Faculty of Science, One Health Research Group, University of Melbourne, Victoria, Australia.
Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, United States of America.
PLoS One. 2024 Aug 29;19(8):e0309192. doi: 10.1371/journal.pone.0309192. eCollection 2024.
Realistic and modifiable infection models are required to study the pathogenesis of amphibian chytridiomycosis. Understanding the mechanism by which Batrachochytrium dendrobatidis (Bd) can infect and kill diverse amphibians is key to mitigating this pathogen and preventing further loss of biodiversity. In vitro studies of Bd typically rely on a tryptone based growth media, whereas the recent development of a kidney cell-line infection model has provided a more realistic alternative, without the need for live animals. Here we use expression of a fluorescent reporter to enhance the in vitro cell-line based growth assay, and show that transformed Bd cells are able to invade and grow in an amphibian kidney epithelial cell line (A6) as well as in a new system using a lung fibroblast cell line (DWJ). Both Bd and host cells were modified to express reporter fluorescent proteins, enabling immediate and continuous observation of the infection process without the need for destructive sampling for fixation and staining. Plasmid DNA conferring hygromycin resistance and TdTomato (RFP) expression was delivered to Bd zoospores via electroporation, and continuous antibiotic selection after recovery produced stable fluorescent Bd transformants. Host cells (A6 and DWJ) were transfected before each assay using lipofection to deliver plasmid DNA conferring green fluorescent protein (GFP) and containing an empty shRNA expression cassette. Bd RFP expression allowed easy localisation of fungal cells and identification of endobiotic growth was assisted by host GFP expression, by allowing visualization of the space in the host cell occupied by the invading fungal body. In addition to enabling enhanced live imaging, these methods will facilitate future genetic modification and characterisation of specific genes and their effect on Bd virulence.
为了研究两栖类壶菌病的发病机制，需要建立现实可行且可调整的感染模型。了解棘口吸虫（Batrachochytrium dendrobatidis，Bd）感染和杀死各种两栖类动物的机制是减轻这种病原体并防止生物多样性进一步丧失的关键。Bd 的体外研究通常依赖于基于蛋白胨的生长培养基，而最近开发的肾脏细胞系感染模型提供了一种更现实的替代方法，而无需使用活体动物。在这里，我们使用荧光报告基因的表达来增强基于细胞系的体外生长测定，并表明转化的 Bd 细胞能够在两栖类肾脏上皮细胞系（A6）以及使用肺成纤维细胞系（DWJ）的新系统中入侵和生长。Bd 和宿主细胞都经过修饰以表达报告荧光蛋白，从而能够立即和连续观察感染过程，而无需进行破坏性采样进行固定和染色。赋予潮霉素抗性和 TdTomato（RFP）表达的质粒 DNA 通过电穿孔递送至 Bd 游动孢子，并且在恢复后进行连续抗生素选择可产生稳定的荧光 Bd 转化体。在每次测定之前，使用脂质体转染来转染宿主细胞（A6 和 DWJ），以递送赋予绿色荧光蛋白（GFP）的质粒 DNA，并包含空 shRNA 表达盒。Bd RFP 表达允许轻松定位真菌细胞，并且通过宿主 GFP 表达辅助识别内生物生长，从而允许可视化入侵真菌体占据的宿主细胞空间。除了能够增强实时成像外，这些方法还将促进未来对特定基因及其对 Bd 毒力的影响进行遗传修饰和表征。
