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Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages
流式细胞术定量测定巨噬细胞中结核分枝杆菌对脂肪酸的摄取   

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eLIFE
Jun 2017

Abstract

Mycobacterium tuberculosis (Mtb) has evolved to assimilate fatty acids from its host. However, until recently, there was no reliable way to quantify fatty acid uptake by the bacteria during host cell infection. Here we describe a new method to quantify fatty acid uptake by intracellular bacilli. We infect macrophages with Mtb constitutively expressing mCherry and then metabolically label them with Bodipy-palmitate. Following the labeling procedure, we isolate Mtb-containing phagosomes on a sucrose cushion and disrupt the phagosomes with detergent. After extensive washes, the isolated bacteria are analyzed by flow cytometry to determine the level of Bodipy-palmitate signal associated with the bacteria. Using a Mtb mutant strain defective in fatty acid uptake in liquid culture we determined that this mutant assimilated 10-fold less Bodipy-palmitate than the wild type strain during infection in macrophages. This quantitative method of fatty acid uptake can be used to further identify pathways involved in lipid uptake by intracellular Mtb and possibly other bacteria.

Keywords: Fatty acid (脂肪酸), Uptake (摄取), Mycobacterium tuberculosis (结核分枝杆菌), Macrophage (巨噬细胞), Intracellular (细胞内), Mycobacteria (分枝杆菌), Bodipy (Bodipy)

Background

The ability of Mycobacterium tuberculosis (Mtb) to assimilate host-derived lipids (fatty acids and cholesterol) enables survival of the pathogen within its host (Russell et al., 2010; Lovewell et al., 2016). This idea is supported by upregulation of cholesterol and fatty acid metabolism-related genes by Mtb inside of macrophage, during mouse infection and in human lung tissue (Schnappinger et al., 2003; Rachman et al., 2006; Rohde et al., 2007; Fontán et al., 2008; Tailleux et al., 2008; Homolka et al., 2010; Rohde et al., 2012). The importance of cholesterol metabolism for Mtb during infection is supported by genetic studies and by identification of new antituberculosis compounds targeting cholesterol metabolism (Pandey and Sassetti, 2008; Wipperman et al., 2014; VanderVen et al., 2015). However, discovery of the specific machinery devoted to fatty acid uptake in Mtb is hindered not only by apparent redundancy of dedicated genes (Cole et al., 1998) but also by a paucity of reliable assays. Metabolic labeling with radioactive substrate has been the accepted approach to assess efficiency of fatty acid intake by bacterium in broth culture (Forrellad et al., 2014). This method is extremely challenging to apply for intracellular Mtb, and few groups have reported successful use of this approach (Daniel et al., 2011). Alternatively, TEM and staining with lipophilic dyes such as Bodipy 493/503, Nile Red, Oil Red can facilitate detection of lipids inside of mycobacteria during infection (Daniel et al., 2011; Podinovskaia et al., 2013; Caire-Brändli et al., 2014). However, neither of these labeling approaches directly assess the active import of substrate, but instead indicate the total amount of accumulated lipids. Therefore, a means of metabolic labeling of active fatty acid import by Mtb during infection that is tractable to downstream characterization is sorely needed.

Recently, it was shown that fluorescent fatty acids can be delivered effectively to intracellular bacteria and can be detected by microscopy (Podinovskaia et al., 2013). These observations led us to develop a new method of flow-cytometry based quantification of fluorescent fatty acid uptake by Mtb within its host cell. This assay allowed us to demonstrate that a ∆lucA::hyg Mtb strain is defective in fatty acid uptake during macrophage infection (Nazarova et al., 2017) (Figure 1). We believe that this methodology opens the door to genetic screens to further understand mechanisms involved in fatty acid uptake by Mtb and possibly other intracellular pathogens during infection in host cells.


Figure 1. Overview of the method used to quantify fatty acid uptake by M. tuberculosis during infection in macrophages

Materials and Reagents

  1. Serological pipets, 5 ml, 10 ml, 25 ml and 50 ml (Corning, Costar®, catalog numbers: 4487 , 4488 , 4489 and 4490 )
  2. Pipette tips, 20 μl, 100 μl, 200 μl, 1 ml (Biotix, Neptune®, catalog numbers: BT20 , BT100 , BT200 ; Thermo Fisher Scientific, Thermo Scientific, catalog number: 2079-HR )
  3. T25 (sterile 25 cm2 tissue culture flasks with filtered cap) (TPP, catalog number: 90026 )
  4. T75 or T150 (sterile 75 cm2 or 150 cm2 tissue culture flasks with filtered cap) (TPP, catalog numbers: 90076 or 90151 )
  5. Sterile 1 ml tuberculin syringe with 25 gauge needle (BD, catalog number: 309626 )
  6. Cell scrapers, 25 cm (SARSTEDT, catalog number: 83.1830 )
  7. 15 ml conical tube (SARSTEDT, catalog number: 62.554.100 )
  8. 50 ml conical tube (SARSTEDT, catalog number: 62.547.100 )
  9. Glasstic® slides with grids (KOVA International, catalog number: 87144 )
  10. 150 x 15 mm Petri dish (VWR, catalog number: 25384-326 )
  11. 2 ml screw-cap tubes (VWR, catalog number: 16466-042 )
  12. FACS tubes (VWR, catalog number: 60818-496 )
  13. 3 ml syringe (with Luer-LokTM tip, BD, catalog number: 309657 )
  14. Disposable plastic OD cuvettes with square caps (Fisher Scientific, FisherbrandTM catalog numbers: 14-955-128 and 14-385-999 )
  15. Mycobacterium tuberculosis constitutively expressing mCherry (pMV306 smyc’::mCherry (Kanr)) Source: Russell and VanderVen lab (Nazarova et al., 2017)
  16. Bone marrow-derived murine macrophages (BALB/c mice, THE JACKSON LABORATORIES, catalog number: 000651 )
    Note: Differentiation is described in detail in Nazarova et al. (2017).
  17. L cells (NCTC clone 929 [L cell, L-929, derivative of Strain L]) (ATCC, catalog number: CCL-1 )
  18. Phosphate-buffered saline (PBS) 1x (Mediatech, catalog number: 21-040 )
  19. Tyloxapol (Acros Organics, catalog number: 422370050 )
  20. Middlebrook 7H9 Broth Base (BD, DifcoTM, catalog number: 271310 )
  21. Distilled water
  22. Glycerol (VWR, catalog number: 97062-452 )
  23. Middlebrook OADC Enrichment (BD, BBLTM, catalog number: 212351 )
  24. Kanamycin sulfate (IBI Scientific, catalog number: IB02120 )
  25. Heat inactivated fetal bovine serum (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
    Note: Heat inactivated at 56 °C for 30 min.
  26. 200 mM L-glutamine (100x) (Mediatech, catalog number: 25-005 )
  27. 100 mM sodium pyruvate (Mediatech, catalog number: 25-000 )
  28. Penicillin-streptomycin solution 100x (Mediatech, catalog number: 30-002 )
  29. Dulbecco’s modification of Eagle’s medium (DMEM) 1x (Mediatech, catalog number: 10-017 )
  30. Glucose (dextrose) (Fisher Scientific, catalog number: BP350-1 )
  31. Bovine serum albumin (BSA) (Roche Diagnostics, catalog number: 03116964001 )
  32. Gelatin from cold water fish skin (Sigma-Aldrich, catalog number: G7765 )
  33. Calcium chloride (CaCl2) (Sigma-Aldrich, catalog number: C4901 )
  34. Magnesium chloride (MgCl2) (AMRESCO, catalog number: J364 )
  35. Fatty acid-free BSA (Roche Diagnostics, catalog number: 03117057001 )
  36. 100% ethanol, 200 Proof (Decon Labs, catalog number: V1016 )
  37. BodipyTM FL C16 (Bodipy-palmitate) (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3821 )
  38. BodipyTM FL C12 (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3822 )
  39. BodipyTM 558/568 C12 (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3835 )
  40. Potassium chloride (KCl) (Mallinckrodt Chemicals or Avantor Performance Materials, MACRON, catalog number: 6858-04 )
  41. Sucrose (Avantor Performance Materials, J.T. Baker, catalog number: 4097 )
  42. Ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) (Avantor Performance Materials, J.T. Baker, catalog number: L657 )
  43. HEPES (VWR, catalog number: BDH4162 )
  44. TweenTM 80 (Fisher Chemical, catalog number: T164-500 )
  45. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127 )
  46. 20% tyloxapol (see Recipes)
  47. 7H9 OADC media(see Recipes)
  48. Kanamycin 25 mg/ml (see Recipes)
  49. D10 media (see Recipes)
  50. L-cell conditioned media (see Recipes)
  51. BMDM media (see Recipes)
  52. Basal uptake buffer (BUB) (see Recipes)
  53. 1% fatty acid-free BSA (see Recipes)
  54. 4 mM Bodipy-palmitate (see Recipes)
  55. Cuvette buffer (see Recipes)
  56. Homogenization buffer (see Recipes)
  57. 20% Tween 80 (see Recipes)
  58. 0.05% tyloxapol (see Recipes)
  59. 4% PFA (see Recipes)

