Rappleye Chad A, Engle Jacquelyn T, Goldman William E
Department of Molecular Microbiology, Campus Box 8230, Washington University, St Louis, MO 63110, USA.
Mol Microbiol. 2004 Jul;53(1):153-65. doi: 10.1111/j.1365-2958.2004.04131.x.
Histoplasma capsulatum is a fungal pathogen that causes respiratory and systemic disease by proliferating within macrophages. While much is known about histoplasmosis, only a single virulence factor has been defined, in part because of the inefficiency of Histoplasma reverse genetics. As an alternative to allelic replacement, we have developed a telomeric plasmid-based system for silencing gene expression in Histoplasma by RNA interference (RNAi). Episomal expression of long RNAs that form stem-loop structures triggered gene silencing. To test the effectiveness of RNAi in Histoplasma, we depleted expression of a gfp transgene as well as two endogenous genes, ADE2 and URA5, and showed significant reductions in corresponding gene function. Silencing was target gene specific, stable during macrophage infection and reversible. We used RNAi targeting AGS1 (encoding alpha-(1,3)-glucan synthase) to deplete levels of alpha-(1,3)-glucan, a cell wall polysaccharide. Loss of alpha-(1,3)-glucan by RNAi yielded phenotypes indistinguishable from an AGS1 deletion: attenuation of the ability to kill macrophages and colonize murine lungs. This demonstrates for the first time that alpha-(1,3)-glucan is an important contributor to Histoplasma virulence.
荚膜组织胞浆菌是一种真菌病原体，可通过在巨噬细胞内增殖引发呼吸道和全身性疾病。尽管人们对组织胞浆菌病了解很多，但仅确定了一种毒力因子，部分原因是荚膜组织胞浆菌反向遗传学效率低下。作为等位基因替换的替代方法，我们开发了一种基于端粒质粒的系统，用于通过RNA干扰（RNAi）使荚膜组织胞浆菌中的基因表达沉默。形成茎环结构的长RNA的附加型表达引发了基因沉默。为了测试RNAi在荚膜组织胞浆菌中的有效性，我们耗尽了gfp转基因以及两个内源性基因ADE2和URA5的表达，并显示相应基因功能显著降低。沉默具有靶基因特异性，在巨噬细胞感染期间稳定且可逆。我们使用靶向AGS1（编码α-（1,3）-葡聚糖合酶）的RNAi来降低细胞壁多糖α-（1,3）-葡聚糖的水平。通过RNAi使α-（1,3）-葡聚糖缺失产生的表型与AGS1缺失无法区分：杀死巨噬细胞和在小鼠肺部定殖的能力减弱。这首次证明α-（1,3）-葡聚糖是荚膜组织胞浆菌毒力的重要贡献因素。
