Method for assaying gliotoxin production in Aspergillus fumigatus

Introduction

Gliotoxin is a metabolite of Aspergillus fumigatus that exhibits immunosuppressive activity against certain cells of the immune system. Secretion of gliotoxin during infection has been suggested as being a factor in the pathogenesis of aspergillosis. Gliotoxin secretion can be assayed in a number of ways by thin layer chromatography (TLC) high performance liquid chromatography (HPLC) or bioassay using the effect of gliotoxin on human cells¹. This is a summary of a procedure described in Belkacemi et al. (1999)².

Materials and equipment

To prepare extracts

• Haemocytometer
• 250ml flask
• Culture plates containing Sabouraud glucose agar
• 0.5% Tween 20 (Sigma)
• 100ml Czapek-Dox medium (30g sucrose, 3g Na₂NO₃, 0.5g MgSO₄.7H₂O, 0.5g KCl, 0.01g FSO₄  in 1l distilled water - all from Sigma)
• 100ml glass flask for extraction
• Microfuge
• Shaking incubator
• Buchner funnel and Whatman No 1 filter paper to fit
• Chloroform & methanol (Hyper Solv, BDH)
• Rotary evaporator + suitable tubes to fit
• -70°C freezer
• Deionised-distilled water

For TLC detection

• 1mgml^-1 gliotoxin standard in methanol
• Silicagel 60 plate with fluorescence indicator (Merck, Damstadt, Germany)
• inlined chromatography tank
• Sulphuric Acid (Sigma)
• 110°C oven

For HPLC detection

• HPLC apparatus

For fibroblast assay

• L929 Lung fibroblast cells (ATTC)
• Dulbecco's modification of Eagles medium ( Hepes buffered, no sodium pyruvate - DMEM - Sigma)
• 10x DMEM (Sigma)
• Foetal calf serum (FCS - Sigma)
• Hank's balanced salt solution (HBSS - Sigma)
• Waterbath set to 37°C
• Sterile plastic Eppendorf tubes
• Tissue culture plastic flasks, 50ml and 200ml
• Trypsin-EDTA (0.05% (w:v) trypsin, 0.02% (w:v) EDTA-Sigma)
• Sterile Pasteur pipettes
• Incubator for tissue culture running at 37°C with 5%CO₂
• Sterile Phosphate buffered saline pH 7.3 (PBS - Sigma)
• 10ml sterile plastic pipettes
• Trypan blue (Sigma)
• Neutral red (Sigma)
• Ethylacetate (Sigma)
• 96-well flat bottomed tissue culture plate (Millipore, UK)
• Acetic acid (Sigma)
• Ethanol (BDH)
• Spectrophotometer

Procedure

Culture and extraction

1. Culture Aspergillus fumigatus  on Sabouraud agar plates for 2 days at 37°C and harvest the conidia using sterile 0.5% Tween 20.
2. Adjust the concentration of conidia 10⁷ml^-1 in distilled water using a haemocytometer.
3. Use 1ml of this conidial suspension to inoculate 100ml of Czapek-dox broth in a 250ml flask.
4. Incubate culture at 37°C in a shaking incubator at 1400rpm for 2 days.
5. Harvest fungal biomass by filtration through Whatman No 1 filter paper in a Buchner funnel.
6. Extract the filtrate three times by shaking for 10 min with 50 ml chloroform at 25°C.
7. Pool the chloroform fractions and evaporate to dryness on a rotary evaporator at reduced pressure and at 47°C.
8. Dissolve dried extracts in 200µl methanol and store at -70°C until use.

Detection of gliotoxin by TLC

1. Spot 10µl of the chloroform extract and a 1mgml^-1 gliotoxin standard in methanol onto a Silicagel 60 plate with fluorescence indicator approximately 3cm from the bottom edge.
2. Develop for 10cm in an inlined tank with a methanol:chloroform (10:90, v:v) solvent system.
3. Air dry the plates and spray with 25% sulphuric acid in distilled water and heat at 110°C for 15min to reveal the gliotoxin spots.

Detection of gliotoxin by HPLC

(based on method by Richard et al. 1989)

1. Dilute the gliotoxin extract 1/50 in 50% methanol 50% deionized-distilled water (the mobile phase) and inject 20µl into the HPLC.
2. Construct a standard curve of peak height versus gliotoxin by injection of 20µl of gliotoxin standards (50, 100 and 200ngml^-1 dissolved in the mobile phase) into the HPLC
3. Calculate the amount of gliotoxin in the samples from the standard curve.

Lung fibroblast (L929) cell assay:

Resurrection and preparation of cells

1. Culture mouse lung fibroblast cells (L929) in Dulbecco's modified eagle's medium (DMEM) containing 10% foetal calf serum (FCS).
2. When resurrecting L929 cells from liquid nitrogen thaw the frozen vial gently at 37°C and add with a sterile Pasteur pipette to 9 volumes of Hank's balanced salt solution (HBSS) at 37°C.
3. Centrifuge the cells at 12000rpm for 5min and resuspend the pellet in 1ml DMEM (10x concentrated), and transfer into a 50ml flask with 10ml freshly prepared culture medium (DMEM).
4. Gas with 5% CO₂ in air and incubate for 48h at 37°C.
5. Wash resurrected cells three times with sterile phosphate-buffered saline (PBS) pH 7.3.
6. Add to the culture 1ml of pre-warmed trypsin-EDTA and 1ml PBS and incubate at 37°C for 2min.
7. When the cells detach from the plastic pipette up and down to dissociate clumps and transfer to 1 ml of culture medium.
8. Remove a small volume of cells to count and assess viability with Trypan blue.
9. Transfer cells to a 200ml flask containing three times the original volume DMEM to a final concentration of 5x10⁶ L929 cells ml^-1.
10. Gas the cells with 5% CO₂ in air and incubate at 37°C for 2-3 days.

L929 lung fibroblast assay

1. Label the L929 cells prepared as described above (steps 5 - 7, then do final centrifugation at 12 000rpm and resuspension in 1ml HBSS) with neutral red. To do this add 10 µl of neutral red (0.04% (w/v) in 1% ethylacetate in HBSS) to 90µl of cells in HBSS and incubate for 15 min at 37°C.
2. Centrifuge the cells at 12000 rpm for 5 min and wash twice in DMEM containing 1% FCS
3. Resuspend the cells in the same medium to a concentration of 5x10⁵ cellsml^-1.
4. Add 100µl of this suspension to the wells of a 96-well flat bottomed tissue culture plate.
5. Add an equal volume of A. fumigatus culture supernatant, extracted gliotoxin or pure gliotoxin, serially diluted from 0.5-10µgml^-1 in 1% ethylacetate.
6. Incubate the plates in 5% CO₂ for 2h at 37°C.
7. Discard the culture medium and wash the cell monolayers by adding excess PBS pH 7.3 at room temperature to the wells of the microtitre plate.
8. Discard the PBS and release the neutral red by adding 100µl of 0.05M acetic acid in 50% ethanol to each well.
9. Measure the absorbance in each well at 570nm.

References

1. Richard JL, Lyon RL, Fichtner RE, Ross PF. Use of thin layer chromatography for detection and high performance liquid chromatography for quantitating gliotoxin from rice cultures of Aspergillus fumigatus Fresenius. Mycopathol 1989; 107: 145-151.
2. Belkacemi, L., Barton, R. C., Hopwood, V. and Evans, E. G. V. (1999) Determination of optimum growth conditions for gliotoxin production by Aspergillus fumigatus and development of a novel method for gliotoxin production. Medical Mycology 37:227-233

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