Extreme genome diversity in the hyper-prevalent parasitic eukaryote Blastocystis.

Blastocystis is the most prevalent eukaryotic microbe colonizing the human gut, infecting approximately 1 billion individuals worldwide. Although Blastocystis has been linked to intestinal disorders, its pathogenicity remains controversial because most carriers are asymptomatic. Here, the genome sequence of Blastocystis subtype (ST) 1 is presented and compared to previously published sequences for ST4 and ST7. Despite a conserved core of genes, there is unexpected diversity between these STs in terms of their genome sizes, guanine-cytosine (GC) content, intron numbers, and gene content. ST1 has 6,544 protein-coding genes, which is several hundred more than reported for ST4 and ST7. The percentage of proteins unique to each ST ranges from 6.2% to 20.5%, greatly exceeding the differences observed within parasite genera. Orthologous proteins also display extreme divergence in amino acid sequence identity between STs (i.e., 59%-61% median identity), on par with observations of the most distantly related species pairs of parasite genera. The STs also display substantial variation in gene family distributions and sizes, especially for protein kinase and protease gene families, which could reflect differences in virulence. It remains to be seen to what extent these inter-ST differences persist at the intra-ST level. A full 26% of genes in ST1 have stop codons that are created on the mRNA level by a novel polyadenylation mechanism found only in Blastocystis. Reconstructions of pathways and organellar systems revealed that ST1 has a relatively complete membrane-trafficking system and a near-complete meiotic toolkit, possibly indicating a sexual cycle. Unlike some intestinal protistan parasites, Blastocystis ST1 has near-complete de novo pyrimidine, purine, and thiamine biosynthesis pathways and is unique amongst studied stramenopiles in being able to metabolize α-glucans rather than ß-glucans. It lacks all genes encoding heme-containing cytochrome P450 proteins. Predictions of the mitochondrion-related organelle (MRO) proteome reveal an expanded repertoire of functions, including lipid, cofactor, and vitamin biosynthesis, as well as proteins that may be involved in regulating mitochondrial morphology and MRO/endoplasmic reticulum (ER) interactions. In sharp contrast, genes for peroxisome-associated functions are absent, suggesting Blastocystis STs lack this organelle. Overall, this study provides an important window into the biology of Blastocystis, showcasing significant differences between STs that can guide future experimental investigations into differences in their virulence and clarifying the roles of these organisms in gut health and disease.

Longin and GAF domains: structural evolution and adaptation to the subcellular trafficking machinery.

Endomembrane trafficking is one of the most prominent cytological features of eukaryotes. Given their widespread distribution and specialization, coiled-coil domains, coatomer domains, small GTPases and Longin domains are considered primordial 'building blocks' of the membrane trafficking machineries. Longin domains are conserved across eukaryotes and were likely to be present in the Last Eukaryotic Common Ancestor. The Longin fold is based on the α-ß-α sandwich architecture and a unique topology, possibly accounting for the special adaptation to the eukaryotic trafficking machinery. The ancient Per ARNT Sim (PAS) and cGMP-specific phosphodiesterases, Adenylyl cyclases and FhlA (GAF) family domains show a similar architecture, and the identification of prokaryotic counterparts of GAF domains involved in trafficking provides an additional connection for the endomembrane system back into the pre-eukaryotic world. Proteome-wide, comparative bioinformatic analyses of the domains reveal three binding regions (A, B and C) mediating either specific or conserved protein-protein interactions. While the A region mediates intra- and inter-molecular interactions, the B region is involved in binding small GTPases, thus providing an evolutionary connection among major building blocks in the endomembrane system. Finally, we propose that the peculiar interaction surface of the C region of the Longin domain allowed it to extensively integrate into the endomembrane trafficking machinery in the earliest stages of building the eukaryotic cell.

Ancient complexity, opisthokont plasticity, and discovery of the 11th subfamily of Arf GAP proteins.

