Hydrogenosomes in the diplomonad Spironucleus salmonicida.
ABSTRACT: 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.
Project description:Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II-FLAG tandem-affinity purification tag or streptavidin binding peptide-glutathione S-transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr), and neomycin phosphotransferase (nptII), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleus.
Project description:BACKGROUND:Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia, small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans-spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. RESULTS:We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida, as well as relatives, including 6 verified cis-spliceosomal introns in S. vortens. Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia. Similar to 'long' G. lamblia introns, RNA secondary structural potential is evident for 'long' (>?50?nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis. Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens, and U2 and U5 snRNAs in S. salmonicida; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia, Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata. CONCLUSIONS:Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and common sets of spliceosomal proteins point to a more streamlined splicing mechanism, where intron sequences and structures may be functionally compensating for the minimalization of spliceosome components.
Project description: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.
Project description:Giardia intestinalis parasites contain mitosomes, one of the simplest mitochondrion-related organelles. Strategies to identify the functions of mitosomes have been limited mainly to homology detection, which is not suitable for identifying species-specific proteins and their functions. An in vivo enzymatic tagging technique based on the Escherichia coli biotin ligase (BirA) has been introduced to G. intestinalis; this method allows for the compartment-specific biotinylation of a protein of interest. Known proteins involved in the mitosomal protein import were in vivo tagged, cross-linked, and used to copurify complexes from the outer and inner mitosomal membranes in a single step. New proteins were then identified by mass spectrometry. This approach enabled the identification of highly diverged mitosomal Tim44 (GiTim44), the first known component of the mitosomal inner membrane translocase (TIM). In addition, our subsequent bioinformatics searches returned novel diverged Tim44 paralogs, which mediate the translation and mitosomal insertion of mitochondrially encoded proteins in other eukaryotes. However, most of the identified proteins are specific to G. intestinalis and even absent from the related diplomonad parasite Spironucleus salmonicida, thus reflecting the unique character of the mitosomal metabolism. The in vivo enzymatic tagging also showed that proteins enter the mitosome posttranslationally in an unfolded state and without vesicular transport.
Project description:The fornicata (fornicates) is a eukaryotic group known to consist of free-living and parasitic organisms. Genome datasets of two model fornicate parasites Giardia intestinalis and Spironucleus salmonicida are well annotated, so far. The nuclear genomes of G. intestinalis assemblages and S. salmonicida are small in terms of the genome size and simple in genome structure. However, an ancestral genomic structure and gene contents, from which genomes of the fornicate parasites have evolved, remains to be clarified. In order to understand genome evolution in fornicates, here, we present the draft genome sequence of a free-living fornicate, Kipferlia bialata, the divergence of which is earlier than those of the fornicate parasites, and compare it to the genomes of G. intestinalis and S. salmonicida. Our data show that the number of protein genes and introns in K. bialata genome are the most abundant in the genomes of three fornicates, reflecting an ancestral state of fornicate genome evolution. Evasion mechanisms of host immunity found in G. intestinalis and S. salmonicida are absent in the K. bialata genome, suggesting that the two parasites acquired the complex membrane surface proteins on the line leading to the common ancestor of G. intestinalis and S. salmonicida after the divergence from K. bialata. Furthermore, the mitochondrion related organelles (MROs) of K. bialata possess more complex suites of metabolic pathways than those in Giardia and in Spironucleus. In sum, our results unveil the process of reductive evolution which shaped the current genomes in two model fornicate parasites G. intestinalis and S. salmonicida.
Project description: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 Ca(2+) coordination sites. Two annexins were confirmed to bind phospholipids in a Ca(2+)-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 Ca(2+)-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 G. intestinalis. We found that S. salmonicida has a large repertoire of annexins and that the gene family has expanded separately across diplomonads, with members showing sequence diversity similar to that seen across kingdom-level groups such as plants and animals. S. salmonicida annexins are prominent components of the cytoskeleton and membrane. Two annexins are associated with a previously unrecognized structure in the anterior of the cell.
