Department of Parasitology, National Institute of Infectious Diseases, Tokyo, Japan.
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Front Cell Infect Microbiol. 2022 Oct 3;12:961645. doi: 10.3389/fcimb.2022.961645. eCollection 2022.
Autophagy is one of the bulk degradation systems and is conserved throughout eukaryotes. In the enteric protozoan parasite , the causative agent of human amebiasis, Atg8 is not exclusively involved in autophagy per se but also in other membrane traffic-related pathways such as phagosome biogenesis. We previously reported that repression of gene expression by antisense small RNA-mediated transcriptional gene silencing (gs) resulted in growth retardation, delayed endocytosis, and reduced acidification of endosomes and phagosomes. In this study, to better understand the role of Atg8 in phagocytosis and trogocytosis, we conducted a comparative proteomic analysis of phagosomes isolated from wild type and -gs strains. We found that 127 and 107 proteins were detected >1.5-fold less or more abundantly, respectively, in phagosomes isolated from -gs strain, compared to the control strain. Among 127 proteins whose abundance was reduced in phagosomes from -gs, a panel of proteins related to fatty acid metabolism, phagocytosis, and endoplasmic reticulum (ER) homeostasis was identified. Various lysosomal hydrolases and their receptors also tend to be excluded from phagosomes by -gs, reinforcing the notion that Atg8 is involved in phagosomal acidification and digestion. On the contrary, among 107 proteins whose abundance increased in phagosomes from -gs strain, ribosome-related proteins and metabolite interconversion enzymes are enriched. We further investigated the localization of several representative proteins, including adenylyl cyclase-associated protein and plasma membrane calcium pump, both of which were demonstrated to be recruited to phagosomes and trogosomes an Atg8-dependent mechanism. Taken together, our study has provided the basis of the phagosome proteome to further elucidate molecular events in the Atg8-dependent regulatory network of phagosome/trogosome biogenesis in .
自噬是一种批量降解系统，在真核生物中普遍存在。在肠道原生动物寄生虫中，引起人类阿米巴病的病原体，Atg8 不仅专门参与自噬本身，还参与其他与膜运输相关的途径，如吞噬体的生物发生。我们之前报道过，通过反义小 RNA 介导的转录基因沉默（gs）抑制基因表达会导致生长迟缓、内吞作用延迟以及内体和吞噬体酸化减少。在这项研究中，为了更好地理解 Atg8 在吞噬作用和 trogocytosis 中的作用，我们对来自野生型和-gs 菌株的分离的吞噬体进行了比较蛋白质组学分析。我们发现，与对照菌株相比，从-gs 菌株分离的吞噬体中分别有 127 种和 107 种蛋白质的丰度减少或增加了 1.5 倍以上。在吞噬体丰度降低的 127 种蛋白质中，鉴定出一组与脂肪酸代谢、吞噬作用和内质网（ER）稳态相关的蛋白质。各种溶酶体水解酶及其受体也倾向于被-gs 排除在吞噬体之外，这进一步证明了 Atg8 参与了吞噬体的酸化和消化。相反，在从-gs 菌株分离的吞噬体中丰度增加的 107 种蛋白质中，核糖体相关蛋白质和代谢物相互转化酶丰富。我们进一步研究了几种代表性蛋白质的定位，包括腺苷酸环化酶相关蛋白和质膜钙泵，这两种蛋白质都被证明通过 Atg8 依赖性机制被募集到吞噬体和 trogosomes 中。综上所述，我们的研究为吞噬体蛋白质组提供了基础，以进一步阐明在-gs 依赖的调节网络中吞噬体/ trogosome 生物发生的分子事件。
