Multimodal regulation of encystation in Giardia duodenalis revealed by deep proteomics

Cyst formation in the parasitic protist Giardia duodenalis is critical to its transmission. Existing proteomic data quantifies only 17% of coding genes transcribed during encystation and does not cover the complete process from trophozoite to mature cyst. Using high-resolution mass spectrometry, we have quantified proteomic changes across encystation and compared this to published transcriptomic data. We reproducibly identified 3,863 (64.5% of Giardia proteins) and quantified 3,382 proteins (56.5 % of Giardia proteins) over standard trophozoite growth, during low-bile encystation priming, 16hrs into encystation, and at cyst maturation. This work provides the first expanded observation of encystation at the proteomic level and triples the coverage of previous encystation proteomes. One-third (1,169 proteins) of the quantified proteome is differentially expressed in the mature cyst relative to the trophozoite, including proteasomal machinery, metabolic pathways and secretory proteins. Changes in lipid metabolism indicated a shift in lipid species dependency during encystation. Consistent with this, we identified the first, putative lipid transporters in this species, representing the StARkin (steroidogenic acute regulatory protein–related lipid transfer), ORP/Osh (Oxysterol Binding protein related protein) and GLTP (Glycosphingolipid transfer protein) families, and follow their differential expression over cyst formation. Lastly, we undertook correlation analyses of the transcriptome and proteome of trophozoites and cysts and find evidence of post-transcriptional regulation of key protein classes (RNA binding proteins) and stage-specific genes (encystation markers) implicating translation-repression in encystation. We provide the most extensive proteomic analysis of encystation in Giardia to date and the first exploration across its complete duration. This work identifies encystation as highly coordinated, involving major changes in proteostasis, metabolism and membrane dynamics, and indicates a potential role for post-transcriptional regulation, mediated through RNA-binding proteins. Together our work provides a valuable resource for Giardia research and the development of transmission blocking anti-giardials.十二指肠贾第虫（Giardia duodenalis）的囊形成对其传播至关重要。现有蛋白质组学数据仅量化了包囊化（encystation）过程中转录的编码基因的17%，且未覆盖从滋养体（trophozoite）到成熟囊包的完整过程。我们借助高分辨率质谱（mass spectrometry）技术，对包囊化全过程的蛋白质组变化进行了定量分析，并将其与已发表的转录组学数据进行比对。在标准滋养体培养、低胆汁包囊化诱导、包囊化16小时以及囊包成熟这四个阶段，我们可重复鉴定出3863种贾第虫蛋白质（占贾第虫总蛋白的64.5%），并定量到3382种蛋白质（占总蛋白的56.5%）。本研究首次在蛋白质组层面全面解析了包囊化过程，将既往包囊化蛋白质组的覆盖度提升至原先的三倍。相较于滋养体，成熟囊包中三分之一的定量蛋白质（共1169种）存在差异表达，其中包括蛋白酶体系统、代谢通路及分泌蛋白。脂质代谢的变化提示包囊化过程中脂质物种依赖模式发生了转变。基于此，我们首次在该物种中鉴定出推定的脂质转运蛋白，涵盖StARkin（steroidogenic acute regulatory protein–related lipid transfer）、ORP/Osh（Oxysterol Binding protein related protein）以及GLTP（Glycosphingolipid transfer protein）家族，并追踪了它们在囊形成过程中的差异表达模式。最后，我们对滋养体和囊包的转录组与蛋白质组进行了相关性分析，发现关键蛋白类群（RNA结合蛋白，RNA binding proteins）及阶段特异性基因（包囊化标志物）存在转录后调控的证据，表明翻译抑制参与了包囊化过程。本研究提供了目前为止最为全面的贾第虫包囊化蛋白质组分析，且首次覆盖了包囊化的完整周期。研究揭示包囊化过程具有高度协同性，涉及蛋白质稳态（proteostasis）、代谢及膜动态的显著变化，并提示由RNA结合蛋白介导的转录后调控可能发挥潜在作用。综上，本研究为贾第虫相关研究以及开发阻断传播的抗贾第虫药物提供了极具价值的资源。