Insights into translocation mechanism and ribosome evolution from cryo-EM structures of translocation intermediates of Giardia intestinalis.

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·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 thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery.

Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis-infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.

Characterization of Metronidazole-Resistant Giardia intestinalis Lines by Comparative Transcriptomics and Proteomics.

Metronidazole (MTZ) is a clinically important antimicrobial agent that is active against both bacterial and protozoan organisms. MTZ has been used extensively for more than 60 years and until now resistance has been rare. However, a recent and dramatic increase in the number of MTZ resistant bacteria and protozoa is of great concern since there are few alternative drugs with a similarly broad activity spectrum. To identify key factors and mechanisms underlying MTZ resistance, we utilized the protozoan parasite Giardia intestinalis, which is commonly treated with MTZ. We characterized two in vitro selected, metronidazole resistant parasite lines, as well as one revertant, by analyzing fitness aspects associated with increased drug resistance and transcriptomes and proteomes. We also conducted a meta-analysis using already existing data from additional resistant G. intestinalis isolates. The combined data suggest that in vitro generated MTZ resistance has a substantial fitness cost to the parasite, which may partly explain why resistance is not widespread despite decades of heavy use. Mechanistically, MTZ resistance in Giardia is multifactorial and associated with complex changes, yet a core set of pathways involving oxidoreductases, oxidative stress responses and DNA repair proteins, is central to MTZ resistance in both bacteria and protozoa.

A Detailed Gene Expression Map of Giardia Encystation.

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.