Equipment

  1. Pipette controller (Drummond Scientific, model: Pipet-Aid®, catalog number: 4-000-101 )
  2. Pipettors (MIDSCI, Alphapette, models: A-20 , A-100 , A-200 , A-1000 )
  3. 37 °C, 6% CO2 incubator
  4. 56 °C water bath
  5. Refrigerator (4 °C)
  6. -20 °C freezer
  7. Spectrophotometer compatible with absorbance measurements at 600 nm (Cole-Parmer, Jenway, model: 6320D )
  8. Beckman Allegra 6KR centrifuge (Beckman Coulter, model: Allegra® 6KR KneewellTM)
  9. GH-3.8 rotor (Beckman Coulter, model: GH-3.8 Rotor )
  10. Inverted microscope with enough resolution to detect cells of macrophage size (Olympus, model: IMT-2 with 10x objective)
  11. Beckman Microfuge® 18 Centrifuge (Beckman Coulter, model: Microfuge® 18 )
  12. Flow cytometer (BD, model: FACS LSR II )

Software

  1. FlowJo (BD)

Procedure

  1. Bacterial culture
    Mycobacterium tuberculosis strains are grown at 37 °C in 7H9 OADC media (see Recipes) in standing culture in T25 flasks until mid-log phase (OD600 ~0.6). Bacterial cultures are started from frozen stocks and maintained for no more than 5 passages. Constitutive expression of fluorescent protein by Mtb is required for further detection. We used Mtb Erdman strains with an integrated pMV306 plasmid expressing mCherry from smyc promoter (smyc’::mCherry). This plasmid confers kanamycin resistance, therefore growth media contains kanamycin at a final concentration of 25 μg/ml (see Recipes).
    Notes:
    1. Do not grow more than 10 ml of bacterial culture in a T25 flask to ensure sufficient amount of oxygen available to Mtb. To estimate timing for bacterial culture growth, consider that Mtb divides approximately once in two days when grown in standing culture. More details on bacterial culture growth for macrophage infection can be found in (Nazarova and Russell, 2017).
    2. We advise testing your wild type strain constitutively expressing fluorescent protein for ability to efficiently intake Bodipy-palmitate during macrophage infection by microscopy. We have noticed that in Mtb Erdman strain expression of mCherry under strong promoter (smyc’ or hsp60’) from replicating plasmid impacts Bodipy-palmitate uptake. However CDC1551 strain with the same plasmid demonstrated high level of fatty acid uptake. Additionally, care should be taken to determine by confocal microscopy if Bodipy-palmitate accumulates within bacteria versus on their surface.

  2. Macrophage isolation and culturing
    Macrophages of various origins can be used. We differentiated macrophages from bone marrow cells from BALB/c mice and maintained in BMDM media (see Recipes) with antibiotics at 37 °C and 6.0% CO2 for 10 days before infection.
    Infection
    1. The day before infection seed macrophages in antibiotic-free BMDM media into T150 tissue culture flasks (3 x 107 cells and 40-50 ml of BMDM media per flask) to obtain confluent monolayers. If macrophages are of limited numbers one T75 flask with 1 x 107 cells would provide sufficient results, however, two T150 flasks (6 x 107 cells) would give easily detectable pellet of bacteria at the end of the experiment.
    2. On the day of infection measure optical density of mid-log Mtb culture. Assuming that OD600 of 0.6 equals 108 bacteria/ml, centrifuge required amount of culture at 3,300 rpm (~2,500 x g) for 12 min in Beckman Allegra 6KR centrifuge, GH-3.8 rotor. We infect macrophages at MOI of 4:1, therefore to infect 6 x 107 cells we need 2.4 x 108 bacteria. To ensure that sufficient numbers of bacteria are pelleted we routinely centrifuge 3 ml of bacteria at OD600 = 0.6.
      Notes: As estimation of bacterial numbers may vary, one may want to determine it on their own. However, to achieve higher replicability in terms of MOI, we advise adhering to our estimation. More details on macrophage infection can be found in (Nazarova and Russell, 2017).
    3. Following centrifugation, remove the supernatant and resuspend the bacterial pellet in 1.5 ml of BUB (see Recipes). Pass bacterial suspension through a 1 ml tuberculin syringe with 25 gauge needle 12-20 times the same syringe and needle. Add 3.5 ml of BUB to the suspension to obtain 5 ml in total and mix thoroughly.
    4. Add 2.4 ml of bacterial suspension to each T150 flask containing 3 x 107 cells. Mix well, but gently. Incubate at 37 °C and 6% CO2 for 4 h.
    5. Remove extracellular bacteria by replacing with fresh pre-warmed antibiotic free BMDM media. Infected macrophages are maintained in BMDM medium at 37 °C and 6% CO2 for 3 days. Changing media on the second day of infection is optional.

    Labeling
    1. On the day of labeling (third day of infection) pre-warm sterile 1% fatty acid-free BSA (see Recipes) in PBS at 37 °C for 30-60 min. Add 4 mM stock of Bodipy-palmitate (see Recipes) to obtain 100 μM concentration. For labeling one T75 flask add 50 μl of 4 mM Bodipy-palmitate stock to 1.95 ml of 1% fatty acid-free BSA. Mix well by vortexing until the solution turns green. Keep at 37 °C and protect from light.
      Notes:
      1. The day of labeling can be chosen with consideration of the questions you are trying to address. We tested uptake of Bodipy-palmitate at various stages of macrophage infection, and noted that before the third day of infection the uptake levels are not sufficient to be detected.
      2. In addition to Bodipy-palmitate, we have tested Bodipy FL C12 and Bodipy 558/568 C12. Bodipy FL C12 accumulated in intracellular bacteria as efficiently as Bodipy-palmitate, while Bodipy 558/568 C12 was poorly assimilated by Mtb. We chose to use Bodipy-palmitate as fatty acids of the length (16 carbons) are more commonly found in the membranes of macrophages infected with Mtb.
    2. For labeling one T75 flask add 1.6 ml of 100 μM Bodipy-palmitate in 1% fatty acid-free BSA (from Step 1 of Labeling) to 18.4 ml of pre-warmed cuvette buffer (see Recipes) such that the final concentration of the labeled lipid is 8 μM, mix well. Use 15 ml for labeling one T75 flask, and 30 ml for labeling one T150 flask. Increase volumes in Steps 1 and 2 (Labeling) accordingly for larger infections.
    3. Remove media from infected macrophages and replace with cuvette buffer containing Bodipy-palmitate (volumes are described in Step 2 of Labeling). Incubate the infected macrophages with label at 37 °C and 6.0% CO2 for 1 h.
    4. After the 1 h labeling period, remove the cuvette buffer containing the Bodipy-palmitate and add fresh pre-warmed cuvette buffer without label for 1 h. Use 15 ml for one T75 flask, and 30 ml for one T150 flask. Proceed to the next step right after 1 h incubation without label.
      Note: Alternatively, Bodipy-palmitate in 1% fatty acid-free BSA can be added directly to infected macrophages cultured in BMDM media. Label chase can be performed in fresh pre-warmed BMDM medium as well. No cuvette buffer is needed in this case. From our experience, either way of labeling gives comparable results.

