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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.Importance The Giardia genus comprises eukaryotic single-celled parasites that infect many animals. The Giardia intestinalis species complex, which can colonize and cause diarrheal disease in humans and different animal hosts has been extensively explored at the genomic an

The identification of ancestral traits is essential to understand the evolution of any group. In the case of parasitic groups, this helps to understand the adaptation to this lifestyle and a particular host. The last Diplomonadida common ancestor has been considered a parasite because most diplomonads are found associated with various animals. However, most of the Fornicata relatives are free-living and there are free-living diplomonads, making the lifestyle of the Diplomonadida ancestor elusive. Here, we present an exhaustive metabolic analysis of diplomonads and the first description of the putative metabolism of the last Diplomonadida common ancestor. Our study suggests that the synthesis of UDP-N-acetyl-D-galactosamine necessary for the cyst wall and most of the virulence factors identified in diplomonads evolved in a pre-parasitic lifestyle. This last Diplomonadida common ancestor was able to utilize available metabolites, but had a reduced capacity to synthesize nucleotides, lipids and amino acids de novo, suggesting that most likely, it was an obligate host-associated.

Many anaerobic microbial parasites possess highly modified mitochondria known as mitochondrion-related organelles (MROs). The best-studied of these are the hydrogenosomes of Trichomonas vaginalis and Spironucleus salmonicida, which produce ATP anaerobically through substrate-level phosphorylation with concomitant hydrogen production; and the mitosomes of Giardia intestinalis, which are functionally reduced and lack any role in ATP production. Howewer, to understand the metabolic specializations that these MROs underwent in adaptation to parasitism, data from their free-living relatives are needed. Here, we present a large-scale comparative transcriptomic study of MROs across a major eukaryotic group, Metamonada, examining lineage-specific gain and loss of metabolic functions in the MROs of Trichomonas, Giardia, Spironucleus and their free-living relatives. Our analyses uncover a complex history of ATP production machinery in diplomonads such as Giardia, and their closest relative, Dysnectes; and a correlation between the glycine cleavage machinery and lifestyles. Our data further suggest the existence of a previously undescribed biochemical class of MRO that generates hydrogen but is incapable of ATP synthesis.

Annexins are multifunctional, calcium-binding proteins found in organisms across all kingdoms. Most studies of annexins from single-celled eukaryotes have focused on the alpha-giardins, proteins assigned to the group E annexins, expressed by the diplomonad Giardia intestinalis. We have characterized the annexin gene family in another diplomonad parasite, Spironucleus salmonicida, by phylogenetic and experimental approaches. We constructed a comprehensive phylogeny of the diplomonad group E annexins and found that they are abundant across the group with frequent gene duplications and losses. The annexins of S. salmonicida were found to be related to alpha-giardins but with better-preserved type II Ca2+ coordination sites. Two annexins were confirmed to bind phospholipids in a Ca2+-dependent fashion but with different specificities. Superresolution and confocal microscopy of epitope-tagged S. salmonicida annexins revealed localization to distinct parts of the cytoskeleton and membrane. The ultrastructural details of the localization of several annexins were determined by proximity labeling and transmission electron microscopy. Two annexins localize to a novel cytoskeletal structure in the anterior of the cell. Our results show that the annexin gene family is expanded in diplomonads and that these group E annexins are associated mostly with cytoskeletal and membrane structures. IMPORTANCE Annexins are proteins that associate with phospholipids in a Ca2+-dependent fashion. These proteins have been intensely studied in animals and plants because of their importance in diverse cellular processes, yet very little is known about annexins in single-celled eukaryotes, which represent the largest diversity of organisms. The human intestinal parasite Giardia intestinalis is known to have more annexins than humans, and they contribute to its pathogenic potential. In this study, we investigated the annexin complement in the salmon pathogen Spironucleus salmonicida, a relative of

Background: It is generally thought that the evolutionary transition to parasitism is irreversible because it is associated with the loss of functions needed for a free-living lifestyle. Nevertheless, free-living taxa are sometimes nested within parasite clades in phylogenetic trees, which could indicate that they are secondarily free-living. Herein, we test this hypothesis by studying the genomic basis for evolutionary transitions between lifestyles in diplomonads, a group of anaerobic eukaryotes. Most described diplomonads are intestinal parasites or commensals of various animals, but there are also free-living diplomonads found in oxygen-poor environments such as marine and freshwater sediments. All these nest well within groups of parasitic diplomonads in phylogenetic trees, suggesting that they could be secondarily free-living. Results: We present a transcriptome study of Trepomonas sp. PC1, a diplomonad isolated from marine sediment. Analysis of the metabolic genes revealed a number of proteins involved in degradation of the bacterial membrane and cell wall, as well as an extended set of enzymes involved in carbohydrate degradation and nucleotide metabolism. Phylogenetic analyses showed that most of the differences in metabolic capacity between free-living Trepomonas and the parasitic diplomonads are due to recent acquisitions of bacterial genes via gene transfer. Interestingly, one of the acquired genes encodes a ribonucleotide reductase, which frees Trepomonas from the need to scavenge deoxyribonucleosides. The transcriptome included a gene encoding squalene-tetrahymanol cyclase. This enzyme synthesizes the sterol substitute tetrahymanol in the absence of oxygen, potentially allowing Trepomonas to thrive under anaerobic conditions as a free-living bacterivore, without depending on sterols from other eukaryotes. Conclusions: Our findings are consistent with the phylogenetic evidence that the last common ancestor of diplomonads was dependent on a host and that, Correction in: BMC Biology, vol. 14, article number 77DOI: 10.1186/s12915-016-0302-1

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

Acquisition of the mitochondrion is a key event in the evolution of the eukaryotic cell, but diversification of the organelle has occurred during eukaryotic evolution. One example of such mitochondria-related organelles (MROs) are hydrogenosomes, which produce ATP by substrate- level phosphorylation with hydrogen as a byproduct. The diplomonad parasite Giardia intestinalis harbours mitosomes, another type of MRO. Here we identify MROs in the salmon parasite Spironucleus salmonicida with similar protein import and Fe-S cluster assembly machineries as in Giardia mitosomes. We find that hydrogen production is prevalent in the diplomonad genus Spironucleus, and that S. salmonicida MROs contain enzymes characteristic of hydrogenosomes. Evolutionary analyses of known hydrogenosomal components indicate their presence in the diplomonad ancestor, and subsequent loss in Giardia. Our results suggest that hydrogenosomes are metabolic adaptations predating the split between parabasalids and diplomonads, which is deeper than the split between animals and fungi in the eukaryotic tree.