Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA

Abstract

A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system.Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar fr-equencies. Examination of randomly selected transformants by biolistics showed that 15 to 40%O were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.Cryptococcus neoformans is a pathogenic yeast with an unexplained predilection for infecting the central nervous system. There has been a significant increase in the number of human infections with this yeast over the last decade as the immunocompromised population has enlarged because of AIDS. Although epidemiological studies suggest that the increase in cryptococcal infection is due to more-susceptible hosts rather than increased virulence, the pathobiology of the organism needs further investigation. Several phenotypic factors associated with virulence have been identified for C. neoformans: the polysaccharide capsule (10,16,20), melanin production (19,20,26), and growth at 370C (20,26 Recent studies of C neofonnans have established basic molecular techniques for performing site-directed mutagenesis (9, 31). A C. neoformans uraS auxotrophic strain (serotype D) was recently reported to be transformed at a low efficiency by complementation with a cloned uraS gene delivered by electroporation. However, the number of integrative events was low and generally nonhomologous (9, 31). A total of 80 to 90% of the transformants were unstable and apparently contained extrachromosomal DNA, consisting of sequences of rearranged uraS-vector DNA with the addition of cryptococcal genomic DNA (8,9,31

Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis

“…PCR products were cloned into the pHYG-7KB plasmid 50 , between the SacI and XbaI sites using the In-Fusion PCR cloning kit (Clontech). The cloned plasmids were used to transform C. neoformans strains by biolistic transformation 51 . Strains were isolated, and the RNA expression levels of CTR1 and CTR4 were confirmed using real-time PCR.…”

Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of Cryptococcus neoformans

“…A plasmid containing a disrupted tps1 gene was generated. (52). Transformants were then transferred to YNB-uracil dropout-glucose and YNB-uracil dropout-galactose plates in order to identify those unable to grow at 37°C on glucose.…”

Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans The GenBank accession number for the RAS2 sequence of C. neoformans H99 is AF294349.

“…The linear, dideoxy-tailed ras2∆ ::URA5 disruption construct was transformed into strains H99-ura5 and LCC70 by biolistic DNA delivery, and transformants were selected on synthetic medium lacking uracil with 1 M sorbitol as described by Toffaletti et al (1993). Transformants were screened using PCR to identify putative ras2 mutants.…”