues
    All experiments should be performed in compliance with national and institutional guidelines (for example, National Institute of Health).
    1. General description
      To study CW distribution to the placenta and fetus in mice, FITC labeled CW is injected intravenously into gestating mice. Male and female mice are purchased and bred in the animal facility on-site. Healthy mice at a body weight of 15-20 g and no signs of dehydration or illness are anesthetized by isofluorane. The mice are kept under isofluorane throughout the duration of the procedure (~5-10 min). Mice recover from the 10 min procedure in ~15-20 min.
    2. CW injection of the mouse
      1. Personnel wear shoe covers, gown, hat, mask upon entering the surgical suite.
      2. Pregnancy is dated by ultrasound by a trained animal husbandry technician to target E10-E15 dams. Unless otherwise noted (such as knockout mice), C57Bl6 mice are used.
      3. In the surgical suite, the mouse is warmed on a heating pad 10-15 min before injection to dilate blood vessels. The mouse is then restrained with tail access.
      4. Bacterial equivalents of 2 x 107 of FITC CW (20 μl of the FITC CW preparation) is diluted into a total volume of 100 μl sterile PBS and injected into the tail vein. The injection is done using a 25 gauge needle.
      5. For recovery, the mice are moved to a recovery table and monitored by respiration rate, toe pinch for capillary refill time and a heating pad for maintaining body temp. They are monitored visually for at least 30 min post-operationally until they are moving freely in the cage to assure they can access water and food.
    3. Harvesting tissues at Embryonic Day E18-20
      1. Mice are euthanized and placenta and embryo heads are excised and fixed overnight at 4 °C in 4% paraformaldehyde (PFA) in a 50 ml conical tube.
      2. Drain the PFA and fill the tube with 30% sucrose. Tissue should initially float at the top of the tube.
      3. Keep tissue in sucrose at 4 °C for 3-5 days until tissue rests at or near the bottom of the tube.
      4. Remove tissue one at a time to a clean 10 cm dish. Cut with a razor blade according to the diagram in Figure 4 for either coronal or sagittal sectioning.
    4. Visualization of CW in the placenta and fetal tissues


      Figure 4. Cross sections of mouse brain. For sectioning, make incision along dotted line. Coronal section is preferred to remove cerebellum and create flat surface for embedding the cortex.

      1. Place in embedding mold with cut surface on bottom of mold. Add OCT compound slowly to cover, avoiding bubbles.
      2. Place on dry ice to freeze for 30 min.
      3. Wrap each mold in aluminum foil and label. Store at -80 °C.
      4. Cut 20 μm sections with a cryostat microtome using superfrost slides, 3-5 serial sections per slide.
      5. Once dried, apply a thin layer of Prolong Gold anti-fade mounting medium with DAPI and carefully cover with a glass coverslip to avoid introducing air bubbles.
      6. Allow slides to cure overnight at room temperature and analyze the following day with confocal microscope.
        Fluorescent images were acquired with an inverted confocal microscope (LSM510; Carl Zeiss MicroImaging, Inc.). Argon laser (excitation 488 nm) and a filter set to detect FITC emission (BP500 – 550) for imaging FITC; Chameleon laser (excitation 740 nm) and a filter set to detect DAPI emission (BP435 – 485) for imaging DAPI. Cells were observed using a 20x/0.75 Plan Neofluor objective with the confocal pinhole set to one Airy unit. Imaging parameters, such as gain and offset levels, and line averaging, are optimized to avoid oversaturation of pixels and to improve signal:noise ratio. Images were acquired using Zen 2008 software package (Carl Zeiss MicroImaging, Inc.). CW pieces per section were counted using the particle analysis tool in ImageJ (imagej.net/Particle_Analysis)

Data analysis



Figure 5. FITC CW in the fetal brain or placenta is documented and quantified by microscopy. Confocal images of CW distribution in placental and fetal brain tissue (arrows) are analyzed using ImageJ.

Three independent experiments are carried out for each treatment condition and the results are combined. Data are analyzed using Graphpad Prism (Graphpad) or any other statistical software with two-tailed t-test with Welch’s correction used for statistical analysis. For example, within 24 h of injection into the maternal circulation, CW crossed into the embryonic brain (Figure 5). A CW density of ~400 CW pieces/mm2 corresponds to 4 x 105 CW pieces per entire cortical plate indicating that ~2% of the inoculum into mother trafficked to the fetal brain.

Recipes

  1. Solutions used to treat CW during purification
    1. 50 mM Tris-HCl, pH 7.0 (7.88 g/L Trizma HCl)
    2. 5% (v/v) SDS (50 g/L)
    3. 1 M NaCl (58.44 g/L)
    4. 100 mM Tris, pH 7.5 (12.11 g/L Tris-base)
    5. 1 M MgSO4 (246.47 g/L)
    6. 1 M CaCl2 (147 g/L)
    7. 1% (v/v) SDS (10 g/L)
    8. 8 M LiCl (339.12 g/L)
    9. 100 mM EDTA (37.22 g/L)
    10. 50 mM carbonate buffer, pH 9.2 (5.5 ml 0.1 M Na2CO3, 34 ml 0.1 M NaHCO3, 60.5 ml Milli-Q H2O)

