ABSTRACT

Although less prevalent than its relative Candida albicans, the yeast Candida glabrata is a successful pathogen of humans, which causes life-threatening candidiasis. It is thus vital to understand the pathogenicity mechanisms and contributing genes in C. glabrata. However, gene complementation as a tool for restoring the function of a previously deleted gene is not standardized in C. glabrata, and it is less frequently used than in C. albicans.In this study, we established a gene complementation strategy using genomic integration at the TRP1 locus. We prove that our approach can not only be used for integration of complementation cassettes, but also for overexpression of markers like fluorescent proteins and the antigen ovalbumin, or of potential pathogenicity-related factors like the biotin transporter gene VHT1. With urea amidolyase Dur1,2 as an example, we demonstrate the application of the gene complementation approach for the expression of sequence-modified genes. With this approach, we found that a lysine-to-arginine mutation in the biotinylation motif of Dur1,2 impairs urea-dependent growth of C. glabrata and C. albicans. Taken together, the TRP1-based gene complementation approach is a valuable tool for investigating novel gene functions and for elucidating their role in the pathobiology of C. glabrata.

中文翻译：

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用于平滑念珠菌中基因互补、过表达、报告基因表达和基因修饰的基于 TRP1 标记的系统

摘要

虽然不如其相对的白色念珠菌流行，但酵母光滑念珠菌是一种成功的人类病原体，它会导致危及生命的念珠菌病。因此，了解C. glabrata的致病机制和相关基因至关重要。然而，作为恢复先前删除基因功能的工具的基因互补在光滑念珠菌中没有标准化，并且它的使用频率低于白色念珠菌。在这项研究中，我们建立了使用基因组整合的基因互补策略。在TRP1轨迹。我们证明我们的方法不仅可以用于互补盒的整合，还可以用于荧光蛋白和抗原卵清蛋白等标记物的过度表达，或生物素转运蛋白基因VHT1等潜在致病相关因素的过度表达。以尿素酰胺酶 Dur1,2 为例，我们展示了基因互补方法在序列修饰基因表达中的应用。通过这种方法，我们发现 Dur1,2 的生物素化基序中的赖氨酸到精氨酸突变会损害C. glabrata和C. albicans 的尿素依赖性生长。综合来看，TRP1基于基因互补的方法是研究新基因功能和阐明它们在光滑念珠菌病理生物学中的作用的宝贵工具。