Project description:Mitochondria are archetypal organelles of endosymbiotic origin in eukaryotic cells. Some unicellular eukaryotes (protists) were considered to be primarily amitochondrial organisms that diverged from the eukaryotic lineage before the acquisition of the premitochondrial endosymbiont, but their amitochondrial status was recently challenged by the discovery of mitochondria-like double membrane-bound organelles called mitosomes. Here, we report that proteins targeted into mitosomes of Giardia intestinalis have targeting signals necessary and sufficient to be recognized by the mitosomal protein import machinery. Expression of these mitosomal proteins in Trichomonas vaginalis results in targeting to hydrogenosomes, a hydrogen-producing form of mitochondria. We identify, in Giardia and Trichomonas, proteins related to the component of the translocase in the inner membrane from mitochondria and the processing peptidase. A shared mode of protein targeting supports the hypothesis that mitosomes, hydrogenosomes, and mitochondria represent different forms of the same fundamental organelle having evolved under distinct selection pressures.
Project description:BACKGROUND: Microbial eukaryotes show large variations in genome structure and content between lineages, indicating extensive flexibility over evolutionary timescales. Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments. Approximately 5,000 expressed sequence tag (EST) sequences were generated from the fish commensal Spironucleus barkhanus and compared to sequences from the morphologically indistinguishable fish parasite Spironucleus salmonicida, and other diplomonads. The ESTs were complemented with sequence variation studies in selected genes and genome size determinations. RESULTS: Many genes detected in S. barkhanus and S. salmonicida are absent in the human parasite Giardia intestinalis, the most intensively studied diplomonad. For example, these fish diplomonads show an extended metabolic repertoire and are able to incorporate selenocysteine into proteins. The codon usage is altered in S. barkhanus compared to S. salmonicida. Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes. Conversely, no allelic variation was found in a previous genome survey of S. salmonicida. This difference was confirmed by sequencing of genomic DNA. Up to five alleles were identified for the cloned S. barkhanus genes, and at least nineteen highly expressed S. barkhanus genes are represented by more than four alleles in the EST dataset. This could be explained by the presence of a non-clonal S. barkhanus population in the culture, by a ploidy above four, or by duplications of parts of the genome. Indeed, genome size estimations using flow cytometry indicated similar haploid genome sizes in S. salmonicida and G. intestinalis (approximately 12 Mb), whereas the S. barkhanus genome is larger (approximately 18 Mb). CONCLUSIONS: This study indicates extensive divergent genome evolution within diplomonads. Genomic traits such as codon usage, frequency of allelic sequence variation, and genome size have changed considerably between S. barkhanus and S. salmonicida. These observations suggest that large genomic differences may accumulate in morphologically indistinguishable eukaryotic microbes.
Project description:BACKGROUND:Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive. RESULTS:To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis. CONCLUSION:While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.
Project description:Diplomonads are common free-living inhabitants of anoxic aquatic environments and are also found as intestinal commensals or parasites of a wide variety of animals. Spironucleus vortens is a putatively commensal diplomonad of angelfish that grows to high cell densities in axenic culture. Genomic sequencing of S. vortens is in progress, yet little information is available regarding molecular and cellular aspects of S. vortens biology beyond descriptive ultrastructural studies. To facilitate the development of S. vortens as an additional diplomonad experimental model, we have constructed and stably transformed an episomal plasmid containing an enhanced green fluorescent protein (GFP) tag, an AU1 epitope tag, and a tandem affinity purification (TAP) tag. This construct also contains selectable antibiotic resistance markers for both S. vortens and E. coli.Stable transformants of S. vortens grew relatively rapidly (within 7 days) after electroporation and were maintained under puromycin selection for over 6 months. We expressed the enhanced GFP variant, eGFP, under transcriptional control of the S. vortens histone H3 promoter, and visually confirmed diffuse GFP expression in over 50% of transformants. Next, we generated a histone H3::GFP fusion using the S. vortens conventional histone H3 gene and its native promoter. This construct was also highly expressed in the majority of S. vortens transformants, in which the H3::GFP fusion localized to the chromatin in both nuclei. Finally, we used fluorescence in situ hybridization (FISH) of the episomal plasmid to show that the transformed plasmid localized to only one nucleus/cell and was present at roughly 10-20 copies per nucleus. Because S. vortens grows to high densities in laboratory culture, it is a feasible diplomonad from which to purify native protein complexes. Thus, we also included a TAP tag in the plasmid constructs to permit future tagging and subsequent purification of protein complexes by affinity chromatography via a two-step purification procedure.Currently, progress in protistan functional and comparative genomics is hampered by the lack of free-living or commensal protists in axenic culture, as well as a lack of molecular genetic tools with which to study protein function in these organisms. This stable transformation protocol combined with the forthcoming genome sequence allows Spironucleus vortens to serve as a new experimental model for cell biological studies and for comparatively assessing protein functions in related diplomonads such as the human intestinal parasite, Giardia intestinalis.