Enhancing the chemical transformation of Candida parapsilosis

Abstract

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.