Christen M. Klinger

Chromerid genomes reveal the evolutionary path from photosynthetic algae to obligate intracellular parasites

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga. DOI: http://dx.doi.org/10.7554/eLife.06974.001

Tracing the archaeal origins of eukaryotic membrane-trafficking system building blocks

In contrast to prokaryotes, eukaryotic cells are characterized by a complex set of internal membrane-bound compartments. A subset of these, and the protein machineries that move material between them, define the membrane-trafficking system (MTS), the emergence of which represents a landmark in eukaryotic evolution. Unlike mitochondria and plastids, MTS organelles have autogenous origins. Much of the MTS machinery is composed of building blocks, including small GTPase, coiled-coil, beta-propeller + alpha-solenoid, and longin domains. Despite the identification of prokaryotic proteins containing these domains, only few represent direct orthologues, leaving the origins and early evolution of the MTS poorly understood. Here, we present an in-depth analysis of MTS building block homologues in the composite genome of Lokiarchaeum, the recently discovered archaeal sister clade of eukaryotes, yielding several key insights. We identify two previously unreported Eukaryotic Signature Proteins; orthologues of the Gtr/Rag family GTPases, involved in target of rapamycin complex signaling, and of the RLC7 dynein component. We could not identify golgin or SNARE (coiled-coil) or beta-propeller + alpha-solenoid orthologues, nor typical MTS domain fusions, suggesting that these either were lost from Lokiarchaeum or emerged later in eukaryotic evolution. Furthermore, our phylogenetic analyses of lokiarchaeal GTPases support a split into Ras-like and Arf-like superfamilies, with different prokaryotic antecedents, before the advent of eukaryotes. While no GTPase activating proteins or exchange factors were identified, we show that Lokiarchaeum encodes numerous roadblock domain proteins and putative longin domain proteins, confirming the latters origin from Archaea. Altogether, our study provides new insights into the emergence and early evolution of the eukaryotic membrane-trafficking system.

Comparative Genomic Analysis of Multi-Subunit Tethering Complexes Demonstrates an Ancient Pan-Eukaryotic Complement and Sculpting in Apicomplexa

Apicomplexa are obligate intracellular parasites that cause tremendous disease burden world-wide. They utilize a set of specialized secretory organelles in their invasive process that require delivery of components for their biogenesis and function, yet the precise mechanisms underpinning such processes remain unclear. One set of potentially important components is the multi-subunit tethering complexes (MTCs), factors increasingly implicated in all aspects of vesicle-target interactions. Prompted by the results of previous studies indicating a loss of membrane trafficking factors in Apicomplexa, we undertook a bioinformatic analysis of MTC conservation. Building on knowledge of the ancient presence of most MTC proteins, we demonstrate the near complete retention of MTCs in the newly available genomes for Guillardia theta and Bigelowiella natans . The latter is a key taxonomic sampling point as a basal sister taxa to the group including Apicomplexa. We also demonstrate an ancient origin of the CORVET complex subunits Vps8 and Vps3, as well as the TRAPPII subunit Tca17. Having established that the lineage leading to Apicomplexa did at one point possess the complete eukaryotic complement of MTC components, we undertook a deeper taxonomic investigation in twelve apicomplexan genomes. We observed excellent conservation of the VpsC core of the HOPS and CORVET complexes, as well as the core TRAPP subunits, but sparse conservation of TRAPPII, COG, Dsl1, and HOPS/CORVET-specific subunits. However, those subunits that we did identify appear to be expressed with similar patterns to the fully conserved MTC proteins, suggesting that they may function as minimal complexes or with analogous partners. Strikingly, we failed to identify any subunits of the exocyst complex in all twelve apicomplexan genomes, as well as the dinoflagellate Perkinsus marinus. Overall, we demonstrate reduction of MTCs in Apicomplexa and their ancestors, consistent with modification during, and possibly pre-dating, the move from free-living marine algae to deadly human parasites.

Cryptic organelle homology in apicomplexan parasites: insights from evolutionary cell biology.

The economic and clinical significance of apicomplexan parasites drives interest in their many evolutionary novelties. Distinctive intracellular organelles play key roles in parasite motility, invasion, metabolism, and replication, and understanding their relationship with the organelles of better-studied eukaryotic systems suggests potential targets for therapeutic intervention. Recent work has demonstrated divergent aspects of canonical eukaryotic components in the Apicomplexa, including Golgi bodies and mitochondria. The apicoplast is a relict plastid of secondary endosymbiotic origin, harboring metabolic pathways distinct from those of host species. The inner membrane complex (IMC) is derived from the cortical alveoli defining the superphylum Alveolata, but in apicomplexans functions in parasite motility and replication. Micronemes and rhoptries are associated with establishment of the intracellular niche, and define the apical complex for which the phylum is named. Morphological, cell biological and molecular evidence strongly suggest that these organelles are derived from the endocytic pathway.

