Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Summary

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into C. neoformans.

Abstract

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide. Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only biolistic delivery provides an effective means to transform linear DNA that can be integrated into the genome by homologous recombination. 

Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known. A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into C. neoformans. We then confirm integration of the ACK-mCherry fusion into the ACK locus.

Introduction

Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis resulting in more than 625,000 deaths per year worldwide ¹. Acetate has been shown to be a major fermentation product during cryptococcal infection ^2,3,4, and genes encoding enzymes from three putative acetate-producing pathways have been shown to be upregulated during infection ⁵. This suggests that acetate production and transport may be a necessary and required part of the pathogenic process; however, the significance of this is not yet understood. One possible pathway for acetate production is the xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) - acetate kinase (Ack), a pathway previously thought to be present only in bacteria but recently identified in both euascomycete as well as basidiomycete fungi, including C. neoformans ⁶.

To determine the localization of these enzymes of this pathway in the cell, a construct carrying a neomycin resistance gene downstream of an ACK gene fusion to the fluorescent tag mCherry (ACK:mCherry:Neo) will be introduced into C. neoformans using the well-established method of biolistic transformation ^7,8. Although electroporation is an efficient method for transformation of plasmids that will be maintained as episomes into Cryptococcus ⁹, it is not useful in creating stable homologous transformants ⁸. Only biolistic delivery using a gene gun provides an effective means to transform linear DNAs that will be integrated into the genome by homologous recombination. For example, Edman et al. showed that of the transformants resulting from electroporation of a plasmid-borne URA5 selectable marker into C. neoformansura5 mutants, just 0.001 to 0.1% of transformants were stable ⁹. Chang et al. achieved just a 0.25% stable transformation efficiency using electroporation to reconstitute capsule production in an acapsular mutant ¹⁰. Unlike electroporation, biolistic transformation has been shown to result in stable transformation efficiency of 2-50% depending on the gene that is being altered ^7,8,11.

This visual experiment will provide a step-by-step demonstration of biolistic transformation of the linear ACK:mCherry:Neo DNA construct into C. neoformans, and will describe how to confirm its proper integration via homologous recombination into the ack locus. The protocol demonstrated here is a modification of the method developed in the Perfect laboratory ⁸.

Protocol

NOTE: The overall scheme of this protocol is outlined in Figure 1.

1. C. neoformans Preparation

1. For each transformation reaction, grow a 2-3 ml O/N culture of C. neoformans in YPD medium at 30 °C shaking at 250 rpm.
2. Centrifuge the O/N culture for 5 min at 900 x g at 10 °C and discard the supernatant.
3. Resuspend each cell pellet in 300 μl of Yeast Peptone Dextrose (YPD) medium.
4. Using glass beads, gently spread 300 μl of the washed cell suspension onto YPD agar containing 1 M sorbitol.
5. Allow plates to dry at ambient temperature for 3-4 hr.

2. Gold Microcarrier Preparation

1. Resuspend 0.25 g of 0.6 μm gold beads in 1 ml of ddH₂O, centrifuge for 1 min at 900 x g to pellet the beads, and remove the supernatant.
2. Resuspend the gold beads in 1 ml of 100% ethanol.
3. Distribute the beads into 4 tubes, 250 μl each, and add 750 μl of 100% ethanol.
4. Store gold bead aliquots at 4 °C.

3. DNA Preparation

1. Prepare orange macrocarrier biolistic discs by submerging them in 100% ethanol using forceps. Place discs into a large petri dish containing drierite to dry (make sure the drierite does not touch the discs).
2. Once dry, press the macrocarrier discs into the silver disc holders (previously wiped down with 100% ethanol).
3. Vortex gold beads (prepared as in step 2) and aliquot 12 µl into a 1.5 ml microcentrifuge tube, one tube per transformation.
4. Add to each tube in order: 2 µg of DNA (preferably 2 µl of 1 µg/µl of DNA), 10 μl 2.5 M CaCl₂, and 2 μl 1 M spermidine free base.
5. Set up a negative control as in step 3.4 but with no DNA.
6. Vortex each tube and incubate at ambient temperature for 5 min. Gently flick each tube occasionally to resuspend the settled beads during this incubation.
7. Spin tubes at 225 x g for 30 sec to pellet the DNA-coated gold beads. Carefully remove the supernatant (by pipetting or aspiration) and discard.
8. Resuspend beads completely in 600 µl of 100% ethanol by slowly pipetting up and down.
9. Spin tubes at 225 x g for 30 sec to pellet the beads without packing. Carefully remove and discard the supernatant.
10. Resuspend the DNA-coated gold beads in 8 µl of 100% ethanol by slowly pipetting up and down.
11. Pipette the DNA-coated gold beads onto the center of the biolistic disc in a 1 cm diameter and allow to dry.
    NOTE: A dried gold circle visible on the center of the biolistic disc indicates that a sufficient concentration of gold beads is present.
    NOTE: The macrocarrier discs loaded with DNA-coated gold beads are now ready for use with the gene gun.

