Enhancing the chemical transformation of <i>Candida parapsilosis</i>

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.近平滑念珠菌（Candida parapsilosis）是侵袭性真菌病的主要致病菌之一，同时亦是低出生体重、极低出生体重新生儿医院获得性真菌血症的首要致病原因。然而，目前针对该真菌的分子与生理特性研究尚未得到充分探索。为深化对该病原菌致病机制的认知，本研究旨在开发并验证一种适用于近平滑念珠菌的高效化学转化方法。化学转化是将外源DNA导入念珠菌酵母的主流实验方法，因其无需特殊实验设备；但当转化所用DNA片段的长度增大时，该方法的转化效能会迅速降低。为明确近平滑念珠菌化学转化的最优条件，本研究选取了一株亮氨酸营养缺陷型（leucine auxotroph）实验室菌株。我们确定了可使转化效率达到峰值的最优细胞密度、孵育时长、特异性增强化学试剂添加方案，以及转化用DNA片段的最佳长度与投入量。实验结果显示，添加二甲基亚砜（dimethyl sulfoxide, DMSO）可有效提升转化效率，但二硫苏糖醇（dithiothreitol, DTT）预处理会降低菌落回收率。经优化后的实验方案可使转化效率提升20~55倍，提升幅度取决于转化用DNA片段的长度。我们使用原养型分离株对优化后的方法进行了验证，结果表明，在6株分离株中有5株可通过该新方法获得显著更多的转化子。此外，本研究还筛选出一种专用培养基，可将制备好的转化感受态酵母细胞于-80℃条件下安全保存长达6周，该方案可减少实验室工作量并简化整体实验流程。上述优化将为后续开展近平滑念珠菌毒力相关遗传基础的研究提供有力支撑。