Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary.
Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA.
Virulence. 2021 Dec;12(1):937-950. doi: 10.1080/21505594.2021.1893008.
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 . Chemical transformation is the primary procedure for introducing foreign DNA into 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 , 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
是侵袭性真菌感染的主要原因，也是低出生体重和极低出生体重儿医院内真菌感染的主要原因。然而，这种真菌的分子和生理特性仍研究不足。为了增进我们对这种病原体的病理生物学的认识，我们试图开发和验证一种有效的方法来对 进行化学转化。化学转化是将外源 DNA 引入 酵母的主要程序，因为它不需要特殊设备，尽管当转化 DNA 的大小增加时，其性能效果会迅速下降。为了确定 在化学转化中的最佳条件，我们选择了一种亮氨酸营养缺陷型实验室菌株。我们确定了转化的最佳细胞密度、孵育时间、特定增强化学物质的包含、以及导致最大转化效率的 DNA 片段的大小和数量。我们确定包含二甲基亚砜是有益的，但二硫苏糖醇预处理会降低菌落回收率。因此，改良的方案导致转化效率提高了 20-55 倍，具体取决于转化片段的大小。我们用原养型分离株验证了改良的方法，并证明新方法在 6 个分离株中的 5 个中导致了更多转化子的恢复。此外，我们还确定了一种培养基，其中转化的酵母细胞能够在-80°C下安全保存长达 6 周，这减少了实验室工作并缩短了整个过程。这些改进将极大地帮助进一步研究在 中的毒力遗传基础。
