Autophagy in Trypanosoma brucei: Amino Acid Requirement and Regulation during Different Growth Phases

Autophagy in the protozoan parasite, Trypanosoma brucei, may be involved in differentiation between different life cycle forms and during growth in culture. We have generated multiple parasite cell lines stably expressing green fluorescent protein- or hemagglutinin-tagged forms of the autophagy marker proteins, TbAtg8.1 and TbAtg8.2, in T. brucei procyclic forms to establish a trypanosome system for quick and reliable determination of autophagy under different culture conditions using flow cytometry. We found that starvation-induced autophagy in T. brucei can be inhibited by addition of a single amino acid, histidine, to the incubation buffer. In addition, we show that autophagy is induced when parasites enter stationary growth phase in culture and that their capacity to undergo starvation-induced autophagy decreases with increasing cell density.

布氏锥虫（Trypanosoma brucei）作为一种原生动物寄生虫，其细胞自噬（autophagy）可能参与不同生活史形态间的分化以及体外培养的生长过程。本研究构建了多株稳定表达自噬标记蛋白TbAtg8.1与TbAtg8.2的绿色荧光蛋白（green fluorescent protein）或血凝素（hemagglutinin）标记版本的寄生虫细胞系，以布氏锥虫循环前期形态为研究模型，建立了可通过流式细胞术（flow cytometry）快速、可靠地检测不同培养条件下细胞自噬水平的锥虫实验体系。研究发现，向孵育缓冲液中添加单一组氨酸（histidine），即可抑制布氏锥虫的饥饿诱导细胞自噬。此外，本研究证实当寄生虫在体外培养中进入生长静止期时，细胞自噬会被诱导，且其饥饿诱导自噬的能力随细胞密度升高而下降。