    Isolation of intracellular bacteria
    Note: This portion of the protocol is based on phagosome isolation described in (Pethe et al., 2004).
    1. Remove cuvette buffer from labeled infected cells, and quickly rinse with 10 ml of Homogenization buffer (see Recipes).
    2. Add 15 ml of ice-cold Homogenization buffer, incubate at 4 °C for 10-15 min, and harvest macrophages by scraping off from each flask with cell scrapers. Transfer the cells to a 50 ml conical tube and pellet the cells by centrifugation at 1,500 rpm (514 x g) for 10 min in Beckman Allegra 6KR centrifuge, GH-3.8 rotor. This and all the following centrifugations are done at 4 °C to block any further uptake of lipids by bacteria.
    3. Remove supernatant, resuspend pellet in 1.5 ml of Homogenization buffer by pipetting and transfer the cells to a 15 ml conical tube. Lyse the cells by 25-70 passages through a 1 ml tuberculin syringe with 25 gauge needle. Monitor cell lysis under a microscope using glasstic slides with 100 grids. Continue until > 95% of the cells are lysed, when intact cells are replaced by cell debris.
      Note: For safety purposes place glass slide with infected material in 150 x 15 mm Petri dish.
    4. Increase the volume to 5 ml by adding Homogenization buffer, resuspend well. Centrifuge cell lysate at 800 rpm (~146 x g) for 10 min in Beckman Allegra 6KR centrifuge, GH-3.8 rotor.
    5. Transfer the supernatant (suspension of phagosomes) into a new 15 ml conical tube. The pellet mainly consists of nuclei and unlysed cells and is discarded.
    6. To the suspension add 20% Tween 80 (see Recipes) to a final concentration of 0.1%, mix well and leave at 4 °C for 15 min to lyse Mtb containing vacuoles.
    7. Quickly agitate by shaking and isolate the bacteria by centrifugation at 2,500 rpm (1,430 x g) for 15 min in Beckman Allegra 6KR centrifuge, GH-3.8 rotor.
    8. Remove supernatant and resuspend the bacterial pellet in 10 ml of 0.05% tyloxapol in PBS (see Recipes). Centrifuge bacteria down at 3,300 rpm (~2,500 x g) for 15 min in Beckman Allegra 6KR centrifuge, GH-3.8 rotor.
    9. Optional: repeat the previous step to further remove labeled fatty acids adhered to bacterial cell surface.
      Note: Non-specific binding of bodipy-palmitate to the bacterial cell surface produces background signal. However, if tested strains/conditions produce a significant difference in specific uptake of label, this background noise wouldn’t impact results greatly.
    10. Remove supernatant and fix bacteria in 4% PFA (see Recipes) for 24 h in 2 ml screw-cap tube.
      Note: If you have flow cytometer in BSL3 facility, bacteria can be analyzed live immediately after isolation and without fixation.

Data analysis

Isolated bacteria should be analyzed within few days following collection, ideally the next day.

  1. Centrifuge fixed samples at 10,000 rpm (9,000 x g) for 5 min in Beckman Microfuge® 18 Centrifuge.
  2. Remove supernatant, resuspend pellet in 1-2 ml of 0.05% tyloxapol in PBS and transfer into FACS tube.
  3. Pass suspension through 1 ml tuberculin syringe with 25 gauge needle 12-20 times to obtain a single cell bacterial suspension.
  4. Analyze immediately on flow cytometer (BD FACS LSR II ) using described gating strategy (Figure 2). Select population of a medium size in forward and side scatter to exclude clumps and small debris, focus on mCherry(PE-Texas Red)-positive population (bacteria), and compare FITC signal from the Bodipy-palmitate between your samples. Since there is minimal overlap between mCherry and Bodipy signals, compensation is not needed. As a negative control use mCherry-positive bacteria not exposed to labeling.
    Notes:
    1. Bodipy-palmitate also accumulates in membranes of cell organelles, which are excluded from analysis, because they are mCherry-negative.
    2. Collect as many events as possible, minimum 50,000. For analysis in Figure 2 we collected 1,000,000 events.
  5. Quantify acquired data using FlowJo by determining mean fluorescence of Bodipy signal from mCherry-positive bacteria.


    Figure 2. Gating strategy for analysis of Bodipy-palmitate uptake by intracellular Mtb. The population of a medium size is selected in forward and side scatter, and analyzed further for level of mCherry signal. Bodipy palmitate signal is determined for the mCherry-positive population representing bacteria. Panel on the right is a representative of detected Bodipy-palmitate signal associated with three different strains: wild type (black), ∆lucA::hyg (red) and complemented strain (blue). Grey histogram represents mCherry-positive bacteria not exposed to labeling. (Adapted from Nazarova et al., 2017)

Recipes

  1. 20% tyloxapol (20 ml)
    1. Using 3 ml syringe add 4 ml of tyloxapol to 16 ml of distilled water in 50 ml conical tube
    2. Heat up at 56 °C, vortex occasionally, until tyloxapol goes into a viscous but clear solution
    3. Filter-sterilize (0.22 μm), store at room temperature for up to 12 months
  2. 7H9 OADC media (1 L)
    1. Dissolve 4.7 g 7H9 DifcoTM Middlebrook 7H9 Broth Base and 2 ml glycerol in 900 ml of distilled water
    2. Aseptically add 2.5 ml of sterile 20% tyloxapol to a final concentration of 0.05% and 100 ml of BBLTM Middlebrook OADC Enrichment, mix
    3. Filter-sterilize (0.22 μm), store at room temperature
  3. Kanamycin 25 mg/ml (10 ml)
    1. Add 250 mg kanamycin sulfate to 10 ml distilled water, mix by vortexing
    2. Filter-sterilize (0.22 μm), aliquot and store at -20 °C
  4. D10 media (1 L)
    1. Add 100 ml heat inactivated fetal bovine serum (10% final concentration), 10 ml 200 mM L-glutamine (2 mM final), 10 ml 100 mM sodium pyruvate (1 mM final), 10 ml penicillin, 100x streptomycin solution to Dulbecco’s modified Eagle’s medium DMEM so that total volume is 1 L
    2. Filter-sterilize (0.22 μm), store at 4 °C for no longer than 2 months
    3. Preheat at 37 °C before use
  5. L-cell conditioned media
    1. Frozen cells are thawed into D10 media and grown for 12-14 days in T150 tissue culture flasks at 37 °C and 6% CO2
    2. Conditioned media is collected, and cell debris is removed by centrifugation at 1,500 rpm (514 x g) for 10 min on Beckman Allegra 6KR centrifuge, GH-3.8 rotor
    3. Supernatant is aliquoted and stored at -20 °C
  6. BMDM media (1 L)
    1. Add 100 ml heat inactivated fetal bovine serum (10% final concentration), 10 ml 200 mM L-glutamine (2 mM final), 10 ml 100 mM sodium pyruvate (1 mM final), 100 ml L-cell-conditioned media (10% final concentration) to Dulbecco’s modified Eagle’s medium DMEM so that total volume is 1 L. Add 10 ml penicillin-streptomycin solution (100x) for media to culture macrophages before infection
    2. Filter-sterilize (0.22 μm), store at 4 °C for no longer than 2 months
    3. Preheat at 37 °C before use
  7. Basal uptake buffer (BUB)
    1. Add 2.25 g glucose, 2.5 g bovine serum albumin, 0.5 ml gelatin, 50 mg CaCl2, 50 mg MgCl2 to 500 ml PBS, mix
    2. Filter-sterilize (0.22 μm), store at 4 °C for no longer than 6 months
  8. 1% fatty acid-free BSA (50 ml)
    1. Dissolve 500 mg of fatty acid-free BSA in 50 ml PBS
    2. Filter-sterilize (0.22 μm), aliquot and store at -20 °C
  9. 4 mM Bodipy-palmitate
    1. Add 526 μl of 100% ethanol to 1 mg of Bodipy-palmitate, mix by vortexing
    2. Store at -20 °C, protect from light
  10. Cuvette buffer (1 L)
    1. Dissolve 101 mg CaCl2, 200 mg KCl, 102 mg MgCl2, 901 mg glucose in 1 L of PBS
    2. Filter-sterilize (0.22 μm) and store at room temperature
    3. A few days before use add heat inactivated fetal bovine serum to final concentration of 10% to obtain the desired volume, filter-sterilize (0.22 μm) and store at 4 °C
    4. Preheat at 37 °C before use
  11. Homogenization buffer (500 ml)
    1. Dissolve 42.75 g sucrose, 95 mg EGTA, 2.38 g HEPES, 250 μl gelatin in 400 ml of distilled water
    2. Adjust pH to 7.0
    3. Fill up with distilled water to the final volume 500 ml
    4. Filter-sterilize (0.22 μm). Store and keep throughout use at 4 °C
  12. 20% Tween 80 (20 ml)
    1. Using 3 ml syringe add 4 ml of Tween 80 to 16 ml of distilled water in 50 ml conical tube
    2. Heat up at 37 °C, vortex occasionally, until Tween 80 goes into solution
    3. Filter-sterilize (0.22 μm), store at room temperature
  13. 0.05% tyloxapol (250 ml)
    1. Aseptically add 625 μl of sterile 20% tyloxapol to 250 ml PBS, mix
    2. Filter-sterilize (0.22 μm), store at room temperature
  14. 4% PFA (500 ml)
    1. Mix 20 g of PFA in PBS while heating up. Avoid boiling, otherwise formaldehyde will be formed
    2. Aliquot and store at -20 °C. Thaw at room temperature before use