The organelle paralogy hypothesis is one model for the acquisition of nonendosymbiotic organelles, generated from molecular evolutionary analyses of proteins encoding specificity in the membrane traffic system. GTPase activating proteins (GAPs) for the ADP-ribosylation factor (Arfs) GTPases are additional regulators of the kinetics and fidelity of membrane traffic. Here we describe molecular evolutionary analyses of the Arf GAP protein family. Of the 10 subfamilies previously defined in humans, we find that 5 were likely present in the last eukaryotic common ancestor. Of the 3 most recently derived subfamilies, 1 was likely present in the ancestor of opisthokonts (animals and fungi) and apusomonads (flagellates classified as the sister lineage to opisthokonts), while 2 arose in the holozoan lineage. We also propose to have identified a novel ancient subfamily (ArfGAPC2), present in diverse eukaryotes but which is lost frequently, including in the opisthokonts. Surprisingly few ancient domains accompanying the ArfGAP domain were identified, in marked contrast to the extensively decorated human Arf GAPs. Phylogenetic analyses of the subfamilies reveal patterns of single and multiple gene duplications specific to the Holozoa, to some degree mirroring evolution of Arf GAP targets, the Arfs. Conservation, and lack thereof, of various residues in the ArfGAP structure provide contextualization of previously identified functional amino acids and their application to Arf GAP biology in general. Overall, our results yield insights into current Arf GAP biology, reveal complexity in the ancient eukaryotic ancestor and integrate the Arf GAP family into a proposed mechanism for the evolution of nonendosymbiotic organelles.

Eukaryotic systematics: a user's guide for cell biologists and parasitologists.

Single-celled parasites like Entamoeba, Trypanosoma, Phytophthora and Plasmodium wreak untold havoc on human habitat and health. Understanding the position of the various protistan pathogens in the larger context of eukaryotic diversity informs our study of how these parasites operate on a cellular level, as well as how they have evolved. Here, we review the literature that has brought our understanding of eukaryotic relationships from an idea of parasites as primitive cells to a crystallized view of diversity that encompasses 6 major divisions, or supergroups, of eukaryotes. We provide an updated taxonomic scheme (for 2011), based on extensive genomic, ultrastructural and phylogenetic evidence, with three differing levels of taxonomic detail for ease of referencing and accessibility (see supplementary material at Cambridge Journals On-line). Two of the most pressing issues in cellular evolution, the root of the eukaryotic tree and the evolution of photosynthesis in complex algae, are also discussed along with ideas about what the new generation of genome sequencing technologies may contribute to the field of eukaryotic systematics. We hope that, armed with this user's guide, cell biologists and parasitologists will be encouraged about taking an increasingly evolutionary point of view in the battle against parasites representing real dangers to our livelihoods and lives.

Ancient complement and lineage-specific evolution of the Sec7 ARF GEF proteins in eukaryotes.

Guanine nucleotide exchange factors (GEFs) are the initiators of signaling by every regulatory GTPase, which in turn act to regulate a wide array of essential cellular processes. To date, each family of GTPases is activated by distinct families of GEFs. Bidirectional membrane trafficking is regulated by ADP-ribosylation factor (ARF) GTPases and the development throughout eukaryotic evolution of increasingly complex systems of such traffic required the acquisition of a functionally diverse cohort of ARF GEFs to control it. We performed phylogenetic analyses of ARF GEFs in eukaryotes, defined by the presence of the Sec7 domain, and found three subfamilies (BIG, GBF1, and cytohesins) to have been present in the ancestor of all eukaryotes. The four other subfamilies (EFA6/PSD, IQSEC7/BRAG, FBX8, and TBS) are opisthokont, holozoan, metazoan, and alveolate/haptophyte specific, respectively, and each is derived from cytohesins. We also identified a cytohesin-derived subfamily, termed ankyrin repeat-containing cytohesin, that independently evolved in amoebozoans and members of the SAR and haptophyte clades. Building on evolutionary data for the ARF family GTPases and their GTPase--activating proteins allowed the generation of hypotheses about ARF GEF protein function(s) as well as a better understanding of the origins and evolution of cellular complexity in eukaryotes.