  2. Preparation of C+Y pneumococcal growth medium components
    1. ‘3 in 1’ salts 
      50 g MgCl2·6H2O
      0·25 g CaCl2 anhydrous
      0·1 ml of 1 M MnSO4 solution
      Dissolve in final volume of 500 ml with Milli-Q water
    2. 20% glucose
      100 g/500 ml Milli-Q H2O (dissolve 100 g glucose in 500 ml Milli-Q H2O)
    3. 50% sucrose
      50 g/100 ml Milli-Q H2O
    4. Adenosine (2 mg/ml)
      1 g/500 ml Milli-Q H2O
    5. Uridine (2 mg/ml)
      1 g/500 ml Milli-Q H2O
    6. Glutamine (1 mg/ml)
      0.5 g/500 ml Milli-Q H2O
    7. Adams I
      30 mg Nicotinic acid (Niacin)
      35 mg (B6) pyridoxine HCl
      120 mg Ca-pantothenate (D-pantothenic acid)
      32 mg thiamine HCl
      14 mg riboflavin
      Dissolve in 200 ml final volume Milli-Q H2O
      Add 0·06 ml of biotin (0·5 mg/ml stock, add 1 drop of 1 N NaOH to go into solution)
      Store covered with foil
    8. Adams II
      50 mg FeSO4·7H2O
      50 mg CuSO4·5H2O
      50 mg ZnSO4·7H2O
      20 mg MnCl2·4H2O
      1 ml HCl
      Add Milli-Q H2O up to 100 ml final volume
    9. Prepare 2% pyruvate
      2 g pyruvic acid
      100 ml Milli-Q H2O
    10. 1 M KH2PO4 
      13·6 g/100 ml Milli-Q H2O
      Autoclave at 121 °C for 15 min
    11. 1 M K2HPO4 
      174·2 g/1 L Milli-Q H2O
      Autoclave at 121 °C for 15 min
    12. 5% yeast extract 
      5 g/100 ml Milli-Q H2O
      Autoclave at 121 °C for 15 min
      Notes:
      1. Filter sterilize all components of C+Y and store at 4 °C.
      2. Shelf life for components of C+Y is 3 months.

  3. Media preparation
    1. Prepare PreC media
      Dissolve the following in 800 ml Milli-Q water
      4.83 g sodium acetate
      20 g casamino acids
      20 mg L-tryptophan
      200 mg L-cysteine HCl
      Adjust pH to 7.4-7.6 by adding 10 N NaOH (~14 drops with Pasteur pipet)
      Stir well (30-60 min) at room temperature
      Increase final volume to 4 L with Milli-Q water
      Aliquot 400 ml into 10 x 500 ml Erlenmeyer flasks
      Autoclave for 30 min at 125 °C
    2. Prepare supplement
      60 ml ‘3 in 1’ salts
      120 ml 20% glucose
      6 ml 50% sucrose
      120 ml adenosine (2 mg/ml)
      120 ml uridine (2 mg/ml)
      Mix in a beaker and store in 200 ml portions
    3. Prepare Adams III
      800 mg asparagine
      80 mg choline chloride
      0.64 ml CaCl2 (1% solution)
      64 ml Adams I
      16 ml Adams II
      360 ml Milli-Q H2O
      Mix and store in foil covered flasks
    4. Prepare 1 M potassium phosphate buffer, pH 8.0
      26.5 ml of KH2PO4 (1 M)
      473 ml of KH2PO4 (1 M)
    5. Mix the following amounts of previously described components for C+Y
      400 ml PreC
      13 ml supplement
      10 ml glutamine (1 mg/ml)
      10 ml Adams III
      5 ml 2% pyruvate
      15 ml 1 M phosphate buffer
      9 ml 5% yeast extract
      Filter sterilize and store at 4 °C**
      Warm to 37 °C before inoculating with bacteria
      Note: **Shelf life for prepared C+Y is 1 month.
  4. Acknowledgments

    This work was supported by NIAID 27913 and ALSAC.

    References

    1. Braun, J. S., Novak, R., Herzoq, K. H., Bodmer, S. M., Cleveland, J. L. and Tuomanen, E. I. (1999). Neuroprotection by a caspase inhibitor in acute bacterial meningitis. Nat Med 5:298-302.
    2. Fillon, S., Soulis, K., Rajasekaran, S., Benedict-Hamilton, H., Radin, J.N., Orihuela, C.J., Kasmi, K. C., Murti, G., Kaushal, D., Gaber, M. W., Weber, J. R., Murray, P. J. and Tuomanen, E. I. (2006). Platelet-activating factor receptor and innate immunity: uptake of gram-positive bacterial CW into host cells and cell-specific pathophysiology. J Immunol 177:6182-6191.
    3. Holtje, J. V. and Tomasz, A. (1975). Specific recognition of choline residues in the cell wall teichoic acid by the N-acetylmuramyl-L-alanine amidase of Pneumococcus. J Biol Chem 250(15): 6072-6076.
    4. Humann, J., Mann, B., Gao, G., Moresco, P., Ramahi, J., Loh, L. N., Farr, A., Hu, Y., Durick-Eder, K., Fillon, S. A., Smeyne, R. J. and Tuomanen, E. I. (2016). Bacterial peptidoglycan traverses the placenta to induce fetal neuroproliferation and aberrant postnatal behavior. Cell Host Microbe 19(6): 901.
    5. Orihuela, C. J., Fillon, S., Smithsielicki, S. H., Kasmi, K. C. E., Gao, G., Soulis, K., Patil, A., Murray, J. P. and Tuomanen, I. E. (2006). Cell wall-mediated neuronal damage in early sepsis. Infect Immun 74:3783-3789.
    6. Tuomanen, E., Liu, H., Hengstler, B., Zak, O. and Tomasz, A. (1985a). The induction of meningeal inflammation by components of the pneumococcal cell wall. J Infect Dis 151(5): 859-868.
    7. Tuomanen, E., Tomasz, A., Hengstler, B. and Zak, O. (1985b). The relative role of bacterial cell wall and capsule in the induction of inflammation in pneumococcal meningitis. J Infect Dis 151(3): 535-540.
    8. Yoshimura, A., Lien, E., Ingalls, R. R., Tuomanen, E., Dziarski, R. and Golenbock, D. (1999). Cutting edge: recognition of Gram-positive bacterial cell wall components by the innate immune system occurs via Toll-like receptor 2. J Immunol 163(1): 1-5.