Acknowledgments

We thank Linda Bennett for excellent technical support. This work was supported by the NIH grants (AI099569 and AI119122) to BCV and (AI080651 and AI134183) to DGR. This protocol was developed and reported in the previous publication (Nazarova et al., 2017). The authors declare no conflicts of interest or competing interests.

References

  1. Caire-Brändli, I., Papadopoulos, A., Malaga, W., Marais, D., Canaan, S., Thilo, L. and de Chastellier, C. (2014). Reversible lipid accumulation and associated division arrest of Mycobacterium avium in lipoprotein-induced foamy macrophages may resemble key events during latency and reactivation of tuberculosis. Infect Immun 82(2): 476-490.
  2. Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., 3rd, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S. and Barrell, B. G. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393(6685): 537-544.
  3. Daniel, J., Maamar, H., Deb, C., Sirakova, T. D. and Kolattukudy, P. E. (2011). Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog 7(6): e1002093.
  4. Fontán, P., Aris, V., Ghanny, S., Soteropoulos, P. and Smith, I. (2008). Global transcriptional profile of Mycobacterium tuberculosis during THP-1 human macrophage infection. Infect Immun 76(2): 717-725.
  5. Forrellad, M. A., McNeil, M., Santangelo Mde, L., Blanco, F. C., Garcia, E., Klepp, L. I., Huff, J., Niederweis, M., Jackson, M. and Bigi, F. (2014). Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis. Tuberculosis (Edinb) 94(2): 170-177.
  6. Homolka, S., Niemann, S., Russell, D. G. and Rohde, K. H. (2010). Functional genetic diversity among Mycobacterium tuberculosis complex clinical isolates: delineation of conserved core and lineage-specific transcriptomes during intracellular survival. PLoS Pathog 6(7): e1000988.
  7. Lovewell, R. R., Sassetti, C. M. and VanderVen, B. C. (2016). Chewing the fat: lipid metabolism and homeostasis during M. tuberculosis infection. Curr Opin Microbiol 29: 30-36.
  8. Nazarova, E. V., Montague, C. R., La, T., Wilburn, K. M., Sukumar, N., Lee, W., Caldwell, S., Russell, D. G. and VanderVen, B. C. (2017). Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis. Elife 6.
  9. Nazarova, E. V. and Russell, D. G. (2017). Growing and handling of Mycobacterium tuberculosis for macrophage infection assays. Methods Mol Biol 1519: 325-331.
  10. Pandey, A. K. and Sassetti, C. M. (2008). Mycobacterial persistence requires the utilization of host cholesterol. Proc Natl Acad Sci 105: 4376-4380.
  11. Pethe, K., Swenson, D. L., Alonso, S., Anderson, J., Wang, C. and Russell, D. G. (2004). Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation. Proc Natl Acad Sci U S A 101(37): 13642-13647.
  12. Podinovskaia, M., Lee, W., Caldwell, S. and Russell, D. G. (2013). Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function. Cell Microbiol 15(6): 843-859.
  13. Rachman, H., Strong, M., Ulrichs, T., Grode, L., Schuchhardt, J., Mollenkopf, H., Kosmiadi, G. A., Eisenberg, D. and Kaufmann, S. H. (2006). Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis. Infect Immun 74(2): 1233-1242.
  14. Rohde, K. H., Abramovitch, R. B. and Russell, D. G. (2007). Mycobacterium tuberculosis invasion of macrophages: linking bacterial gene expression to environmental cues. Cell Host Microbe 2(5): 352-364.
  15. Rohde, K. H., Veiga, D. F., Caldwell, S., Balazsi, G. and Russell, D. G. (2012). Linking the transcriptional profiles and the physiological states of Mycobacterium tuberculosis during an extended intracellular infection. PLoS Pathog 8(6): e1002769.
  16. Russell, D. G., VanderVen, B. C., Lee, W., Abramovitch, R. B., Kim, M. J., Homolka, S., Niemann, S. and Rohde, K. H. (2010). Mycobacterium tuberculosis wears what it eats. Cell Host Microbe 8(1): 68-76.
  17. Schnappinger, D., Ehrt, S., Voskuil, M. I., Liu, Y., Mangan, J. A., Monahan, I. M., Dolganov, G., Efron, B., Butcher, P. D., Nathan, C. and Schoolnik, G. K. (2003). Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment. J Exp Med 198(5): 693-704.
  18. Tailleux, L., Waddell, S. J., Pelizzola, M., Mortellaro, A., Withers, M., Tanne, A., Castagnoli, P. R., Gicquel, B., Stoker, N. G., Butcher, P. D., Foti, M. and Neyrolles, O. (2008). Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages. PLoS One 3(1): e1403.
  19. VanderVen, B. C., Fahey, R. J., Lee, W., Liu, Y., Abramovitch, R. B., Memmott, C., Crowe, A. M., Eltis, L. D., Perola, E., Deininger, D. D., Wang, T., Locher, C. P. and Russell, D. G. (2015). Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium's metabolism is constrained by the intracellular environment. PLoS Pathog 11(2): e1004679.
  20. Wipperman, M. F., Sampson, N. S. and Thomas, S. T. (2014). Pathogen roid rage: cholesterol utilization by Mycobacterium tuberculosis. Crit Rev Biochem Mol Biol 49(4): 269-293.