4. Operating the Gene Gun

1. Turn on the vacuum pump.
2. Turn on the helium gas by turning the knob counterclockwise until a pressure of approximately 2,200 psi is reached on the pressure gauge.
3. Turn on the gene gun by flipping the red switch on the left.
4. Be sure that the flow rates for the vacuum and the vent are adjusted so the vacuum will reach 28 inches Hg within 15 sec.
5. Be sure the distance between the rupture disc and macrocarrier is approximately 3/8 inch.
6. Clean the entire chamber by wiping down with ethanol.
7. Submerge the rupture discs in 100% ethanol. Allow to dry on a sterile surface (e.g., Petri dish).
8. Use a torque wrench to loosen the rupture disc holder. Insert a clean rupture disc into the holder. Screw the rupture disc holder back into place and tighten with torque wrench by turning it once to the right.
   NOTE: Rupture discs will be replaced following each shoot.
9. Submerge the mesh screens in 100% ethanol. Allow to dry on a sterile surface (e.g., Petri dish).
10. Once dry, place a washed mesh screen on the white plastic mounting plate. Place the macrocarrier disc holder DNA side down into the disc chamber. Screw on the silver cap, and place the mounting plate in the highest slot.
    NOTE: The mesh screen will be replaced after each shoot.
11. Place a YPD agar plate containing 1 M sorbitol on the bottom plate.
12. Shut chamber door and lock into place.
13. Push and hold the middle red switch up to engage vacuum and allow the vacuum to reach 28 inches Hg. Once proper vacuum level is reached, move this switch to the down position. When ready, hold down the red switch on the right to fire. When the rupture disc pops, immediately release the fire button and push the middle red switch to the middle position to vent the chamber to 0 psi.
14. Clean out rupture disc debris and turn off the gene gun. Then turn off the helium gas by turning the knob clockwise, and finally, turn off the vacuum pump.

5. Plating Transformed Cells

1. Allow the transformation plates to sit at RT for 4 hr to allow the cells to recover.
2. Pipette 700 µl of YPD onto the plate. Use a cell scraper to gently scrape the cells off of the agar and pipette liquid into a sterile 1.5 ml microfuge tube. Repeat this step to ensure all cells have been recovered from the plate.
3. Pellet cells at 225 x g for 30 sec. Remove and discard the supernatant.
4. Resuspend the pellet in 500 µl of YPD.
5. Pipette 100 µl of the cell suspension onto the center of the YPD + antibiotic plates and spread using glass beads.
6. Leave inverted plates at RT for 3-4 days. As colonies appear, patch onto new YPD + antibiotic plates.

6. Genomic DNA Isolation for PCR

NOTE: This is a modified version using reagents from a DNA purification kit (See Table of Materials).