简介

细胞壁是所有革兰氏阳性菌表面上的复杂生物聚合物。在感染期间,细胞壁被先天免疫受体Toll样受体2识别,引起强烈的炎症和组织损伤。在动物模型中,从血流到体内许多器官(包括脑,心脏,胎盘和胎儿)的细胞壁运输。该协议描述了如何从肺炎链球菌制备纯化的细胞壁,检测其在动物组织中的分布,并且使用胎盘和胎脑作为实例研究组织反应。
关键词: 细胞壁,肽聚糖,细菌炎症,神经增殖,胎儿神经发生,胎盘运输,Toll样受体2配体,肺炎链球菌

/strong>宿主对感染的反应涉及许多细菌组分的识别,包括细胞壁(CW),一种形成所有革兰氏阳性细菌表面的复合大分子。革兰氏阳性细菌的CW由肽聚糖和磷壁酸的共价网络形成。肺炎链球菌是肺炎,败血症和脑膜炎的主要原因,已经成为研究对包括CW在内的革兰氏阳性细菌感染的先天免疫反应的重要模式生物体。  当肺炎链球菌(肺炎球菌)感染时,CW成分在生长期或抗生素诱导的死亡期间从细菌释放,它在血流中循环并穿过细胞屏障,包括胎盘和血脑屏障。 CW组分具有等于或大于完整细菌的炎性活性(Tuomanen等人,1985a和1985b)。 CW可以被视为内毒素的革兰氏阳性等价物。在感染期间释放的大量CW片段通过激活先天免疫受体Toll样受体2(TLR2)来极大地刺激宿主炎症反应(Yoshimura等人,1999)。反应取决于感染的器官而不同:产后脑经历凋亡,瘢痕形成在心脏中占优势,并且胎儿脑逃避损伤,显示显着的神经增生(Orihuela等人,2006; Braun等人。,1999; Fillon et al ,2006; Humann et al ,2016)。
   该方案描述了如何从肺炎链球菌中制备纯化的CW(Tuomanen等人,1985b),并且在静脉内注射后遵循其在小鼠中的分布,聚焦于胎盘和胎儿脑作为实例(Humann等人,2016)。该模型产生用于研究对CW组分的组织反应的器官的组织病理切片。我们的模型的重点肺炎球菌CW源于其良好描述的炎症和损伤在许多器官,其广泛已知的化学和作为经典的TLR2病原体相关的分子模式的识别作用。