简介

结核分枝杆菌(Mtb)已经发展为从其宿主吸收脂肪酸。然而,直到最近,还没有可靠的方法来量化宿主细胞感染期间细菌对脂肪酸的摄取。在这里,我们描述了一种新的方法来量化细胞内杆菌对脂肪酸的摄取。我们用Mtb组成性表达mCherry感染巨噬细胞,然后用Bodipy-palmitate代谢标记它们。标记程序后,我们在蔗糖垫上分离含有Mtb的吞噬体,并用去污剂破坏吞噬体。大量洗涤后,通过流式细胞术分析分离的细菌以确定与细菌相关的Bodipy-棕榈酸酯信号的水平。使用液体培养物中脂肪酸摄取缺陷的Mtb突变株,我们确定该突变体在巨噬细胞感染期间同化比野生型菌株少10倍的Bodipy-棕榈酸酯。脂肪酸摄取的这种定量方法可用于进一步鉴定参与细胞内Mtb和可能的其他细菌的脂质摄取的途径。

【背景】结核分枝杆菌(Mtb)同化宿主来源的脂质(脂肪酸和胆固醇)的能力使得病原体能够在其宿主内存活(Russell等人,2010; Lovewell 等人,2016)。在小鼠感染期间和在人肺组织中,通过巨噬细胞内的Mtb上调胆固醇和脂肪酸代谢相关基因来支持该想法(Schnappinger等人,2003; Rachman等人,2006; Rohde等人,2007;Fontán等人,2008; Tailleux等人,2008; Homolka et al。,2010; Rohde et al。,2012)。在感染过程中,胆固醇代谢对Mtb的重要性得到了遗传学研究和鉴定靶向胆固醇代谢的新型抗结核药物的支持(Pandey和Sassetti,2008; Wipperman等人,2014; VanderVen等人,2015)。然而,专门研究Mtb中脂肪酸摄取的具体机制的发现不仅受到专用基因的明显冗余(Cole等人,1998)的阻碍,而且受到缺乏可靠的测定的阻碍。使用放射性底物进行代谢标记已成为评估肉汤培养物中细菌摄入脂肪酸的效率的可接受方法(Forrellad等人,2014年)。该方法对于应用胞内Mtb是非常具有挑战性的,并且很少有团体报告成功使用这种方法(Daniel等人,2011年)。或者,TEM和用亲脂性染料如Bodipy 493/503,尼罗红,油红染色可以促进在感染期间检测分枝杆菌内的脂质(Daniel等人,2011; Podinovskaia等人,2013; Caire-Brändli等人,2014)。然而,这些标记方法都不直接评估底物的主动输入,而是指示累积的脂质的总量。因此,非常需要在感染过程中通过Mtb进行活性脂肪酸代谢标记的方法,该方法易于下游表征。

最近,显示荧光脂肪酸可以有效地递送到胞内细菌中,并且可以通过显微镜检测(Podinovskaia等人,2013)。这些观察结果使我们开发了一种基于流式细胞计量术的基于Mtb在其宿主细胞内的荧光脂肪酸摄取量的新方法。该测定使我们能够证明在巨噬细胞感染过程中,ΔlmA:: hyg Mtb菌株在脂肪酸摄取方面存在缺陷(Nazarova等人,2017)(图1) 。我们相信这种方法打开了基因筛选的大门,以进一步了解在宿主细胞感染期间Mtb和可能的其他细胞内病原体摄取脂肪酸所涉及的机制。

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图1.巨噬细胞感染过程中用于量化结核分枝杆菌脂肪酸摄取的方法概述

关键字:脂肪酸, 摄取, 结核分枝杆菌, 巨噬细胞, 细胞内, 分枝杆菌, Bodipy

材料和试剂

  1. 血清移液管,5ml,10ml,25ml和50ml(Corning,Costar的产品目录号:4487,4488,4489和4490)
  2. (Biotix,Neptune,目录号:BT20,BT100,BT200; Thermo Fisher Scientific,Thermo Scientific,产品目录号:2079-HR),移液管尖端20μl,100μl,200μl,
  3. T25(带有过滤盖的无菌25cm2组织培养瓶)(TPP,目录号:90026)。
  4. T75或T150(无菌75cm2或150cm2具有过滤盖的组织培养瓶)(TPP,目录号:90076或90151)。

  5. 无菌1毫升结核菌素注射器与25号针(BD,目录号:309626)
  6. 细胞刮刀,25厘米(SARSTEDT,目录号:83.1830)

  7. 15毫升锥形管(SARSTEDT,目录号:62.554.100)

  8. 50毫升锥形管(SARSTEDT,目录号:62.547.100)
  9. Glasstic ®带网格的幻灯片(KOVA International,目录号:87144)

  10. 150×15毫米培养皿(VWR,目录号:25384-326)
  11. 2毫升螺帽管(VWR,目录号:16466-042)
  12. FACS管(VWR,目录号:60818-496)
  13. 3毫升注射器(带Luer-Lok TM尖端,BD,目录号:309657)
  14. 带方盖的一次性塑料OD比色皿(Fisher Scientific,Fisherbrand TM TM目录号:14-955-128和14-385-999)
  15. 结核分枝杆菌组成型表达mCherry(pMV306 smyc':: mCherry (Kan r ))来源:Russell和VanderVen实验室(Nazarova ,2017)
  16. 骨髓来源的鼠巨噬细胞(BALB / c小鼠,THE JACKSON LABORATORIES,目录号:000651)
    注意:差异在Nazarova等人中有详细描述。 (2017)。
  17. L细胞(NCTC克隆929 [L细胞,L-929,菌株L的衍生物])(ATCC,目录号:CCL-1)

  18. 磷酸盐缓冲盐水(PBS)1x(Mediatech,目录号:21-040)
  19. Tyloxapol(Acros Organics,目录号:422370050)
  20. Middlebrook 7H9肉汤培养基(BD,Difco TM,产品目录号:271310)
  21. 蒸馏水
  22. 甘油(VWR,目录号:97062-452)
  23. Middlebrook OADC富集(BD,BBL TM ,目录号:212351)
  24. 卡那霉素硫酸盐(IBI Scientific,目录号:IB02120)
  25. 加热灭活的胎牛血清(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
    注意:56°C下热灭活30分钟
  26. 200mM L-谷氨酰胺(100x)(Mediatech,目录号:25-005)
  27. 100 mM丙酮酸钠(Mediatech,目录号:25-000)

  28. 100x青霉素 - 链霉素溶液(Mediatech,目录号:30-002)
  29. Dulbecco对Eagle's培养基(DMEM)1x(Mediatech,目录号:10-017)的修改
  30. 葡萄糖(右旋糖)(Fisher Scientific,目录号:BP350-1)
  31. 牛血清白蛋白(BSA)(Roche Diagnostics,目录号:03116964001)
  32. 来自冷水鱼皮的明胶(Sigma-Aldrich,目录号:G7765)
  33. 氯化钙(CaCl 2)(Sigma-Aldrich,目录号:C4901)
  34. 氯化镁(MgCl 2)(AMRESCO,目录号:J364)
  35. 不含脂肪酸的BSA(Roche Diagnostics,目录号:03117057001)
  36. 100%乙醇,200份Proof(Decon Labs,产品目录号:V1016)
  37. Bodipy TM FL C16(Bodipy-棕榈酸酯)(Thermo Fisher Scientific,Invitrogen TM,目录号:D3821)
  38. Bodipy TM FL C12(Thermo Fisher Scientific,Invitrogen TM,目录号:D3822)
  39. Bodipy TM 558/568 C12(Thermo Fisher Scientific,Invitrogen TM,目录号:D3835)
  40. 氯化钾(KCl)(Mallinckrodt Chemicals或Avantor Performance Materials,MACRON,目录号:6858-04)
  41. 蔗糖(Avantor Performance Materials,J.T.Baker,目录号:4097)
  42. 亚乙基双(氧亚乙基亚硝基)四乙酸(EGTA)(Avantor Performance Materials,J.T.Baker,目录号:L657)
  43. HEPES(VWR,目录号:BDH4162)
  44. Tween TM 80(Fisher Chemical,目录号:T164-500)
  45. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:158127)
  46. 20%泰洛沙泊(见食谱)
  47. 7H9 OADC媒体(见食谱)
  48. 卡那霉素25毫克/毫升(见食谱)
  49. D10媒体(见食谱)
  50. L细胞条件培养基(见食谱)
  51. BMDM媒体(见食谱)
  52. 基础摄取缓冲液(BUB)(见食谱)
  53. 1%无脂肪酸BSA(见食谱)
  54. 4毫米Bodipy棕榈酸酯(见食谱)
  55. 比色杯缓冲液(见食谱)
  56. 匀浆缓冲液(见食谱)
  57. 20%吐温80(见食谱)
  58. 0.05%tyloxapol(见食谱)
  59. 4%PFA(见食谱)

