Functional genomic analysis of genes important for Candida albicans fitness in diverse environmental conditions. Functional genomic analysis of genes important for Candida albicans fitness in diverse environmental conditions

As the limited arsenal of effective antifungals in clinical use is threatened by emerging resistance, the demand for new strategies to combat invasive fungi is urgent. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leveraged a pooled functional genomic screening strategy to identify and characterize genes important for fitness of the human fungal pathogen Candida albicans in diverse conditions. Through this approach we identified a novel essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrated that it encodes subunit 3 of replication factor A, Rfa3. Furthermore, we applied robust computational analyses to leverage profile similarity analysis of mutant phenotypes across conditions to identify functionally-coherent gene clusters and predict gene function. Through this approach, we correctly predicted the cell cycle-associated function of C3_06880W, a previously uncharacterized genes required for fitness specifically at elevated temperatures. Protein structure predictions coupled with genetic and biochemical assays confirmed C3_06880W encodes Iml3, a component of the C. albicans kinetochore with key roles in C. albicans virulence in vivo. Overall, this work reveals novel insights into the vulnerabilities of C. albicans. Overall design: To enable systematic fitness comparisons between diverse growth conditions, we utilized a pooled functional genomics approach for massively parallel analysis of Candida albicans GRACE strains to assess each strain’s fitness profile through quantification of strain-specific barcodes by next-generation sequencing. Specifically, a pooled collection of 2,238 barcoded GRACE strains covering ~35% of the genome was grown in triplicate in the presence and absence of DOX for 24 hours, then sub-cultured into matching fresh medium with or without DOX for an additional 48-hour incubation. ). Screens in all conditions were performed in technical triplicate and biological duplicate apart from the baseline condition (YNB at 30 ˚C), which was tested in five biological replicates. After growth of the cultures, strain-specific molecular barcodes were amplified from extracted gDNA, and pooled barcode PCR products were subjected to next-generation sequencing.

临床可用的有效抗真菌药物储备库正受新兴耐药性的威胁，开发对抗侵袭性真菌的新策略已迫在眉睫。拓展治疗靶点范围的一种有效途径，是鉴定在宿主相关环境中对病原体生长至关重要的基因。本研究采用混合功能基因组筛选（pooled functional genomic screening）策略，对人类真菌病原体白色念珠菌（Candida albicans）在多种培养条件下的适应性相关基因进行鉴定与表征。通过该方法，我们鉴定得到一个新的必需基因C1_09670C，该基因在酿酒酵母（Saccharomyces cerevisiae）中暂无已知同源基因，并证实其编码复制因子A（replication factor A）的3号亚基Rfa3。此外，我们通过严谨的计算分析，利用不同条件下突变体表型的特征相似性分析，鉴定出功能相关的基因簇并预测基因功能。通过该策略，我们准确预测了C3_06880W的细胞周期相关功能——该基因此前未被表征，且仅在高温环境下对适应性至关重要。结合蛋白质结构预测与遗传、生化实验，我们证实C3_06880W编码Iml3，它是白色念珠菌动粒（kinetochore）的组成成分，在体内对白色念珠菌的毒力发挥关键作用。总体而言，本研究为白色念珠菌的致病易感机制提供了全新见解。整体实验设计：为实现不同生长条件下的系统性适应性比较，我们采用混合功能基因组学方法，对白色念珠菌GRACE菌株进行大规模平行分析，通过下一代测序（next-generation sequencing）定量菌株特异性条形码（barcode）以评估各菌株的适应性特征。具体而言，我们将覆盖约35%基因组的2238株带条形码的GRACE菌株集合，在添加与不添加多西环素（DOX）的培养基中分别进行三次重复培养24小时，随后转接至匹配的新鲜含/不含DOX的培养基中，继续孵育培养48小时。除基础条件（30℃的酵母氮源基础培养基，YNB）设置了五次生物学重复外，所有条件下的筛选均设置三次技术重复与两次生物学重复。培养结束后，从提取的基因组DNA（genomic DNA，gDNA）中扩增菌株特异性分子条形码，将混合的条形码PCR产物进行下一代测序。