关键字:细胞壁, 肽聚糖, 细菌炎症, 神经增殖, 胎儿神经发生, 胎盘运输, Toll样受体2配体, 肺炎链球菌

材料和试剂

  1. 0.22μm瓶顶过滤器(Corning,目录号:431096)
  2. 玻璃管(Thermo Fisher Scientific,Fisher Scientific,目录号:14-961-32)
  3. 1,000ml离心瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3120-1000)
  4. 30ml离心瓶(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3119-0050)
  5. 血清移液管,1例5 ml,10 ml,25 ml
  6. 微量离心管(Eppendorf,目录号:022364111)
  7. 铝箔(Thermo Fisher Scientific,Fisher Scientific,目录号:01-213-100)
  8. 25号针(BD,目录号:305122)
  9. 50ml聚丙烯管(SARSTEDT,目录号:62.547.254)
  10. 培养皿(Thermo Fisher Scientific,Fisher Scientific,目录号:FB0875712)
  11. 小电剃刀刮动物
  12. 超级微滑块(VWR,目录号:48311-703)
  13. 保费盖玻璃(Thermo Fisher Scientific,Fisher Scientific,目录号:12-548-5P)
  14. 磁力搅拌棒
  15. 接种环(Thermo Fisher Scientific,Fisher Scientific,目录号:22-363-595)
  16. 1ml注射器(BD,目录号:309628)
  17. 酸洗的106μm玻璃珠(Sigma-Aldrich,目录号:G4649)
  18. 用于组织学的塑料包埋模具(Polysciences,目录号:18646D-1)
  19. 肺炎链球菌菌株-CW更容易从未包封的菌株如R6(ATCC,目录号:BAA-255)中纯化。
  20. C57B16小鼠,雄性和雌性的混合物(THE JACKSON LABORATORY,目录号:000664)
  21. 水(Sigma-Aldrich,目录号:W3500-1L)
  22. 超纯水(例如:Milli-Q,EMD Millipore)
  23. 胰蛋白酶大豆琼脂(TSA)(EMD Millipore,目录号:105458)
  24. 无菌脱纤维绵羊血(i-Tek Medical Technologies,目录号:103-100-3)
  25. 甘油
  26. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4390)
    注意:此产品已停产。
  27. 氯化钠(NaCl)(Sigma-Aldrich,目录号:71382)
  28. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:208094)
  29. DNase I(Sigma-Aldrich,目录号:DN25-10MG)
  30. RNA酶A(Sigma-Aldrich,目录号:R6513-10MG)
  31. 氯化钙脱水物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C-3881)
  32. 胰蛋白酶(AMRESCO,目录号:M150)
  33. α-淀粉酶(Sigma-Aldrich,目录号:A3176)
  34. 氯化锂(LiCl)(Sigma-Aldrich,目录号:L9650)
  35. 乙二胺四乙酸二钠二水合物(EDTA)(Sigma-Aldrich,目录号:E5134)
  36. 丙酮(Thermo Fisher Scientific,Fisher Scientific,目录号:A946-4)
  37. Pierce LAL生色内毒素定量试剂盒(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:88282)
  38. 荧光素异硫氰酸酯异构体I(FITC)(Sigma-Aldrich,目录号:F7250)
  39. Dulbecco's磷酸盐缓冲盐水(DPBS)(Mediatech,目录号:21-030-CV)
  40. 多聚甲醛,16%w/v(Alfa Aesar,目录号:43368)
  41. 蔗糖(Sigma-Aldrich,目录号:S9378)
  42. Tissue Tek OCT复合物(SAKURA FINETEK USA,目录号:4583)
  43. 使用DAPI的Prolong Gold Antifade(Thermo Fisher Scientific,Molecular Probes TM ,目录号:P36931)
  44. HCl(Sigma-Aldrich,目录号:T5941)
  45. Tris-base(Sigma-Aldrich,目录号:1070897600)
  46. 盐酸(HCl)(Sigma-Aldrich,目录号:320331-500ml)
  47. 碳酸钠(Na 2 CO 3)(Sigma-Aldrich,目录号:451614)
  48. 碳酸氢钠(NaHCO 3)(Sigma-Aldrich,目录号:S-6014
  49. 氯化镁六水合物(MgCl 2)(Sigma-Aldrich,目录号:M0250)
  50. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C1016)
  51. MnSO 4·一水合物(Sigma-Aldrich,目录号:M7634)
  52. 葡萄糖(Sigma-Aldrich,目录号:G7528)
  53. 腺苷(Oakwood Products,目录号:093333)
  54. 尿苷(EMD Millipore,目录号:6680)
  55. 谷氨酰胺(Sigma-Aldrich,目录号:G8540)
  56. 烟酸(Sigma-Aldrich,目录号:N4126)
  57. (B6)盐酸吡哆醇(Sigma-Aldrich,目录号:P9755)
  58. 泛酸钙(D-泛酸)(Sigma-Aldrich,目录号:P3161)
    注意:此产品已停产。
  59. 盐酸硫胺素(Sigma-Aldrich,目录号:5871-100GM)
  60. 核黄素(Sigma-Aldrich,目录号:R4500)
  61. 生物素(Sigma-Aldrich,目录号:B4639)
  62. 10N NaOH溶液(Thermo Fisher Scientific,Fisher Scientific,目录号:SS255-1)
  63. 硫酸亚铁七水合物(FeSO 4·7H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:I146-500)
  64. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Sigma-Aldrich,目录号:C8027)
  65. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z4750)
  66. 氯化锰四水合物(MnCl 2·4H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:M87-500)
  67. 丙酮酸(Sigma-Aldrich,目录号:107360-25g)
  68. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  69. 磷酸氢二钾(K 2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  70. 酵母提取物(BD,Bacto TM ,目录号:212750)
  71. 无水乙酸钠(Sigma-Aldrich,目录号:S8625)
  72. 酪氨酸氨基酸技术(BD,Bacto TM ,目录号:223120)
  73. L-色氨酸(Sigma-Aldrich,目录号:T8941)
  74. L-半胱氨酸HCL(Thermo Fisher Scientific,Fisher Scientific,目录号:BP376-100)
  75. 天冬酰胺(Sigma-Aldrich,目录号:A4284)
  76. 氯化胆碱(Sigma-Aldrich,目录号:C7527)
  77. 干冰
  78. 在净化过程中用于治疗CW的溶液
    1. 50 mM Tris-HCl,pH 7.0(参见配方)
    2. 5%(v/v)SDS(见配方)
    3. 1 M NaCl(见配方)
    4. 100mM Tris,pH 7.5(参见Recipes)
    5. 1M MgSO 4(参见配方)
    6. 1 M CaCl 2(参见配方)
    7. 1%(v/v)SDS(见配方)
    8. 8 M LiCl(见配方)
    9. 100mM EDTA(参见配方)
    10. 碳酸盐缓冲液,pH 9.2(参见配方)
  79. 准备C + Y组件
    1. '3合1'盐(参见食谱)
    2. 20%葡萄糖(请参阅食谱)
    3. 50%蔗糖(请参阅食谱)
    4. 腺苷(2 mg/ml)(请参阅食谱) br />
    5. 尿苷(2 mg/ml)(见食谱) br />
    6. 谷氨酰胺(1 mg/ml)(请参阅食谱) br />
    7. Adams I(请参阅食谱)
    8. Adams II(请参阅配方)
    9. 2%丙酮酸(请参阅食谱)
    10. 1 M KH 2 sub> (请参阅配方)
    11. 1 MK HPO 4 (见配方)
    12. 5%酵母提取物(请参阅食谱
  80. 媒体准备
    1. PreC媒体(请参阅配方)
    2. 补充(见配方)
    3. Adams III(请参阅食谱)
    4. 1 M磷酸钾缓冲液(见配方) >
    5. C + Y medium(请参阅食谱)

