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 produc....

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....

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