A chromosome-scale reference genome for Spironucleus salmonicida.

Spironucleus salmonicida is a diplomonad causing systemic infection in salmon. The first S. salmonicida genome assembly was published 2014 and has been a valuable reference genome in protist research. However, the genome assembly is fragmented without assignment of the sequences to chromosomes. In our previous Giardia genome study, we have shown how a fragmented genome assembly can be improved with long-read sequencing technology complemented with optical maps. Combining Pacbio long-read sequencing technology and optical maps, we are presenting here this new S. salmonicida genome assembly in nine near-complete chromosomes with only three internal gaps at long repeats. This new genome assembly is not only more complete sequence-wise but also more complete at annotation level, providing more details into gene families, gene organizations and chromosomal structure. This near-complete reference genome will aid comparative genomics at chromosomal level, and serve as a valuable resource for the diplomonad community and protist research.

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.

Metabolic Reconstruction Elucidates the Lifestyle of the Last Diplomonadida Common Ancestor.

The identification of ancestral traits is essential to understanding the evolution of any group. In the case of parasitic groups, this helps us understand the adaptation to this lifestyle and a particular host. Most diplomonads are parasites, but there are free-living members of the group nested among the host-associated diplomonads. Furthermore, most of the close relatives within Fornicata are free-living organisms. This leaves the lifestyle of the ancestor unclear. Here, we present metabolic maps of four different diplomonad species. We identified 853 metabolic reactions and 147 pathways present in at least one of the analyzed diplomonads. Our study suggests that diplomonads represent a metabolically diverse group in which differences correlate with different environments (e.g., the detoxification of arsenic). Using a parsimonious analysis, we also provide a description of the putative metabolism of the last Diplomonadida common ancestor. Our results show that the acquisition and loss of reactions have shaped metabolism since this common ancestor. There is a net loss of reaction in all branches leading to parasitic diplomonads, suggesting an ongoing reduction in the metabolic capacity. Important traits present in host-associated diplomonads (e.g., virulence factors and the synthesis of UDP-N-acetyl-d-galactosamine) are shared with free-living relatives. The last Diplomonadida common ancestor most likely already had acquired important enzymes for the salvage of nucleotides and had a reduced capacity to synthesize nucleotides, lipids, and amino acids de novo, suggesting that it was an obligate host-associated organism.IMPORTANCE Diplomonads are a group of microbial eukaryotes found in oxygen-poor environments. There are both parasitic (e.g., Giardia intestinalis) and free-living (e.g., Trepomonas) members in the group. Diplomonads are well known for their anaerobic metabolism, which has been studied for many years. Here, we reconstructed whole metabolic networks of four extant diplomonad species as well as their ancestors, using a bioinformatics approach. We show that the metabolism within the group is under constant change throughout evolutionary time, in response to the environments that the different lineages explore. Both gene losses and gains are responsible for the adaptation processes. Interestingly, it appears that the last Diplomonadida common ancestor had a metabolism that is more similar to extant parasitic than free-living diplomonads. This suggests that the host-associated lifestyle of parasitic diplomonads, such as the human parasite G. intestinalis, is an old evolutionary adaptation.

The compact genome of Giardia muris reveals important steps in the evolution of intestinal protozoan parasites.

Diplomonad parasites of the genus Giardia have adapted to colonizing different hosts, most notably the intestinal tract of mammals. The human-pathogenic Giardia species, Giardia intestinalis, has been extensively studied at the genome and gene expression level, but no such information is available for other Giardia species. Comparative data would be particularly valuable for Giardia muris, which colonizes mice and is commonly used as a prototypic in vivo model for investigating host responses to intestinal parasitic infection. Here we report the draft-genome of G. muris. We discovered a highly streamlined genome, amongst the most densely encoded ever described for a nuclear eukaryotic genome. G. muris and G. intestinalis share many known or predicted virulence factors, including cysteine proteases and a large repertoire of cysteine-rich surface proteins involved in antigenic variation. Different to G. intestinalis, G. muris maintains tandem arrays of pseudogenized surface antigens at the telomeres, whereas intact surface antigens are present centrally in the chromosomes. The two classes of surface antigens engage in genetic exchange. Reconstruction of metabolic pathways from the G. muris genome suggest significant metabolic differences to G. intestinalis. Additionally, G. muris encodes proteins that might be used to modulate the prokaryotic microbiota. The responsible genes have been introduced in the Giardia genus via lateral gene transfer from prokaryotic sources. Our findings point to important evolutionary steps in the Giardia genus as it adapted to different hosts and it provides a powerful foundation for mechanistic exploration of host-pathogen interaction in the G. muris-mouse pathosystem.

Lateral Acquisitions Repeatedly Remodel the Oxygen Detoxification Pathway in Diplomonads and Relatives.

Oxygen and reactive oxygen species (ROS) are important stress factors for cells because they can oxidize many large molecules. Fornicata, a group of flagellated protists that includes diplomonads, have anaerobic metabolism but are still able to tolerate fluctuating levels of oxygen. We identified 25 protein families putatively involved in detoxification of oxygen and ROS in this group using a bioinformatics approach and propose how these interact in an oxygen detoxification pathway. These protein families were divided into a central oxygen detoxification pathway and accessory pathways for the synthesis of nonprotein thiols. We then used a phylogenetic approach to investigate the evolutionary origin of the components of this putative pathway in Diplomonadida and other Fornicata species. Our analyses suggested that the diplomonad ancestor was adapted to low-oxygen levels, was able to reduce O2 to H2O in a manner similar to extant diplomonads, and was able to synthesize glutathione and l-cysteine. Several genes involved in the pathway have complex evolutionary histories and have apparently been repeatedly acquired through lateral gene transfer and subsequently lost. At least seven genes were acquired independently in different Fornicata lineages, leading to evolutionary convergences. It is likely that acquiring these oxygen detoxification proteins helped anaerobic organisms (like the parasitic Giardia intestinalis) adapt to low-oxygen environments (such as the digestive tract of aerobic hosts).