设备

  1. 37℃CO 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:3110)中。
  2. 台式微量离心机(Eppendorf,型号:5417C)
  3. 1,000 ml锥形瓶
  4. 4,000ml锥形瓶
  5. Sorvall离心机RC 5C Plus和适用于离心管或瓶子的转子
  6. 500 ml烧杯
  7. 搅拌加热板
  8. 涡流搅拌器
  9. Speed-vac(Savant,型号:SC110A)
  10. 水浴超声仪(Thermo Fisher Scientific,Fisher Scientific,型号:FS20)
  11. 加热垫
  12. 低温切片机(Microme,型号:HM505E)
  13. Zeiss LSM 510 NLO Meta共聚焦显微镜
  14. 分光光度计(Turner,型号:340)
  15. Spectra MAX340酶标仪在405 nm处测量吸光度(Molecular Device,型号:Spectra MAX340

软件

  1. Zen 2008软件包(Carl Zeiss MicroImaging,Inc。)
  2. ImageJ( imagej.net/Particle_Analysis
  3. Graphpad Prism(图形板)

程序

  1. 肺炎球菌CW:肽聚糖 - 磷壁酸复合物的制备
    注意:
    1. 制备所有溶液,并用蒸馏水洗涤(使用来自Sigma [#W3500]的细胞培养蒸馏水,具有低内毒素浓度)。
    2. 在使用前过滤所有溶液(过滤器0.22μm)。
    3. 最好使用新的干净的玻璃器皿和设备,以避免任何内毒素(LPS)污染。
    4. 在大规模生长之前,检查血小板上的肺炎球菌菌种,无抗生素以确认没有污染。
    5. 这里描述的方案使用C + Y培养基(参见Recipes)作为部分确定的生长培养基。其它市售可得的培养基也是合适的,但是具有更多样的污染组分,例如需要除去以获得纯制剂的内毒素。
    1. 制备肺炎链球菌的原液
      CW可以从任何肺炎球菌菌株纯化。然而,对于未封装的菌株如R6(Tuomanen等人,1985b),该方法是最成功的。
      1. 在含有3%(v/v)无菌去纤维蛋白的绵羊血液的TSA琼脂(每500ml水20g TSA,高压灭菌器)上的条纹菌株,并将板在37℃,5%CO 2下孵育16小时-18小时(图1)。在血琼脂上的生长允许溶血的可视化,这是S的特征。肺炎。


