Emily K. Herman

Algal genomes reveal evolutionary mosaicism and the fate of nucleomorphs

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have >21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Pan genome of the phytoplankton Emiliania underpins its global distribution

Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.

A Eukaryote without a Mitochondrial Organelle

The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

Intracellular trafficking and protein sorting are mediated by various protein complexes, with the retromer complex being primarily involved in retrograde traffic from the endosome or lysosome to the Golgi complex. Here, comparative genomics, cell biology and phylogenetics were used to probe the early evolution of retromer and its function. Retromer subunits Vps26, Vps29 and Vps35 are near universal, and, by inference, the complex was an ancient feature of eukaryotic cells. Surprisingly, we found DSCR3, a Vps26 paralogue in humans associated with Downs syndrome, in at least four eukaryotic supergroups, implying a more ancient origin than previously suspected. By contrast, retromer cargo proteins showed considerable interlineage variability, with lineage-specific and broadly conserved examples found. Vps10 trafficking probably represents an ancestral role for the complex. Vps5, the BAR-domain-containing membrane-deformation subunit, was found in diverse eukaryotes, including in the divergent eukaryote Trypanosoma brucei, where it is the first example of a BAR-domain protein. To determine functional conservation, an initial characterisation of retromer was performed in T. brucei; the endosomal localisation and its role in endosomal targeting are conserved. Therefore retromer is identified as a further feature of the sophisticated intracellular trafficking machinery of the last eukaryotic common ancestor, with BAR domains representing a possible third independent mechanism of membrane-deformation arising in early eukaryotes.

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.

Multivesicular bodies in the enigmatic amoeboflagellate Breviata anathema and the evolution of ESCRT 0

Endosomal sorting complexes required for transport (ESCRTs) are heteromeric protein complexes required for multivesicular body (MVB) morphogenesis. ESCRTs I, II, III and III-associated are ubiquitous in eukaryotes and presumably ancient in origin. ESCRT 0 recruits cargo to the MVB and appears to be opisthokont-specific, bringing into question aspects of the current model of ESCRT mechanism. One caveat to the restricted distribution of ESCRT 0 was the previous limited availability of amoebozoan genomes, the supergroup closest to opisthokonts. Here, we significantly expand the sampling of ESCRTs in Amoebozoa. Our electron micrographic and bioinformatics evidence confirm the presence of MVBs in the amoeboflagellate Breviata anathema. Searches of genomic databases of amoebozoans confirm the ubiquitous nature of ESCRTs I–III-associated and the restriction of ESCRT 0 to opisthokonts. Recently, an alternate ESCRT 0 complex, centering on Tom1 proteins, has been proposed. We determine the distribution of Tom1 family proteins across eukaryotes and show that the Tom1, Tom1L1 and Tom1L2 proteins are a vertebrate-specific expansion of the single Tom1 family ancestor, which has indeed been identified in at least one member of each of the major eukaryotic supergroups. This implies a more widely conserved and ancient role for the Tom1 family in endocytosis than previously suspected.

The mitochondrial genome and a 60-kb nuclear DNA segment from Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis.

Naegleria fowleri is a unicellular eukaryote causing primary amoebic meningoencephalitis, a neuropathic disease killing 99% of those infected, usually within 7–14 days. Naegleria fowleri is found globally in regions including the US and Australia. The genome of the related nonpathogenic species Naegleria gruberi has been sequenced, but the genetic basis for N. fowleri pathogenicity is unclear. To generate such insight, we sequenced and assembled the mitochondrial genome and a 60‐kb segment of nuclear genome from N. fowleri. The mitochondrial genome is highly similar to its counterpart in N. gruberi in gene complement and organization, while distinct lack of synteny is observed for the nuclear segments. Even in this short (60‐kb) segment, we identified examples of potential factors for pathogenesis, including ten novel N. fowleri‐specific genes. We also identified a homolog of cathepsin B; proteases proposed to be involved in the pathogenesis of diverse eukaryotic pathogens, including N. fowleri. Finally, we demonstrate a likely case of horizontal gene transfer between N. fowleri and two unrelated amoebae, one of which causes granulomatous amoebic encephalitis. This initial look into the N. fowleri nuclear genome has revealed several examples of potential pathogenesis factors, improving our understanding of a neglected pathogen of increasing global importance.

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 β-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.

Resolving the homology-function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology.

With advances in DNA sequencing technology, it is increasingly common and tractable to informatically look for genes of interest in the genomic databases of parasitic organisms and infer cellular states. Assignment of a putative gene function based on homology to functionally characterized genes in other organisms, though powerful, relies on the implicit assumption of functional homology, i.e. that orthology indicates conserved function. Eukaryotes reveal a dazzling array of cellular features and structural organization, suggesting a concomitant diversity in their underlying molecular machinery. Significantly, examples of novel functions for pre-existing or new paralogues are not uncommon. Do these examples undermine the basic assumption of functional homology, especially in parasitic protists, which are often highly derived? Here we examine the extent to which functional homology exists between organisms spanning the eukaryotic lineage. By comparing membrane trafficking proteins between parasitic protists and traditional model organisms, where direct functional evidence is available, we find that function is indeed largely conserved between orthologues, albeit with significant adaptation arising from the unique biological features within each lineage.

An ER‐directed transcriptional response to unfolded protein stress in the absence of conserved sensor‐transducer proteins in Giardia lamblia

The protozoan Giardia lamblia has a minimized organelle repertoire, and most strikingly lacks a classical stacked Golgi apparatus. Nevertheless, Giardia trophozoites constitutively secrete variant surface proteins, and dramatically increase the volume of protein secretion during differentiation to cysts. Eukaryotic cells have evolved an elaborate system for quality control (QC) of protein folding and capacity in the endoplasmic reticulum (ER). Upon ER‐overload, an unfolded protein response (UPR) is triggered on transcriptional/translational level aiming at alleviating ER stress. In Giardia, a minimized secretory machinery and absence of glycan‐dependent QC suggests that a genetically conserved UPR (or functional equivalent) to cope with insults to the secretory system has been eliminated. We tested this hypothesis of UPR elimination by profiling the transcriptional response during induced ER‐folding stress. We show that on the contrary, ER‐folding stress triggers a stressor‐specific, ER‐directed response with upregulation of only ∼ 30 genes, with different kinetics and scope compared with the UPR of other eukaryotes. Computational genomics revealed conserved cis‐acting motifs in upstream regions of responder genes capable of stressor‐specific gene regulation in transfected cells. Interestingly, the sensors/transducers of folding stress, well conserved in model eukaryotes, are absent in Giardia suggesting the presence of a novel version of this essential eukaryotic function.