Department Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Adolf-Reichwein-Straße 23, 07745 Jena, Germany.
Institute of Microbiology, Friedrich Schiller University, Neugasse 25, 07743 Jena, Germany.
FEMS Yeast Res. 2021 Jan 6;20(8). doi: 10.1093/femsyr/foaa066.
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.
虽然不像其相对的白色念珠菌那样普遍，但光滑念珠菌是一种成功的人类病原体，可引起危及生命的念珠菌病。因此，了解光滑念珠菌的致病性机制和相关基因至关重要。然而，基因互补作为恢复先前删除基因功能的工具，在光滑念珠菌中尚未标准化，其使用频率也低于白色念珠菌。在本研究中，我们建立了一种使用 TRP1 基因座基因组整合进行基因互补的策略。我们证明，我们的方法不仅可用于互补盒的整合，还可用于标记物（如荧光蛋白和抗原卵清蛋白）或潜在致病性相关因素（如生物素转运基因 VHT1）的过表达。以尿素酰胺酶 Dur1,2 为例，我们展示了基因互补方法在表达序列修饰基因中的应用。通过该方法，我们发现 Dur1,2 生物素化基序中的赖氨酸到精氨酸突变会损害光滑念珠菌和白色念珠菌依赖尿素的生长。总之，基于 TRP1 的基因互补方法是研究新基因功能及其在光滑念珠菌病理生物学中作用的有价值的工具。