        图1.未封装的菌株R6在TSA血琼脂上的过夜生长。肺炎球菌的纯培养物是步骤A1b中制备的种子的细菌的来源。

      2. 用接种环将所有菌落从琼脂平板上刮下,并接种到试管中的10ml C + Y培养基中。在37℃,5%CO 2孵育培养物。当培养物在620nm(OD 620)的光密度达到0.4时,通过在微量离心机中在1500×g,4℃下离心10分钟收获培养物。
      3. 小心地取出培养物上清液。将细菌沉淀重悬于5ml冷冻培养基(2.5 ml C + Y培养基和2.5 ml 80%[v/v]甘油)中,制成1 ml等分试样。将股票储存在-80°C的冷冻管中。这些原液含有约1×10 7 cfu/ml。
    2. 大规模培养肺炎球菌
      1. 准备10升C + Y培养基(见Recipes),并在三个4,000毫升锥形瓶(每瓶3,300毫升)中等分。在37℃,5%CO 2培养基中培养过夜,以确认无污染。
      2. 将1ml /烧瓶的肺炎球菌原种(10 7 cfu/ml)接种到培养基中。在37℃,5%CO 2下孵育培养物,不摇动 注意:
        1. 为了估计烧瓶中的细菌生长,当培养物开始出现混浊时,将一个10ml等分试样(使用无菌血清移液管)从烧瓶转移到无菌玻璃管中。读取此转移培养物在620nm的培养物浊度(OD 620)。保持细菌培养在玻璃管,并孵育在培养箱与烧瓶培养。随时间监测玻璃管培养物的OD <620>。
        2. 在烧瓶中的OD <620>将比在玻璃管中的OD620高大约0.1。
      3. 当烧瓶中的OD 620达到0.7-0.8时,从培养箱中取出烧瓶并将其浸入冰中15分钟。每2分钟旋转烧瓶以快速冷却培养物
      4. 将冷却的培养物倒入预冷的1,000ml离心瓶中,并通过在Sorvall离心机中在4℃,4,000xg离心10分钟收获培养物。
      5. 通过倾倒小心倾析上清液。颗粒可以储存在-80°C直到使用
    3. 细菌细胞的初步收获和机械破碎
      注意:粗制CW的操作定义是可以通过在SDS中煮沸而精制的物质。
      1. 用10 ml /瓶冰冷的50 mM Tris-HCl,pH 7.0重悬每个沉淀(见Recipes)。 注意:尽快执行此步骤以避免细菌自溶。
      2. 在500ml烧杯中在热板上煮沸200ml 5%(v/v)SDS(参见Recipes)。仔细监测温度控制,以防止沸腾在烧杯的边缘。
      3. 合并重悬的细菌沉淀并将细菌悬浮液缓慢加入沸腾的SDS中。
      4. 将悬浮液煮沸15分钟,并冷却至室温。
      5. 将煮沸的悬浮液等分到30ml离心管中。在Sorvall离心机中在室温下将悬浮液12,000×g离心10分钟。小心弃去上清液,避免移出沉淀。
      6. 将沉淀重悬在20ml /管1M NaCl中(见Recipes)。在Sorvall离心机中在室温下将悬浮液12,000×g离心10分钟。轻轻小心地弃去上清液。重复此步骤另外2次。
      7. 将沉淀重悬在20ml /管水中。在Sorvall离心机中在室温下将悬浮液12,000×g离心10分钟。轻轻小心地弃去上清液。重复此步骤另外7次,以去除洗涤剂。
      8. 将沉淀重悬于2 ml水中,加入等体积的玻璃珠。
      9. 涡旋球粒玻璃珠悬浮液在4°C,最大速度16-18小时。
    4. 消除DNA,RNA和蛋白质
      1. 通过在Sorvall离心机中在室温下离心5,000×10 5分钟收获断裂的CWs 10分钟。
      2. 小心地将上清液(包含CW)转移到新鲜的30ml离心管中
      3. 向沉淀中加入10ml水并剧烈涡旋。在室温下孵育悬浮液20分钟,通过沉降分离上清液。然后,用无菌血清移液管小心去除上清液。在步骤A4b中将上清液转移到离心管中。重复此步骤至少5次,直到上清液澄清
      4. 在Sorvall离心机中在室温下离心含有CW的上清液27,000×g 15分钟。
      5. 将上清液轻轻弃去,小心地将沉淀重悬于10ml 100mM Tris,pH7.5中(参见Recipes)。
      6. 将0.2ml 1M MgSO 4(参见Recipes)加入悬浮液中,使最终浓度为20mM。
      7. 加入10μlDNase I和50μlRNase A到悬浮液中,最终浓度分别为10和50μg/ml。在37℃下孵育悬浮液2小时
      8. 将0.1ml的1M CaCl 2(参见Recipes)加入悬浮液中,使最终浓度为10mM。
      9. 加入0.1ml胰蛋白酶(10mg/ml)使终浓度为100μg/ml和0.3U的α-淀粉酶。
      10. 孵育悬浮液在37℃下12-15小时。
    5. 去除非共价加合物和污染内毒素
      1. 将沉淀重悬在10ml 8M LiCl中(见Recipes),37℃孵育15分钟。
      2. 在Sorvall离心机中在室温下离心悬浮液27,000×g 15分钟。弃去上清液。
      3. 用10ml 100mM EDTA重悬沉淀(见Recipes),并在37℃下孵育15分钟。
      4. 将悬浮液在室温下,27,000×g离心15分钟。弃去上清液。
      5. 将沉淀重悬在10ml水中,在Sorvall离心机中在室温下离心悬浮液27,000×g 15分钟。弃去上清液。
      6. 将沉淀重悬在10ml丙酮中,在Sorvall离心机中在室温下离心悬浮液27,000×g 15分钟。丢弃上清液非常仔细,因为颗粒是非常松散的
      7. 将沉淀重悬在10ml水中,在Sorvall离心机中在室温下离心悬浮液27,000×g 15分钟。弃去上清液。重复此步骤5次。
      8. 将沉淀重悬于2ml水中。
      9. 根据制造商的说明,通过Pierce TM LAL生色内毒素定量试剂盒测试CW悬浮液的内毒素。
      10. 记录微量离心管的重量,并将CW悬浮液分装到试管中
      11. 在Speed-vac中冻干CW悬浮液。
      12. 记录含有冻干CW的管的重量。减去空管的重量以获得CW的质量。通过每46mg CW材料加入348μlH 2 O来重建干燥的CW,这导致CW原料相当于10 6 cfu /μl。将CW原液储存在4°C。
      13. 已经描述了这种CW材料的组成(Holtje等人,1975)。
        注意:CW股票将有一个"乳白色"外观(图2)。


        图2.纯化的肺炎球菌CW的最终外观

    6. FITC标签
      1. 将所需的量(100-200μl,取决于小鼠的数量)从CW原液转移到新鲜的微量离心管中。
      2. 在水浴超声仪中超声处理CW 30分钟。
      3. 将超声处理的CW在500μlFITC溶液(1mg/ml,溶解于无菌碳酸盐缓冲液中;参见Recipes)中重悬。通过包装在铝箔中保护制剂免受光照。将悬浮液在室温下孵育1小时
      4. 在室温下将FITC标记的CW离心,在微量离心机中17,000×g离心2分钟。弃去上清液,并重悬于1ml DPBS中。重复此步骤2次。
      5. 将在DPBS中的FITC标记的CW沉淀物重悬浮至浓度为1×10 6个细菌当量/μl。


        图3. FITC标记的肺炎球菌CW 的最终外观

  2. 胎盘和胎儿组织中CW分布的检测
    所有实验应按照国家和机构指南(例如,国家卫生研究所)进行。
    1. 一般说明
      为了研究CW分布到小鼠的胎盘和胎儿,将FITC标记的CW静脉内注射到妊娠小鼠中。购买雄性和雌性小鼠,并在动物设施中就地繁殖。通过异氟烷麻醉体重15-20g并且没有脱水或疾病迹象的健康小鼠。在整个操作过程中将小鼠保持在异氟烷下(约5-10分钟)。小鼠在〜15-20分钟内从10分钟程序恢复。
    2. CW注射的小鼠
      1. 人员在进入手术室时穿鞋套,长袍,帽子,面罩
      2. 怀孕是由训练有素的畜牧技术人员用超声波进行定位以瞄准E10-E15大坝。除非另有说明(例如敲除小鼠),使用C57B16小鼠
      3. 在手术套件中,在注射前10-15分钟,在加热垫上使小鼠温热以扩张血管。然后用尾部访问限制鼠标。
      4. 将2×10 7个细菌当量的FITC CW(20μl的FITC CW制备物)稀释到总体积为100μl的无菌PBS中,并注射到尾静脉中。使用25号针进行注射。
      5. 为了恢复,将小鼠移至恢复表并通过呼吸速率,用于毛细血管再充填时间的趾压缩和用于维持体温的加热垫进行监测。它们在操作后直观地监测至少30分钟,直到它们在笼中自由移动,以确保它们能够获得水和食物。
    3. 在胚胎日E18-20收获组织
      1. 将小鼠安乐死,切下胎盘和胚胎头,并在4℃下在50ml锥形管中的4%多聚甲醛(PFA)中固定过夜。
      2. 排空PFA并用30%蔗糖填充管。组织应该最初浮在管的顶部。
      3. 保持组织在蔗糖在4℃下3-5天,直到组织休息在管或其底部附近
      4. 一次移除一个组织到一个干净的10厘米的菜。按照图4所示的剃刀刀片切割冠状或矢状切片
    4. CW在胎盘和胎儿组织中的可视化