设备

  1. 移液器控制器(Drummond Scientific,型号:Pipet-Aid ,目录号:4-000-101)
  2. 移液器(MIDSCI,Alphapette,型号:A-20,A-100,A-200,A-1000)
  3. 37°C,6%CO 2培养箱

  4. 56°C水浴
  5. 冰箱(4°C)
  6. -20°C冷冻机
  7. 分光光度计兼容600nm处的吸光度测量(Cole-Parmer,Jenway,型号:6320D)
  8. Beckman Allegra 6KR离心机(Beckman Coulter,型号:Allegra 6KR Kneewell TM))
  9. GH-3.8转子(Beckman Coulter,型号:GH-3.8转子)
  10. 具有足够分辨率的倒置显微镜检测巨噬细胞大小的细胞(奥林巴斯,型号:10倍物镜的IMT-2)
  11. Beckman Microfuge 18离心机(Beckman Coulter,型号:Microfuge 18)
  12. 流式细胞仪(BD,型号:FACS LSR II)

软件

  1. FlowJo(BD)

程序

  1. 细菌培养
    结核分枝杆菌菌株在37℃下在7H9 OADC培养基(参见配方)中在T25瓶中静置培养直至对数中期(OD 600-6〜0.6)。细菌培养物从冷冻库中开始并保持不超过5代。进一步检测需要Mtb荧光蛋白的组成型表达。我们使用来自smyc启动子( smyc ':: mCherry )的表达来自mCherry 的整合pMV306质粒的Mtb Erdman菌株) 。该质粒赋予卡那霉素抗性,因此生长培养基含有最终浓度为25μg/ ml的卡那霉素(参见食谱)。
    注意:
    1. 不要在T25烧瓶中生长超过10毫升的细菌培养物,以确保Mtb有足够的氧气供应量。为了估计细菌培养生长的时机,可以考虑在站立培养中生长两天大约一次的Mtb分裂。 (Nazarova and Russell,2017)可以找到关于巨噬细胞感染细菌培养生长的更多细节。
    2. 我们建议测试你的野生型菌株组成型表达荧光蛋白的能力,通过显微镜检查在巨噬细胞感染期间有效地摄取Bodipy-palmitate。我们已经注意到,Mtb Erdman菌株在强启动子(smyc'或hsp60')下mCherry表达来自复制质粒影响Bodipy-棕榈酸酯摄取。然而,具有相同质粒的CDC1551菌株显示出高水平的脂肪酸摄取。此外,应注意通过共聚焦显微镜确定Bodipy-palmitate是否在细菌内与其表面上积聚。

  2. 巨噬细胞分离和培养
    可以使用各种来源的巨噬细胞。在感染之前,我们用来自BALB / c小鼠的骨髓细胞分化巨噬细胞并在37℃和6.0%CO 2下用抗生素维持在BMDM培养基中(参见食谱)10天。
    的 感染
    1. 感染前一天,将无抗生素BMDM培养基中的巨噬细胞种入T150组织培养瓶(3×10 7细胞和每瓶40-50ml BMDM培养基)以获得融合单层。如果巨噬细胞的数量是有限的,则一个含有1×10 7个细胞的T75培养瓶将提供足够的结果,然而,两个T150培养瓶(6×10 7细胞)可以容易地检测到
      。在实验结束时,细菌会沉淀
    2. 在感染当天测量中对数Mtb培养物的光密度。假设0.6的OD 600等于10 8细菌/ ml,则以3300rpm(〜2500×gg)离心所需量的培养物12分钟在Beckman Allegra 6KR离心机中,GH-3.8转子。我们以4:1的MOI感染巨噬细胞,因此感染6×10 7个细胞需要2.4×10 8个细菌。为了确保足够数量的细菌沉淀,我们常规地在OD 600 = 0.6下离心3ml细菌。
      注:由于细菌数量的估计可能会有所不同,可能需要自行确定。但是,为了在MOI方面实现更高的可复制性,我们建议坚持我们的估计。关于巨噬细胞感染的更多细节可参见(Nazarova and Russell,2017)。
    3. 离心后,取出上清液并用1.5ml BUB重悬细菌沉淀(参见食谱)。通过1毫升结核菌素注射器与25号针12-20倍相同的注射器和针的细菌悬液。
      加入3.5毫升BUB到悬浮液中,共5毫升,并充分混合。
    4. 向每个含有3×10 7个细胞的T150烧瓶中加入2.4ml细菌悬浮液。充分混合,但温和。
      在37°C和6%CO 2下孵育4小时。
    5. 用新鲜的预热无抗生素BMDM培养基代替细胞外细菌。受感染的巨噬细胞在37℃和6%CO 2下在BMDM培养基中维持3天。在感染的第二天更换媒体是可选的。

    的 标签
    1. 在标记(感染的第三天)前预热的无菌1%无脂肪酸BSA(参见食谱)在37℃PBS中30-60分钟。加入4mM Bodipy-palmitate储备液(见食谱)以获得100μM浓度。为了标记一个T75烧瓶,将50μl的4mM Bodipy-棕榈酸酯储液添加到1.95ml的不含1%无脂肪酸的BSA中。涡旋混合,直至溶液变绿。保持在37°C,避光。
      注意:
      1. 标签的日期可以考虑您试图解决的问题来选择。我们在巨噬细胞感染的不同阶段测试了Bodipy-palmitate的摄取量,并注意到在感染第三天之前摄入量不足以被检测到。
      2. 除了Bodipy棕榈酸酯,我们还测试了Bodipy FL C12和Bodipy 558/568 C12。 Bodipy FL C12在胞内细菌中积累,与Bodipy-palmitate一样有效,而Bodipy 558/568 C12被Mtb弱化同化。我们选择使用Bodipy棕榈酸酯作为长度(16碳)的脂肪酸更常见于感染Mtb的巨噬细胞膜中。
    2. 为了标记一个T75烧瓶,将1.6ml在1%不含脂肪酸的BSA(来自 Labeling 的步骤1)中的100μMBodipy-棕榈酸酯加入18.4ml预热比色杯缓冲液(参见配方),使标记的脂质的最终浓度为8μM,充分混合。使用15毫升标记一个T75烧瓶,30毫升标记一个T150烧瓶。对于较大的感染,相应地增加第1步和第2步中的体积( 标签 )。
    3. 从感染的巨噬细胞中除去培养基,用含有Bodipy-palmitate的比色杯缓冲液代替(体积描述于 Labeling 的步骤2)。在37℃和6.0%CO 2下孵育感染的巨噬细胞1小时。
    4. 1小时标记期后,取出含有Bodipy-palmitate的比色杯缓冲液,并加入新鲜预热的无标记的比色杯缓冲液1小时。一个T75烧瓶使用15毫升,一个T150烧瓶使用30毫升。
      在孵育1小时后立即进入下一步,不带标签。
      注意:或者,可将1%无脂肪酸BSA中的Bodipy-palmitate直接加入在BMDM培养基中培养的感染的巨噬细胞中。标签追逐也可以在新鲜的预热BMDM介质中进行。在这种情况下不需要比色皿缓冲液。根据我们的经验,任何一种标签方式都可以得出相似的结果。

