Data_Sheet_1_RNAi as a Tool to Study Virulence in the Pathogenic Yeast Candida glabrata.pdf

The yeast Candida glabrata is a major opportunistic pathogen causing mucosal and systemic infections in humans. Systemic infections caused by this yeast have high mortality rates and are difficult to treat due to this yeast’s intrinsic and frequently adapting antifungal resistance. To understand and treat C. glabrata infections, it is essential to investigate the molecular basis of C. glabrata virulence and resistance. We established an RNA interference (RNAi) system in C. glabrata by expressing the Dicer and Argonaute genes from Saccharomyces castellii (a budding yeast with natural RNAi). Our experiments with reporter genes and putative virulence genes showed that the introduction of RNAi resulted in 30 and 70% gene-knockdown for the construct-types antisense and hairpin, respectively. The resulting C. glabrata RNAi strain was used for the screening of a gene library for new virulence-related genes. Phenotypic profiling with a high-resolution quantification of growth identified genes involved in the maintenance of cell integrity, antifungal drugs, and ROS resistance. The genes identified by this approach are promising targets for the treatment of C. glabrata infections.

酿酒酵母毛霉状念珠菌是人类黏膜和系统性感染的主要机会性病原体。由该酵母引起的系统性感染具有高死亡率，且由于该酵母固有的、频繁进化的抗真菌耐药性，治疗难度较大。为了理解和治疗毛霉状念珠菌感染，研究该菌致病性和耐药性的分子基础至关重要。本研究通过表达来自酵母菌酿酒酵母城堡菌（一种具有天然RNA干扰能力的芽生酵母）的Dicer和Argonaute基因，在毛霉状念珠菌中建立了RNA干扰（RNAi）系统。我们的实验表明，通过报道基因和推测的致病基因，RNAi的引入导致了抗义和发夹型构建体中基因敲低率分别达到30%和70%。由此得到的毛霉状念珠菌RNAi菌株被用于筛选新的致病相关基因的基因库。通过高分辨率生长量化的表型分析，识别出参与维持细胞完整性、抗真菌药物和活性氧（ROS）抵抗的基因。该研究方法所识别的基因有望成为治疗毛霉状念珠菌感染的治疗靶点。