A Multifunctional, Synthetic Gaussia princeps Luciferase Reporter for Live Imaging of Candida albicans Infections
A Multifunctional, Synthetic Gaussia princeps Luciferase Reporter for Live Imaging of Candida albicans Infections
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DOI：
10.1128/iai.00223-09
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发表时间：
2009-11-01
影响因子：
3.1
通讯作者：
d'Enfert, Christophe
中科院分区：
文献类型：
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作者：
Enjalbert, Brice;Rachini, Anna;d'Enfert, Christophe
实时监测动物感染/基因表达的时空进展将大大有助于我们理解宿主-病原体相互作用，同时减少产生统计学显著数据集所需的动物数量。灵敏的体内成像技术可以检测体内荧光素酶报告基因发出的低水平光，但现有的报告基因对于真菌感染不是最佳的。因此，我们的目标是开发一种新的报告系统成像白色念珠菌感染，克服了目前的荧光素酶报告这种主要的真菌病原体的局限性。这种荧光素酶报告基因是通过将合成的、密码子优化的Gaussia princeps荧光素酶基因与C.白色念珠菌PGA 59，其编码糖基磷脂酰肌醇连接的细胞壁蛋白。从该PGA 59-gLUC融合体（称为gLUC 59）表达的荧光素酶定位于C.白色念珠菌细胞表面，允许检测完整细胞中的荧光素酶。对强（ACT 1和EFT 3）、氧化应激诱导（TRX 1、TRR 1和IPF 9996）和形态发生依赖（HWP 1）启动子的融合分析证实，gLUC 59是酵母或菌丝细胞基因调控研究的方便和敏感的报告基因，也是一种灵活的筛选工具。此外，ACT 1-gLUC 59融合是浅表和皮下C.白色念珠菌感染gLUC 59和相关的细胞表面暴露的荧光素酶报告基因可能在分子生物学、细胞生物学、病理生物学和高通量筛选中找到广泛的应用。
Real-time monitoring of the spatial and temporal progression of infection/gene expression in animals will contribute greatly to our understanding of host-pathogen interactions while reducing the number of animals required to generate statistically significant data sets. Sensitive in vivo imaging technologies can detect low levels of light emitted from luciferase reporters in vivo, but the existing reporters are not optimal for fungal infections. Therefore, our aim was to develop a novel reporter system for imaging Candida albicans infections that overcomes the limitations of current luciferase reporters for this major fungal pathogen. This luciferase reporter was constructed by fusing a synthetic, codon-optimized version of the Gaussia princeps luciferase gene to C. albicans PGA59, which encodes a glycosylphosphatidylinositol-linked cell wall protein. Luciferase expressed from this PGA59-gLUC fusion (referred to as gLUC59) was localized at the C. albicans cell surface, allowing the detection of luciferase in intact cells. The analysis of fusions to strong (ACT1 and EFT3), oxidative stress-induced (TRX1, TRR1, and IPF9996), and morphogenesis-dependent (HWP1) promoters confirmed that gLUC59 is a convenient and sensitive reporter for studies of gene regulation in yeast or hyphal cells, as well as a flexible screening tool. Moreover, the ACT1-gLUC59 fusion represented a powerful tool for the imaging of disease progression in superficial and subcutaneous C. albicans infections. gLUC59 and related cell surface-exposed luciferase reporters might find wide applications in molecular biology, cell biology, pathobiology, and high-throughput screens.