    细胞内细菌的分离
    注意:该方案的这一部分基于(Pethe等,2004)中描述的吞噬体分离。
    1. 从标记的感染细胞中取出比色杯缓冲液,并用10毫升匀浆缓冲液快速冲洗(见食谱)。
    2. 加入15毫升冰冷匀浆缓冲液,在4°C孵育10-15分钟,并用细胞刮刀从每个烧瓶中刮下来收获巨噬细胞。将细胞转移到50ml锥形管中,并通过在Beckman Allegra 6KR离心机,GH-3.8转子中以1,500rpm(514×g)离心10分钟使细胞沉淀。这个和所有下面的离心是在4°C完成的,以阻止细菌进一步吸收脂质。
    3. 除去上清液,用1.5 ml匀浆缓冲液通过移液重新悬浮沉淀,并将细胞转移到15 ml锥形管中。用25号针头通过1ml结核菌素注射器使细胞溶解25-70次。在显微镜下使用100格栅玻片监测细胞裂解。继续直至>
      。当完整的细胞被细胞碎片取代时,95%的细胞被裂解 注意:为了安全起见,将载玻片用感染材料放入150 x 15 mm培养皿中。
    4. 加入均化缓冲液使体积增加至5毫升,重悬。在Beckman Allegra 6KR离心机,GH-3.8转子中以800rpm离心细胞裂解物(〜146μgx g)10分钟。
    5. 将上清液(吞噬体悬液)转移到新的15ml锥形管中。颗粒主要由核和未经处理的细胞组成,并被丢弃。
    6. 向悬浮液中加入20%吐温80(见配方)至最终浓度为0.1%,充分混合并在4℃放置15分钟以裂解含有空泡的Mtb。
    7. 通过在贝克曼Allegra 6KR离心机,GH-3.8转子以2500转/分钟(1,430 em x em)离心15分钟,通过摇动和分离细菌快速搅拌。
    8. 去除上清液并重新悬浮在PBS中的10ml 0.05%泰洛沙泊中的细菌沉淀物(参见食谱)。在Beckman Allegra 6KR离心机,GH-3.8转子中,以3,300rpm(〜2,500×g克)的速度离心细菌15分钟。
    9. 可选:重复上一步以进一步去除粘附在细菌细胞表面的标记脂肪酸。
      注意:bodipy-palmitate与细菌细胞表面的非特异性结合产生背景信号。然而,如果测试的菌株/条件在标记物的特定摄取方面产生显着差异,则该背景噪音不会显着影响结果。
    10. 除去上清液并将细菌用4%PFA(参见食谱)在2 ml螺帽管中固定24 h。
      注意:如果您在BSL3设施中使用流式细胞仪,可以在分离后立即分析并且无需固定即可分析细菌。

数据分析

分离后的细菌应在收集后几天内分析,最好在第二天分析。

  1. 在Beckman Microfuge 18-18离心机中以10,000rpm(9,000×g g)离心固定样品5分钟。
  2. 去除上清液,重悬沉淀在1-2毫升0.05%tyloxapol在PBS中,并转移到FACS管。

  3. 。将悬浮液通过1毫升结核菌素注射器与25号针12-20次,以获得单细胞菌悬液。
  4. 使用所描述的门控策略(图2)立即在流式细胞仪(BD FACS LSR II)上分析。选择正向和侧向散布的中等大小的群体以排除团块和小碎片,关注mCherry(PE-Texas Red)阳性群体(细菌),并比较样本间Bodipy-palmitate的FITC信号。由于mCherry和Bodipy信号之间的重叠最小,因此不需要补偿。作为阴性对照使用未暴露于标记的mCherry阳性细菌。
    注意:
    1. 由于它们是mCherry阴性,所以Bodipy-palmitate也积累在细胞器细胞膜中,这些细胞器被排除在分析之外。
    2. 尽可能收集尽可能多的活动,最低限额为50,000人。对于图2中的分析,我们收集了1,000,000个事件。
  5. 通过确定来自mCherry阳性细菌的Bodipy信号的平均荧光,使用FlowJo量化获得的数据。


    图2.用于分析胞内Mtb对Bodipy-棕榈酸酯摄取的门控策略在正向和侧向散射中选择中等大小的群体,并进一步分析mCherry信号的水平。对于代表细菌的mCherry阳性群体确定Bodipy棕榈酸酯信号。右图是与三种不同菌株相关的检测到的Bodipy-棕榈酸酯信号的代表:野生型(黑色),△lucA :: hyg(红色)和补充菌株(蓝色)。灰色直方图表示未暴露于标记的mCherry阳性细菌。 (改编自Nazarova等人,2017年)

食谱

  1. 20%泰洛沙泊(20毫升)
    1. 使用3毫升注射器将4毫升泰洛沙泊加入50毫升锥形管中的16毫升蒸馏水中
    2. 在56°C升温,偶尔涡旋,直至泰洛沙泊进入粘稠而清澈的溶液。
    3. 过滤消毒(0.22微米),在室温下储存长达12个月
  2. 7H9 OADC介质(1 L)

    1. 在900毫升蒸馏水中溶解4.7克7H9 Difco TM Middlebrook 7H9肉汤基料和2毫升甘油
    2. 无菌加入2.5ml无菌20%泰洛沙泊,终浓度为0.05%,加入100ml BBL TM Middlebrook OADC Enrichment,混匀。
    3. 过滤消毒(0.22微米),在室温下保存
  3. 卡那霉素25mg / ml(10ml)
    1. 加入250毫克卡那霉素硫酸盐到10毫升蒸馏水中,通过涡旋混合
    2. 过滤消毒(0.22微米),分装并储存在-20°C
  4. D10介质(1 L)
    1. 加入100ml加热灭活的胎牛血清(终浓度10%),10ml 200mM L-谷氨酰胺(终浓度2mM),10ml 100mM丙酮酸钠(终浓度1mM),10ml青霉素,100x链霉素溶液至Dulbecco改良Eagle's培养基DMEM,使总体积为1L
    2. 过滤消毒(0.22μm),4°C储存不超过2个月
    3. 使用前在37°C预热
  5. L-细胞条件培养基
    1. 将冷冻细胞融化到D10培养基中,并在T150组织培养瓶中在37℃和6%CO 2下生长12-14天。
    2. 收集条件培养基,并通过Beckman Allegra 6KR离心机,GH-3.8转子以1,500rpm(514×g)离心10分钟除去细胞碎片。
    3. 上清液分装并储存在-20°C。
  6. BMDM培养基(1 L)
    1. 加入100ml热灭活的胎牛血清(终浓度10%),10ml 200mM L-谷氨酰胺(终浓度2mM),10ml 100mM丙酮酸钠(终浓度1mM),100ml L-细胞条件培养基终浓度%)到Dulbecco改良的Eagle's培养基DMEM中,使总体积为1L。在感染前加入10ml青霉素 - 链霉素溶液(100x)用于培养巨噬细胞的培养基
    2. 过滤消毒(0.22μm),4°C储存不超过2个月

    3. 使用前预热于37°C
  7. 基础摄取缓冲液(BUB)

    1. 加入2.25克葡萄糖,2.5克牛血清白蛋白,0.5毫升明胶,50毫克氯化钙,50毫克MgCl 2至500毫升PBS
    2. 过滤消毒(0.22μm),4°C储存不超过6个月
  8. 1%不含脂肪酸的BSA(50毫升)

    1. 500毫克不含脂肪酸的牛血清白蛋白溶于50毫升PBS中
    2. 过滤消毒(0.22微米),分装并储存在-20°C
  9. 4mM Bodipy-棕榈酸酯

    1. 添加526μl的100%乙醇到1 mg的Bodipy棕榈酸酯中,通过涡旋混合
    2. 在-20°C储存,避光。
  10. 比色杯缓冲液(1L)

    1. 在1L PBS中溶解101mg CaCl 2 2,200mg KCl,102mg MgCl 2,901mg葡萄糖
    2. 过滤消毒(0.22μm)并在室温下保存
    3. 使用前几天加入热灭活的胎牛血清至终浓度为10%以获得所需体积,过滤灭菌(0.22μm)并在4℃下储存。
    4. 使用前在37°C预热
  11. 均化缓冲液(500ml)

    1. 在400毫升蒸馏水中溶解42.75克蔗糖,95毫克EGTA,2.38克HEPES,250微升明胶
    2. 调整pH值至7.0
    3. 填充蒸馏水至最终体积500毫升
    4. 过滤消毒(0.22微米)。
      在4°C储存和使用
  12. 20%吐温80(20ml)
    1. 使用3毫升注射器添加4毫升吐温80至16毫升蒸馏水在50毫升锥形管
    2. 在37°C加热,偶尔涡旋,直到吐温80进入溶液
    3. 过滤消毒(0.22微米),在室温下保存
  13. 0.05%泰洛沙泊(250毫升)

