二倍体白色念珠菌中必需基因的启动子关闭通常不足以产生紧密的,由于泄漏表达而导致的条件无效等位基因,一直是发病机理研究中的绊脚石。此外,由于突变菌株中频繁的非整倍性,非必需基因的纯合缺失通常是有问题的。快速,通过锚定策略对必需基因的有条件消耗已成功用于酿酒酵母和其他模型生物。这里,雷帕霉素介导人FK506结合蛋白(FKBP12)和含有FKBP12-雷帕霉素结合(FRB)结构域的靶蛋白的二聚化,导致重新定位到改变的亚细胞位置。在这项工作中,我们使用核糖体蛋白Rpl13作为锚,并以两个核蛋白作为靶标,以原理证明方法构建了一组突变体.我们首先通过在CaTOR1基因中引入显性突变和RBP1的纯合缺失来构建耐雷帕霉素的白色念珠菌菌株,RBP1是雷帕霉素的主要靶标FKBP12的直系同源物。然后通过定点诱变对FKBP12和FRB编码序列进行CUG密码子调整以适应白色念珠菌。构建表达必需TBP1基因或非必需SPT8基因作为FRB融合体的锚定菌株。我们发现雷帕霉素在15分钟内导致TBP-AA菌株的生长迅速停止,而SPT8-AA菌株则表现出spt8Δ/spt8Δ突变体的组成型丝状表型。因此,这里开发的白色念珠菌锚定工具箱可用于全基因组分析,以快速可靠的方式鉴定基因功能,进一步加速白色念珠菌抗真菌药物的开发。重要分子遗传学研究迄今为止已经确定,在总共6,198个单倍体开放阅读框中,约27%的开放阅读框对于白念珠菌在丰富培养基中的营养生长至关重要。然而,一个主要的限制是构建基本白色念珠菌基因的快速条件等位基因,几乎定量消耗编码的蛋白质。这里,我们开发了一个工具箱,用于快速和有条件地耗尽基因,这将有助于研究必需和非必需基因的基因功能。
Promoter shutoff of essential genes in the diploid Candida albicans has often been insufficient to create tight, conditional null alleles due to leaky expression and has been a stumbling block in pathogenesis research. Moreover, homozygous deletion of non-essential genes has often been problematic due to the frequent aneuploidy in the mutant strains. Rapid, conditional depletion of essential genes by the anchor-away strategy has been successfully employed in Saccharomyces cerevisiae and other model organisms. Here, rapamycin mediates the dimerization of human FK506-binding protein (FKBP12) and FKBP12-rapamycin-binding (FRB) domain-containing target protein, resulting in relocalization to altered sub-cellular locations. In this work, we used the ribosomal protein Rpl13 as the anchor and took two nuclear proteins as targets to construct a set of mutants in a proof-of-principle approach. We first constructed a rapamycin-resistant C. albicans strain by introducing a dominant mutation in the CaTOR1 gene and a homozygous deletion of RBP1, the ortholog of FKBP12, a primary target of rapamycin. The FKBP12 and the FRB coding sequences were then CUG codon-adapted for C. albicans by site-directed mutagenesis. Anchor-away strains expressing the essential TBP1 gene or the non-essential SPT8 gene as FRB fusions were constructed. We found that rapamycin caused rapid cessation of growth of the TBP-AA strain within 15 minutes and the SPT8-AA strain phenocopied the constitutive filamentous phenotype of the spt8Δ/spt8Δ mutant. Thus, the anchor-away toolbox for C. albicans developed here can be employed for genome-wide analysis to identify gene function in a rapid and reliable manner, further accelerating anti-fungal drug development in C. albicans.
OBJECTIVE: Molecular genetic studies thus far have identified ~27% open-reading frames as being essential for the vegetative growth of Candida albicans in rich medium out of a total 6,198 haploid set of open reading frames. However, a major limitation has been to construct rapid conditional alleles of essential C. albicans genes with near quantitative depletion of encoded proteins. Here, we have developed a toolbox for rapid and conditional depletion of genes that would aid studies of gene function of both essential and non-essential genes.