Kinetoplastid Phylogenomics Reveals the Evolutionary Innovations Associated with the Origins of Parasitism.

The evolution of parasitism is a recurrent event in the history of life and a core problem in evolutionary biology. Trypanosomatids are important parasites and include the human pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., which in humans cause African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. Genome comparison between trypanosomatids reveals that these parasites have evolved specialized cell-surface protein families, overlaid on a well-conserved cell template. Understanding how these features evolved and which ones are specifically associated with parasitism requires comparison with related non-parasites. We have produced genome sequences for Bodo saltans, the closest known non-parasitic relative of trypanosomatids, and a second bodonid, Trypanoplasma borreli. Here we show how genomic reduction and innovation contributed to the character of trypanosomatid genomes. We show that gene loss has "streamlined" trypanosomatid genomes, particularly with respect to macromolecular degradation and ion transport, but consistent with a widespread loss of functional redundancy, while adaptive radiations of gene families involved in membrane function provide the principal innovations in trypanosomatid evolution. Gene gain and loss continued during trypanosomatid diversification, resulting in the asymmetric assortment of ancestral characters such as peptidases between Trypanosoma and Leishmania, genomic differences that were subsequently amplified by lineage-specific innovations after divergence. Finally, we show how species-specific, cell-surface gene families (DGF-1 and PSA) with no apparent structural similarity are independent derivations of a common ancestral form, which we call "bodonin." This new evidence defines the parasitic innovations of trypanosomatid genomes, revealing how a free-living phagotroph became adapted to exploiting hostile host environments.

Unexpected ancient paralogs and an evolutionary model for the COPII coat complex.

The coat protein complex II (COPII) is responsible for the transport of protein cargoes from the Endoplasmic Reticulum (ER) to the Golgi apparatus. COPII has been functionally characterized extensively in vivo in humans and yeast. This complex shares components with the nuclear pore complex and the Seh1-Associated (SEA) complex, inextricably linking its evolution with that of the nuclear pore and other protocoatomer domain-containing complexes. Importantly, this is one of the last coat complexes to be examined from a comparative genomic and phylogenetic perspective. We use homology searching of eight components across 74 eukaryotic genomes, followed by phylogenetic analyses, to assess both the distribution of the COPII components across eukaryote diversity and to assess its evolutionary history. We report that Sec12, but not Sed4 was present in the Last Eukaryotic Common Ancestor along with Sec16, Sar1, Sec13, Sec31, Sec23, and Sec24. We identify a previously undetected paralog of Sec23 that, at least, predates the archaeplastid clade. We also describe three Sec24 paralogs likely present in the Last Eukaryotic Common Ancestor, including one newly detected that was anciently present but lost from both opisthokonts and excavates. Altogether, we report previously undescribed complexity of the COPII coat in the ancient eukaryotic ancestor and speculate on models for the evolution, not only of the complex, but its relationship to other protocoatomer-derived complexes.

Missing pieces of an ancient puzzle: evolution of the eukaryotic membrane-trafficking system.

The membrane-trafficking system underpins cellular trafficking of material in eukaryotes and its evolution would have been a watershed in eukaryogenesis. Evolutionary cell biological studies have been unraveling the history of proteins responsible for vesicle transport and organelle identity revealing both highly conserved components and lineage-specific innovations. Recently, endomembrane components with a broad, but patchy, distribution have been observed as well, pieces that are missing from our cell biological and evolutionary models of membrane trafficking. These data together allow for new insights into the history and forces that shape the evolution of this critical cell biological system.

Characterization of TSET, an ancient and widespread membrane trafficking complex.

The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.DOI: http://dx.doi.org/10.7554/eLife.02866.001.