    1. 无菌添加625μl无菌20%泰洛沙泊至250 ml PBS中
    2. 过滤消毒(0.22微米),在室温下保存
  14. 4%PFA(500毫升)
    1. 加热时混合20g PFA的PBS溶液。避免沸腾,否则会形成甲醛
    2. 分装并储存在-20°C。使用前在室温下解冻

致谢

我们感谢Linda Bennett提供出色的技术支持。这项工作得到了NIH拨款(AI099569和AI119122)对BCV和(AI080651和AI134183)对DGR的支持。该协议是在以前的出版物中开发和报告的(Nazarova等人,2017年)。作者声明不存在利益冲突或利益冲突。

参考

  1. Caire-Brändli,I.,Papadopoulos,A.,Malaga,W.,Marais,D.,Canaan,S.,Thilo,L.和de Chastellier,C.(2014)。 脂蛋白诱导的鸟分枝杆菌的可逆性脂质积累和相关的分裂停滞泡沫状巨噬细胞可能类似于潜伏期和结核病重新激活期间的关键事件。 感染免疫 82(2):476-490。
  2. Cole,ST,Brosch,R.,Parkhill,J.,Garnier,T.,Churcher,C.,Harris,D.,Gordon,SV,Eiglmeier,K.,Gas,S.,Barry,CE,3rd,Tekaia ,F.,Badcock,K.,Basham,D.,Brown,D.,Chillingworth,T.,Connor,R.,Davies,R.,Devlin,K.,Feltwell,T.,Gentles,S.,Hamlin N.,Holroyd,S.,Hornsby,T.,Jagels,K.,Krogh,A.,McLean,J.,Moule,S.,Murphy,L.,Oliver,K.,Osborne,J.,Quail MA,Rajandream,MA,Rogers,J.,Rutter,S.,Seeger,K.,Skelton,J.,Squares,R.,Squares,S.,Sulston,JE,Taylor,K.,Whitehead,S.和Barrell,BG(1998)。 从全基因组序列中解读结核分枝杆菌的生物学 a> Nature 393(6685):537-544。
  3. Daniel,J.,Maamar,H.,Deb,C.,Sirakova,T.D和Kolattukudy,P.E。(2011)。 结核分枝杆菌使用宿主三酰甘油积累脂滴并获得休眠 - (PLoS Pathog)7(6):e1002093。
  4. Fontán,P.,Aris,V.,Ghanny,S.,Soteropoulos,P.和Smith,I。(2008)。 THP-1人巨噬细胞感染期间结核分枝杆菌的全球转录概况。 感染免疫 76(2):717-725。
  5. Forrellad,MA,McNeil,M.,Santangelo Mde,L.,Blanco,FC,Garcia,E.,Klepp,LI,Huff,J.,Niederweis,M.,Jackson,M.和Bigi,F。(2014) 。 Mce1转运蛋白在结核分枝杆菌的脂质体内平衡中的作用。< /结核病(Edinb) 94(2):170-177。
  6. Homolka,S.,Niemann,S.,Russell,D.G。和Rohde,K.H。(2010)。 功能性遗传多样性
  7. Homolka,S.,Niemann,S.,Russell,D.G。和Rohde,K.H。(2010)。 结核分枝杆菌复杂临床分离株的功能遗传多样性:保守核心的划分和细胞内存活期间的谱系特异性转录组。 PLoS Pathog 6(7):e1000988。
  8. Lovewell,R.R。,Sassetti,C.M。和VanderVen,B.C。(2016)。 咀嚼脂肪:体内脂质代谢和体内平衡。结核病感染。 Curr Opin Microbiol 29:30-36。
  9. Nazarova,E.V.,Montague,C.R.,La,T.,Wilburn,K.M.,Sukumar,N.,Lee,W.,Caldwell,S.,Russell,D.G。和VanderVen,B.C。(2017)。 Rv3723 / LucA协调结核分枝杆菌中的脂肪酸和胆固醇摄取。< / a> Elife 6。
  10. Nazarova,E.V。和Russell,D.G.(2017)。 生长和处理结核分枝杆菌用于巨噬细胞感染检测。
  11. Pandey,A.K。和Sassetti,C.M。(2008)。 分枝杆菌的持久性需要利用宿主的胆固醇。 Proc Natl Acad Sci 105:4376-4380。
  12. Pethe,K.,Swenson,D.L.,Alonso,S.,Anderson,J.,Wang,C.and Russell,D.G。(2004)。 分离结核分枝杆菌突变体阻滞吞噬体成熟缺陷< / pro>美国国家科学院院刊101(37):13642-13647。
  13. Podinovskaia,M.,Lee,W.,Caldwell,S.和Russell,D.G。(2013)。 结核分枝杆菌感染巨噬细胞引起吞噬功能的全面改变 /细胞微生物 15(6):843-859。
  14. Rachman,H.,Strong,M.,Ulrichs,T.,Grode,L.,Schuchhardt,J.,Mollenkopf,H.,Kosmiadi,G.A.,Eisenberg,D。和Kaufmann,S.H。(2006)。 肺结核中结核分枝杆菌的独特转录组签名感染免疫 74(2):1233-1242。
  15. Rohde,K. H.,Abramovitch,R. B.和Russell,D. G.(2007)。 结核杆菌入侵巨噬细胞:将细菌基因表达与环境线索联系起来。 Cell Host Microbe 2(5):352-364。
  16. Rohde,K. H.,Veiga,D. F.,Caldwell,S.,Balazsi,G.和Russell,D. G.(2012)。 将延伸期间的结核分枝杆菌的转录谱和生理状态联系起来细胞内感染。
  17. Russell,D.G.,VanderVen,B.C.,Lee,W.,Abramovitch,R.B.,Kim,M.J.,Homolka,S.,Niemann,S.and Rohde,K.H。(2010)。 结核分枝杆菌穿着它的食物。 细胞宿主微生物 8(1):68-76。
  18. Schnappinger,D.,Ehrt,S.,Voskuil,MI,Liu,Y.,Mangan,JA,Monahan,IM,Dolganov,G.,Efron,B.,Butcher,PD,Nathan,C.and Schoolnik,GK 2003)。 巨噬细胞内结核分枝杆菌的转录调整:洞察phagosomal环境。 J Exp Med 198(5):693-704。
  19. Tailleux,L.,Waddell,SJ,Pelizzola,M.,Mortellaro,A.,Withers,M.,Tanne,A.,Castagnoli,PR,Gicquel,B.,Stoker,NG,Butcher,PD,Foti,M.和Neyrolles,O。(2008)。 通过转录分析结核分枝杆菌和感染的人类树突状细胞和巨噬细胞。
  20. VanderVen,BC,Fahey,RJ,Lee,W.,Liu,Y.,Abramovitch,RB,Memmott,C.,Crowe,AM,Eltis,LD,Perola,E.,Deininger,DD,Wang,T.,Locher ,CP和Russell,DG(2015)。 结核分枝杆菌中新型胆固醇降解抑制剂揭示了细菌的新陈代谢如何受到细胞内环境的限制。
  21. Wipperman,M.F。,Sampson,N.S。和Thomas,S.T。(2014)。 病原体狂怒:由结核分枝杆菌利用胆固醇。 Crit Rev Biochem Mol Biol 49(4):269-293。
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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. Nazarova, E. V., Podinovskaia, M., Russell, D. G. and VanderVen, B. C. (2018). Flow Cytometric Quantification of Fatty Acid Uptake by Mycobacterium tuberculosis in Macrophages. Bio-protocol 8(4): e2734. DOI: 10.21769/BioProtoc.2734.
  2. Nazarova, E. V., Montague, C. R., La, T., Wilburn, K. M., Sukumar, N., Lee, W., Caldwell, S., Russell, D. G. and VanderVen, B. C. (2017). Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis. Elife 6.
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