An endocytic-secretory cycle participates in Toxoplasma gondii in motility

Apicomplexan parasites invade host cells in an active process involving their ability to move by gliding motility. While the acto-myosin system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions regarding the involvement of other mechanisms in parasite motility. In many eukaryotes, a secretory-endocytic cycle leads to the recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility, and generation of retrograde membrane flow. Here, we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, because inhibition of endocytosis blocks retrograde flow and motility. We demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as labelled phospholipids, nanogold particles (NGPs), antibodies, and Concanavalin A (ConA). Using labelled phospholipids, we observed that the endocytic and secretory pathways of the parasite converge, and endocytosed lipids are subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings, and we propose an additional model, working in parallel to the acto-myosin motor, that reconciles parasite motility with observations in other eukaryotes: an apicomplexan fountain-flow-model for parasite motility

Evolutionary analysis identifies a Golgi pathway and correlates lineage-specific factors with endomembrane organelle emergence in apicomplexans

The organelle paralogy hypothesis (OPH) aims to explain the evolution of non-endosymbiotically derived organelles. It predicts that lineage-specific pathways or organelles should result when identity-encoding membrane-trafficking components duplicate and co-evolve. Here, we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in Apicomplexa, a globally important parasitic lineage. We are able to identify 18 paralogs of known membrane-trafficking machinery, in several cases co-incident with the presence of new endomembrane organelles in apicomplexans or their parent lineage, the Alveolata. Moreover, focused analysis of the apicomplexan Arf-like small GTPases (i.e., ArlX3) revealed a specific post-Golgi trafficking pathway. This pathway appears involved in delivery of proteins to micronemes and rhoptries, with knockdown demonstrating reduced invasion capacity. Overall, our data have identified an unforeseen post-Golgi trafficking pathway in apicomplexans and are consistent with the OPH mechanism acting to produce endomembrane pathways or organelles at various evolutionary stages across the alveolate lineage

Plastid Transcript Editing across Dinoflagellate Lineages Shows Lineage-Specific Application but Conserved Trends.

Dinoflagellates are a group of unicellular protists with immense ecological and evolutionary significance and cell biological diversity. Of the photosynthetic dinoflagellates, the majority possess a plastid containing the pigment peridinin, whereas some lineages have replaced this plastid by serial endosymbiosis with plastids of distinct evolutionary affiliations, including a fucoxanthin pigment-containing plastid of haptophyte origin. Previous studies have described the presence of widespread substitutional RNA editing in peridinin and fucoxanthin plastid genes. Because reports of this process have been limited to manual assessment of individual lineages, global trends concerning this RNA editing and its effect on the biological function of the plastid are largely unknown. Using novel bioinformatic methods, we examine the dynamics and evolution of RNA editing over a large multispecies data set of dinoflagellates, including novel sequence data from the peridinin dinoflagellate Pyrocystis lunula and the fucoxanthin dinoflagellate Karenia mikimotoi. We demonstrate that while most individual RNA editing events in dinoflagellate plastids are restricted to single species, global patterns, and functional consequences of editing are broadly conserved. We find that editing is biased toward specific codon positions and regions of genes, and generally corrects otherwise deleterious changes in the genome prior to translation, though this effect is more prevalent in peridinin than fucoxanthin lineages. Our results support a model for promiscuous editing application subsequently shaped by purifying selection, and suggest the presence of an underlying editing mechanism transferred from the peridinin-containing ancestor into fucoxanthin plastids postendosymbiosis, with remarkably conserved functional consequences in the new lineage

Progressive and biased divergent evolution underpins the origin and diversification of peridinin dinoflagellate plastids

Dinoflagellates are algae of tremendous importance to ecosystems and to public health. The cell biology and genome organization of dinoflagellate species is highly unusual. For example, the plastid genomes of peridinin-containing dinoflagellates encode only a minimal number of genes arranged on small elements termed "minicircles". Previous studies of peridinin plastid genes have found evidence for divergent sequence evolution, including extensive substitutions, novel insertions and deletions, and use of alternative translation initiation codons. Understanding the extent of this divergent evolution has been hampered by the lack of characterized peridinin plastid sequences. We have identified over 300 previously unannotated peridinin plastid mRNAs from published transcriptome projects, vastly increasing the number of sequences available. Using these data, we have produced a well-resolved phylogeny of peridinin plastid lineages, which uncovers several novel relationships within the dinoflagellates. This enables us to define changes to plastid sequences that occurred early in dinoflagellate evolution, and that have contributed to the subsequent diversification of individual dinoflagellate clades. We find that the origin of the peridinin dinoflagellates was specifically accompanied by elevations both in the overall number of substitutions that occurred on plastid sequences, and in the Ka/Ks ratio associated with plastid sequences, consistent with changes in selective pressure. These substitutions, alongside other changes, have accumulated progressively in individual peridinin plastid lineages. Throughout our entire dataset, we identify a persistent bias toward non-synonymous substitutions occurring on sequences encoding photosystem I subunits and stromal regions of peridinin plastid proteins, which may have underpinned the evolution of this unusual organelle.Wellcome Trus

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

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Small Genomes and Big Data: Adaptation of Plastid Genomics to the High-Throughput Era

Plastid genome sequences are becoming more readily available with the increase in high-throughput sequencing, and whole-organelle genetic data is available for algae and plants from across the diversity of photosynthetic eukaryotes. This has provided incredible opportunities for studying species which may not be amenable to in vivo study or genetic manipulation or may not yet have been cultured. Research into plastid genomes has pushed the limits of what can be deduced from genomic information, and in particular genomic information obtained from public databases. In this Review, we discuss how research into plastid genomes has benefitted enormously from the explosion of publicly available genome sequence. We describe two case studies in how using publicly available gene data has supported previously held hypotheses about plastid traits from lineage-restricted experiments across algal and plant diversity. We propose how this approach could be used across disciplines for inferring functional and biological characteristics from genomic approaches, including integration of new computational and bioinformatic approaches such as machine learning. We argue that the techniques developed to gain the maximum possible insight from plastid genomes can be applied across the eukaryotic tree of life

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 latter's origin from Archaea. Altogether, our study provides new insights into the emergence and early evolution of the eukaryotic membrane-trafficking system.</p

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

<div><p>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 <i>Guillardia</i><i>theta</i> and <i>Bigelowiella</i><i>natans</i>. 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 <i>Perkinsus marinus</i>. 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.</p> </div

Comparative genomic survey of selected MTC proteins across the diversity of eukaryotes.

<p>The analysis demonstrates the conserved nature of the newly described TRAPPII subunit Tca17/TRAPPC2L, the CORVET complex, and the Dsl1 complex. In this and subsequent Coulson plots, filled pie sectors indicate an identified homologue, while unfilled indicate that no homologue was identified. Asterisks indicate that Vps39 is also present in these taxa (See Table S2). Infinity symbols represent organisms with divergent peroxisomes, or in which peroxisomes have not been identified. Colour-coding is arbitrary and for visual purposes only.</p