Chimeric origins of ochrophytes and haptophytes revealed through an ancient plastid proteome

Plastids are supported by a wide range of proteins encoded within the nucleus and imported from the cytoplasm. These plastid-targeted proteins may originate from the endosymbiont, the host, or other sources entirely. Here, we identify and characterise 770 plastid-targeted proteins that are conserved across the ochrophytes, a major group of algae including diatoms, pelagophytes and kelps, that possess plastids derived from red algae. We show that the ancestral ochrophyte plastid proteome was an evolutionary chimera, with 25% of its phylogenetically tractable nucleus-encoded proteins deriving from green algae. We additionally show that functional mixing of host and plastid proteomes, such as through dual-targeting, is an ancestral feature of plastid evolution. Finally, we detect a clear phylogenetic signal from one ochrophyte subgroup, the lineage containing pelagophytes and dictyochophytes, in plastid-targeted proteins from another major algal lineage, the haptophytes. This may represent a possible serial endosymbiosis event deep in eukaryotic evolutionary history. DOI: http://dx.doi.org/10.7554/eLife.23717.001

A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfμ1) mediated protein trafficking in P. falciparum. In trophozoites Pfμ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfμ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfμ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfμ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfμ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum.

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.

A Systematic Cell-Based Analysis of Localization of Predicted Drosophila Peroxisomal Proteins

Peroxisomes are membrane‐bound organelles found in almost all eukaryotic cells. They perform specialized biochemical functions that vary with organism, tissue or cell type. Mutations in human genes required for the assembly of peroxisomes result in a spectrum of diseases called the peroxisome biogenesis disorders. A previous sequence‐based comparison of the predicted proteome of Drosophila melanogaster (the fruit fly) to human proteins identified 82 potential homologues of proteins involved in peroxisomal biogenesis, homeostasis or metabolism. However, the subcellular localization of these proteins relative to the peroxisome was not determined. Accordingly, we tested systematically the localization and selected functions of epitope‐tagged proteins in Drosophila Schneider 2 cells to determine the subcellular localization of 82 potential Drosophila peroxisomal protein homologues. Excluding the Pex proteins, 34 proteins localized primarily to the peroxisome, 8 showed dual localization to the peroxisome and other structures, and 26 localized exclusively to organelles other than the peroxisome. Drosophila is a well‐developed laboratory animal often used for discovery of gene pathways, including those linked to human disease. Our work establishes a basic understanding of peroxisome protein localization in Drosophila. This will facilitate use of Drosophila as a genetically tractable, multicellular model system for studying key aspects of human peroxisome disease.

Phylogeny and Evolution

The concept of a phylogeny of parasites is inextricably linked to that of the phylogeny of eukaryotes. Though it can be useful to infer functional principles from similar morphologies and trophic strategies, the evolutionary histories of parasites are most accurately viewed as independent shifts to this lifestyle from a free-living state. This chapter will describe the phylogeny of eukaryotes, the evolutionary positions of various prominent parasites within this framework, and the ways in which genomics has facilitated understanding of the free-living to parasitic transition, both in terms of phylogeny and function. Two major cellular systems of parasitological relevance, mitochondrion-related organelles and endocytic systems, will be explored, highlighting where considering the genomics and molecular cell biology of parasites in the context of their emergence from free-living relatives have helped us to better understand organelle evolution.

Assessment of microbiome changes after rumen transfaunation: implications on improving feed efficiency in beef cattle

BackgroundUnderstanding the host impact on its symbiotic microbiota is important in redirecting the rumen microbiota and thus improving animal performance. The current study aimed to understand how rumen microbiota were altered and re-established after being emptied and receiving content from donor, thus to understand the impact of such process on rumen microbial fermentation and to explore the microbial phylotypes with higher manipulation potentials.ResultsIndividual animal had strong effect on the re-establishment of the bacterial community according to the observed profiles detected by both fingerprinting and pyrosequencing. Most of the bacterial profile recovery patterns and extents at genus level varied among steers; and each identified bacterial genus responded to transfaunation differently within each host. Coriobacteriaceae, Coprococcus, and Lactobacillus were found to be the most responsive and tunable genera by exchanging rumen content. Besides, the association of 18 bacterial phylotypes with host fermentation parameters suggest that these phylotypes should also be considered as the regulating targets in improving host feed efficiency. In addition, the archaeal community had different re-establishment patterns for each host as determined by fingerprint profiling: it was altered after receiving non-native microbiome in some animals, while it resumed its original status after the adaptation period in the other ones.ConclusionsThe highly individualized microbial re-establishment process suggested the importance of considering host genetics, microbial functional genomics, and host fermentation/performance assessment when developing effective and selective microbial manipulation methods for improving animal feed efficiency.