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Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-electron microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal proteins, but retain some bacterial features. The translocation intermediates, with naturally bound tRNAs and eukaryotic elongation factor 2 (eEF2), display characteristic ribosomal intersubunit rotation and small subunit's head swiveling-universal for translocation. In addition, we observe the eukaryote-specific 'subunit rolling' dynamics, albeit with limited features. Finally, the eEF2 center dot GDP state features a uniquely positioned 'leaving phosphate (Pi)' that proposes hitherto unknown molecular events of Pi and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes from both the structural and mechanistic perspectives.

Giardia intestinalis is a unicellular parasite causing the disease giardiasis. This disease is mostly prevalent in low resource settings, mandating low-cost treatment options. Treatment is mainly based on antiparasitics from the classes of 5-nitroimidazoles and benzimidazoles, which both target parasite DNA and other cellular macromolecules based on radicals generated from the drugs. In recent years resistance to those classes and cross-resistance in between them has become a problem, hence alternative antigiardials are needed. In this thesis we enhanced our understanding of the crucial differentiation process of encystation, which produce the environmentally stable and infective form of the parasite, the cyst (Paper I). Resistance development has been slow in G. intestinalis until now and it has been shown that resistance to the main treatment option, metronidazole, can be lost after en- and excystation so an enhanced understanding of this process can help us to identify the cause of this loss of drug tolerance. In the second study (Paper II) we followed this up by analysing two metronidazole resistant lines and one revertant on their ability to produce infective cysts, while cross checking with growth rates, metronidazole resistance level, transcriptomics and proteomics. We found the resistant cells lines to have deeply disturbed cellular pathways with the main resistance mechanisms being a reduction of uptake, a reduction of activation rate and an upregulation of oxidative stress responses. Cyst production and growth rates were highly reduced giving those lines a clear disadvantage when no drug pressure is applied. Most changes, phenotypically and expression wise, were reset in the revertant. As next step we evaluated alternative antigiardials and their targets. In Paper III we characterised the giardial thymidine kinase, on which this parasite depends completely to supply thymidine for DNA synthesis. We identified a nucleoside analogue, azidothymidine, that is t

Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.

Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-Electron Microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal-proteins, but retain some bacterial features. The translocation intermediates, with naturally-bound tRNAs and eEF2, display characteristic ribosomal intersubunit rotation and small subunit’s head swiveling - universal for translocation. In addition, we observe the eukaryote-specific ‘subunit rolling’ dynamics, albeit with limited features. Finally, the eEF2•GDP state features a uniquely positioned ‘leaving Pi’ that proposes hitherto unknown molecular events of Pi- and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes both from the structural and mechanistic perspectives.