1. Grow a 5 ml culture of each of the C. neoformans transformants in YPD liquid at 30 °C shaking at 250 rpm O/N.
2. Pellet 3 ml of cells at 900 x g, and resuspend in 600 µl of nuclei lysis solution.
3. Add the suspension to a new 1.5 ml microcentrifuge tube with 200 µl of 0.5 mm acid washed glass beads.
4. Homogenize for 45 sec in a mini beadbeater at ambient temperature, cool tube on ice, and repeat.
5. Allow sample to settle on ice for 2 min and transfer supernatant to a new 1.5 ml tube. Add 200 µl of protein precipitation solution to each tube, (100 µl for every 600 µl of supernatant recovered) and vortex vigorously for 20 sec.
6. Allow samples to settle on ice for 5 min, and centrifuge at 11,000 x g for 3 min.
7. Transfer the supernatant to a clean 1.5 ml tube containing 300 µl of RT isopropanol. Gently mix by inversion.
8. Centrifuge samples at 11,000 x g for 2 min, carefully remove the supernatant, and drain the tubes onto paper towels.
9. Add 300 µl of RT 70% ethanol to each tube, and gently invert to wash the pellet.
10. Centrifuge samples at 11,000 x g for 2 min, and carefully remove all of the ethanol.
11. Drain the tube onto clean paper towels, and allow the pellet to air dry for 10-15 min.
12. Add 50 µl of DNA rehydration solution and 1.5 µl of RNase solution to each pellet and vortex.
13. Centrifuge samples for 5 sec to remove all of the liquid from the cap.
14. Incubate samples at 37 °C for 15 min.
15. Rehydrate the DNA by incubating the samples at 65 °C for 1 hr.
16. Quantify DNA spectrophotometrically by measuring the absorbance at 260 nm (an A₂₆₀ reading of 1.0 is equivalent to ~50 µg/ml double-stranded DNA), and use up to 200 ng in each PCR reaction.

7. RNA Isolation for Reverse Transcriptase-PCR.

1. Using a RNA purification kit (See Table of Materials), follow the manufacturer’s instructions to isolate RNA from yeast cells using a minibeadbeater.
2. Quantify the concentration of the RNA by measuring the absorbance at 260 nm (an A₂₆₀ reading of 1.0 is equivalent to ~40 µg/ml single-stranded RNA).
3. Using an RT-PCR kit (See Table of Materials), follow the manufacturer’s instructions to set up RT-PCR reactions with approximately 1 µg of RNA. For the results obtained in this study, use the primers listed in Table 1.

Results

A successful biolistic transformation of C. neoformans can be obtained by following this protocol scheme (Figure 1). With biolistic transformation, a successful shoot of the coated gold beads is indicated by a gold ring visible on the plate after the DNA is shot (Figure 2A). Colonies should appear within 4 to 5 days when left at room temperature after plating the recovered cells from the YPD + 1M sorbitol plates onto selective media. Transforming 2 µg of DNA should result in 20 ...

Discussion

Utilizing this protocol, biolistic transformation can be accomplished in which linear DNA is integrated into a desired locus in the Cryptococcus neoformans genome by homologous recombination. Certain steps in the protocol can have a dramatic effect on the effectiveness/efficiency of the transformation. For a successful transformation, it is imperative that the DNA utilized in the shoot has a concentration of at least 1 µg. However, the volume of DNA added to the gold beads can be increased in the chance the...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.6 μm gold beads	Bio-Rad	165-2262	http://www.bio-rad.com
Spermadine-free base	Sigma- Aldrich	S0266	https://www.sigmaaldrich.com
G418 - Sulfate (Neomycin)	Gold Biotechnology	G-418-10	www.goldbio.com
Hygromycin	Gold Biotechnology	H-270-1	www.goldbio.com
1350 psi Rupture Discs	Bio-Rad	165-2330	http://www.bio-rad.com
Stopping Screens	Bio-Rad	165-2336	http://www.bio-rad.com
Macrocarriers discs	Bio-Rad	165-2335	http://www.bio-rad.com
YPD Broth	Becton Dickinson & Co.	242820	www.bd.com
Agar	Becton Dickinson & Co.	214530	www.bd.com
Sorbitol	Fisher Scientific	BP439	http://www.fishersci.com
PDS-1000/He System	Bio-Rad	165-2257	http://www.bio-rad.com
Microscope	Zeiss	Axio	http://www.zeiss.com/microscopy
KOD One Step PCR Kit	EMD Millipore	71086-4	http://www.emdmillipore.com
One Step RT-PCR Kit	Qiagen	210212	www.qiagen.com
Wizard Genomic DNA Purification Kit	Promega	A1120	www.promega.com
RNeasy Mini Kit	Qiagen	74104	www.qiagen.com
Mini Beadbeater - 1	BioSpecs	3110BX	http://www.biospec.com
Microfuge 18 Centrifuge	Beckman Coulter	F241.5P	www.beckmancoulter.com
Microplate Spectrophotometer	BioTek	EPOCH	www.biotek.com