Enhancing the chemical transformation of Candida parapsilosis

Abstract

Candida parapsilosis is a leading cause of invasive mycoses and the major cause of nosocomial fungaemia amongst low and very low birth weight neonates. However, the molecular and physiological characteristics of this fungus remain understudied. To advance our knowledge about the pathobiology of this pathogen, we sought to develop and validate an effective method for chemical transformation of C. parapsilosis . Chemical transformation is the primary procedure for introducing foreign DNA into Candida yeast as it requires no special equipment, although its performance efficacy drops rapidly when the size of the transforming DNA increases. To define optimal conditions for chemical transformation in C. parapsilosis , we selected a leucine auxotroph laboratory strain. We identified optimal cell density for transformation, incubation times, inclusion of specific enhancing chemicals, and size and amounts of DNA fragments that resulted in maximized transformation efficiency. We determined that the inclusion of dimethyl sulfoxide was beneficial, but dithiothreitol pretreatment reduced colony recovery. As a result, the modified protocol led to a 20–55-fold increase in transformation efficiency, depending on the size of the transforming fragment. We validated the modified methodology with prototrophic isolates and demonstrated that the new approach resulted in the recovery of significantly more transformants in 5 of 6 isolates. Additionally, we identified a medium in which transformation competent yeast cells could safely be maintained at −80°C for up to 6 weeks that reduces laboratory work and shortens the overall procedure. These modifications will significantly aid further investigations into the genetic basis for virulence in C. parapsilosis.

Characterization and functional analysis of zinc trafficking in the human fungal pathogen Candida parapsilosis

“…The previously investigated yeast species S. cerevisiae , C. gattii C. albicans and C. dubliniensis each encode only two Zip-type cellular zinc importers. In S. cerevisiae , C. gattii and C. dubliniensis , the Zap1 transcription factor is responsible for the regulation of Zip importers in response to environmental zinc levels [25–27]. Candida albicans Zap1 also regulates the zinc regulon in this species [35].…”

“…For reintegration, CpZRT21 and CpZRC1 were introduced into the CpNEUT5 L neutral locus using the Gateway cloning method as adapted to C. parapsilosis by Németh et al [26]. For functional restoration, CpZRC1 with a CaTDH3 promoter was introduced into the mutant strain Δ/Δ CpZRC1 by transformation as described by Németh et al [27].…”

“…For phagocytosis experiments, GFP-labelled CLIB 214 RI, Δ/Δ CpZRT21 and Δ/Δ CpZRC1 cells were used, where the CaTDH3 -GFP fusion construction was integrated into the CpNEUT5 L neutral locus as described by Németh et al [27].…”

CRISPR-Cas9 editing induces Loss of Heterozygosity in the pathogenic yeast Candida parapsilosis

“…Yeast cells were transformed with 5 μg of the relevant plasmid and either 25 μL of unpurified repair template for editing the gene (eRT) or 5 μg of purified repair template for deleting the gene (delRT), using the lithium acetate method described in (47), with minor modifications (starting OD600nm of YPD culture 0.1 instead of 0.05). Transformants were selected onto YPD agar plates containing 200 μg/mL nourseothricin (Jena Bioscience GmbH, Germany) and screened by colony PCR to confirm the presence of the mutation (Table S2).…”

“…Yeast cells were transformed with 5 μg of the relevant plasmid and either 25 μL of unpurified repair template for editing the gene (eRT) or 5 μg of purified repair template for deleting the gene (delRT), using the lithium acetate method described in (47), with minor modifications (starting OD 600nm of YPD culture 0.1 instead of 0.05).…”