title: Biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen Cryptococcus 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.
authors: Department of Genetics and Biochemistry, Eukaryotic Pathogens Innovation Center (EPIC), Clemson University.
authors: Tonya
authors: T
authors: Taylor
authors: Department of Biology, Western Carolina University.
authors: Indrani
authors: I
authors: Bose
authors: Department of Genetics and Biochemistry, Eukaryotic Pathogens Innovation Center (EPIC), Clemson University.
authors: Taylor
authors: T
authors: Luckie
authors: Department of Genetics and Biochemistry, Eukaryotic Pathogens Innovation Center (EPIC), Clemson University; kssmith@clemson.edu.
authors: Kerry
authors: K
authors: Smith
mesh_terms: D000084:Acetate Kinase / Q000235:genetics / Q000378:metabolism; D000446:Aldehyde-Lyases / Q000235:genetics / Q000378:metabolism; D019677:Artificial Gene Fusion / Q000379:methods*; D019470:Biolistics / Q000379:methods*; D003455:Cryptococcus neoformans / Q000201:enzymology / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D018274:Electroporation / Q000379:methods; D017930:Genes, Reporter; D059765:Homologous Recombination; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010428:Pentosephosphates / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D000084:Acetate Kinase; D000446:Aldehyde-Lyases; D004271:DNA, Fungal; D008164:Luminescent Proteins; D010428:Pentosephosphates; D000097573:Red Fluorescent Protein; C031625:xylulose-5-phosphate; C065556:fructose-6-phosphate phosphoketolase