      图4.小鼠脑切片。切片,沿虚线切口。冠状切面是优选的,以去除小脑和创建平面嵌入皮层
      1. 放置在嵌入模具中,切割表面在模具底部。慢慢加入OCT化合物以避免气泡
      2. 置于干冰上冷冻30分钟。
      3. 包装每个模具在铝箔和标签。储存于-80℃。
      4. 用冷冻切片机切割20μm切片,使用超冷冻切片,每个切片3-5个连续切片
      5. 一旦干燥,涂上一层薄薄的Prolong Gold防褪色封装介质与DAPI和仔细盖玻璃盖玻片,以避免引入气泡。
      6. 让载玻片在室温下固化过夜,并用共聚焦显微镜分析第二天 使用倒置共焦显微镜(LSM510; Carl Zeiss MicroImaging,Inc。)获取荧光图像。氩激光器(激发488nm)和设置为检测FITC发射(BP500-550)以用于FITC成像的滤波器;变色龙激光器(激发740nm)和用于检测DAPI发射(BP435-485)的滤波器组以用于成像DAPI。使用具有设置为一个Airy单位的共焦针孔的20x/0.75 Plan Neofluor物镜观察细胞。优化成像参数,例如增益和偏移水平以及线平均,以避免像素过饱和并改善信号:噪声比。使用Zen 2008软件包(Carl Zeiss MicroImaging,Inc。)获取图像。使用ImageJ中的粒子分析工具(

数据分析


图5.通过显微镜记录和定量胎儿脑或胎盘中的FITC CW。使用ImageJ分析胎盘和胎儿脑组织中的CW分布的共焦图像(箭头)。

对每种处理条件进行三次独立实验,并将结果组合。使用Graphpad Prism(Graphpad)或具有双尾t检验的任何其它统计软件,用用于统计分析的Welch校正来分析数据。例如,在24小时内注射到母体循环中,CW跨越进入胚胎脑(图5)。约400个CW片/mm 2的CW密度对应于每个皮质板的4×10 5个CW片,表明约2%的接种物进入母亲被贩运到胎儿脑。

食谱

  1. 在净化过程中用于治疗CW的溶液
    1. 50mM Tris-HCl,pH7.0(7.88g/L Trizma HCl)
    2. 5%(v/v)SDS(50g/L)
    3. 1M NaCl(58.44g/L)
    4. 100mM Tris,pH7.5(12.11g/L Tris碱)
    5. 1 M MgSO 4(246.47g/L)
    6. 1 M CaCl 2(147g/L)
    7. 1%(v/v)SDS(10g/L)
    8. 8 M LiCl(339.12g/L)
    9. 100mM EDTA(37.22g/L)
    10. 50mM碳酸盐缓冲液,pH9.2 (5.5ml 0.1M Na 2 CO sub style ="color:#666666; font-family:Arial,Helvetica,sans-serif; text-align:justify; white-space:normal;"> 3 ,34ml 0.1M NaHCO #666666; font-family:Arial,Helvetica,sans-serif; text-align:justify; white-space:normal;"> 3 ,60.5ml Milli-Q H 2 O)

  2. 制备C + Y肺炎球菌生长培养基成分
    1. '3合1'盐
      50g MgCl 2·6H 2 O·dm / 0·25g CaCl 2水溶液
      0·1ml 1M MnSO 4溶液
      用Milli-Q水
      溶解于最终体积为500ml中
    2. 20%葡萄糖 100g/500ml Milli-Q H 2 O(在500ml Milli-Q H 2 O中溶解100g葡萄糖)。
    3. 50%蔗糖 50g/100ml Milli-Q H sub 2 O 2 /
    4. 腺苷(2mg/ml)
      1克/500毫升Milli-Q H sub 2 O 2 /
    5. 尿苷(2mg/ml)
      1克/500毫升Milli-Q H sub 2 O 2 /
    6. 6·谷氨酰胺(1 mg/ml)
      0·5g/500ml Milli-Q H 2 O·v/v
    7. 亚当斯我
      30mg烟酸(烟酸)
      35mg(B6)吡哆醇HCl 120毫克泛酸钙(D-泛酸)
      32mg硫胺素HCl 14mg核黄素
      溶解在200ml终体积Milli-Q H 2 O中 加入0. 06 ml生物素(0.5 mg/ml原液,加入1滴1N NaOH进入溶液)
      店铺覆盖有箔
    8. Adams II
      50mg FeSO 4·7H 2 O·h/v 50mg CuSO 4·5H 2 O·TiO 2 50mg ZnSO 4·7H 2 O·dm / 20mg MnCl 2·4H 2 O·dm/2 1 ml HCl
      添加Milli-Q H sub 2 O至最终体积为100 ml
    9. 准备2%丙酮酸
      2 g丙酮酸
      100ml Milli-Q H sub 2 O 3 /
    10. 1 M KH 2 PO 4
      13·6g/100ml Milli-Q H 2 O·
      121℃高压灭菌15分钟
    11. 1 M K 2 HPO 4
      174·2g/1L Milli-Q H 2 O·
      121℃高压灭菌15分钟
    12. 5%酵母提取物
      5g/100ml Milli-Q H sub 2 O 2 / 121℃高压灭菌15分钟
      注意:
      1. 对C + Y的所有组分进行过滤灭菌,并储存在4℃。
      2. C + Y组件的保质期为3个月。

  3. 媒体准备
    1. 准备PreC介质
      将以下物质溶于800ml Milli-Q水中
      4.83g乙酸钠 20克酪蛋白氨基酸
      20 mg L-色氨酸
      200 mg L-半胱氨酸HCL
      通过加入10N NaOH(用巴斯德吸管约14滴)将pH调节至7.4-7.6 在室温下搅拌(30-60分钟) 用Milli-Q水将最终体积增加到4 L 等分400毫升到10×500毫升锥形瓶中
      在125℃下高压灭菌30分钟
    2. 准备补充
      60ml 3'1'盐
      120ml 20%葡萄糖 6ml 50%蔗糖 120ml腺苷(2mg/ml)
      120ml尿苷(2mg/ml) 在烧杯中混合并储存在200ml份
    3. 准备亚当斯III
      800 mg天冬酰胺
      80 mg氯化胆碱
      0.64ml CaCl 2(1%溶液) 64 ml Adams I
      16 ml Adams II
      360ml Milli-Q H sub 2 O 2 / 混合并储存在带箔的烧瓶中
    4. 制备1M磷酸钾缓冲液,pH 8.0
      26.5ml KH 2 PO 4(1M)
      473ml KH 2 PO 4(1M)
    5. 混合以下量的前述组分用于C + Y
      400 ml PreC
      13 ml补充剂
      10ml谷氨酰胺(1mg/ml) 10ml Adams III
      5ml 2%丙酮酸 15ml 1M磷酸盐缓冲液 9ml 5%酵母提取物
      过滤灭菌并在4°C **
      保存 温育至37°C,然后接种细菌
      注意:**预备的C + Y的保质期为1个月。
  4. 致谢

    这项工作是由NIAID 27913和ALSAC支持的。

    参考文献

    1. Braun,JS,Novak,R.,Herzoq,KH,Bodmer,SM,Cleveland,JLand Tuomanen,EI(1999)。通过半胱天冬酶抑制剂在急性细菌性脑膜炎中的神经保护。 5 5:298-302。
    2. Fillon,S.,Soulis,K.,Rajasekaran,S.,Benedict-Hamilton,H.,Radin,JN,Orihuela,CJ,Kasmi,KC,Murti,G.,Kaushal,D.,Gaber,MW, JR,Murray,PJ和Tuomanen,EI(2006)。& 177:6182-6191。血小板活化因子受体和先天免疫:将革兰氏阳性细菌cw吸收到宿主细胞和细胞特异性病理生理学中。<="">
    3. 177:6182-6191。血小板活化因子受体和先天免疫:将革兰氏阳性细菌cw吸收到宿主细胞和细胞特异性病理生理学中。,JV和Tomasz,A。(1975)。  通过肺炎球菌的Nε - 乙酰胞壁酰-L-丙氨酸酰胺酶特异性识别细胞壁磷壁酸中的胆碱残基 J.Biol.Chem.250(15): 6072-6076。
    4. Humann,J.,Mann,B.,Gao,G.,Moresco,P.,Ramahi,J.,Loh,LN,Farr,A.,Hu,Y.,Durick-Eder,K.,Fillon, Smeyne,RJ和Tuomanen,EI(2016)。  细菌肽聚糖穿过胎盘以诱导胎儿神经增殖和异常的出生后行为。细胞宿主微生物 19(6):901。
    5. Orihuela,CJ,Fillon,S.,Smithsielicki,SH,Kasmi,KCE,Gao,G.,Soulis,K.,Patil,A.,Murray,JP和Tuomanen,IE(2006)。< a class = ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489725/"target ="_ blank">早期脓毒症中细胞壁介导的神经元损伤 Infect Immun 74:3783-3789。
    6. Tuomanen,E.,Liu,H.,Hengstler,B.,Zak,O.和Tomasz,A。(1985a)。  肺炎球菌细胞壁组分引起的脑膜炎症 151 Infect Dis 151(5):859- 868.
    7. Tuomanen,E.,Tomasz,A.,Hengstler,B。和Zak,O。(1985b)。  细菌细胞壁和胶囊在肺炎球菌性脑膜炎的诱导炎症中的相对作用。 Infect Dis 151(3):535-
    8. Yoshimura,A.,Lien,E.,Ingalls,RR,Tuomanen,E.,Dziarski,R。和Golenbock,D。(1999)。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mann, B., Loh, L. N., Gao, G. and Tuomanen, E. (2016). Preparation of Purified Gram-positive Bacterial Cell Wall and Detection in Placenta and Fetal Tissues. Bio-protocol 6(23): e2037. DOI: 10.21769/BioProtoc.2037.
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