Publications by authors named "Michael L Ginger"

55 Publications

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BMC Biol 2021 07 22;19(1):142. Epub 2021 Jul 22.

Division of Infectious Disease, Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada.

Background: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely.

Results: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system.

Conclusions: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.
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http://dx.doi.org/10.1186/s12915-021-01078-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8296547PMC
July 2021

Divergent Cytochrome Maturation System in Kinetoplastid Protists.

mBio 2021 05 4;12(3). Epub 2021 May 4.

School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom

In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes and is catalyzed by either multisubunit cytochrome maturation system I or holocytochrome synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids and parasites, attach heme to mitochondrial -type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists. Cytochromes are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes with a type of heme attachment not seen elsewhere in nature and were the only cytochrome bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.
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http://dx.doi.org/10.1128/mBio.00166-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262978PMC
May 2021

Reductionist Pathways for Parasitism in Euglenozoans? Expanded Datasets Provide New Insights.

Trends Parasitol 2021 02 27;37(2):100-116. Epub 2020 Oct 27.

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budějovice (Budweis), Czech Republic; Faculty of Sciences, University of South Bohemia, České Budějovice (Budweis), Czech Republic. Electronic address:

The unicellular trypanosomatids belong to the phylum Euglenozoa and all known species are obligate parasites. Distinct lineages infect plants, invertebrates, and vertebrates, including humans. Genome data for marine diplonemids, together with freshwater euglenids and free-living kinetoplastids, the closest known nonparasitic relatives to trypanosomatids, recently became available. Robust phylogenetic reconstructions across Euglenozoa are now possible and place the results of parasite-focused studies into an evolutionary context. Here we discuss recent advances in identifying the factors shaping the evolution of Euglenozoa, focusing on ancestral features generally considered parasite-specific. Remarkably, most of these predate the transition(s) to parasitism, suggesting that the presence of certain preconditions makes a significant lifestyle change more likely.
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http://dx.doi.org/10.1016/j.pt.2020.10.001DOI Listing
February 2021

Self-Assembled Anion-Binding Cryptand for the Selective Liquid-Liquid Extraction of Phosphate Anions.

Angew Chem Int Ed Engl 2020 11 2;59(46):20480-20484. Epub 2020 Sep 2.

School of Animal, Rural and Environmental Sciences, Nottingham Trent University, Nottingham, NG25 0QF, UK.

The ligands L and L form trinuclear self-assembled complexes with Cu (i.e. [(L ) Cu ] or [(L ) Cu ] ) both of which act as a host to a variety of anions. Inclusion of long aliphatic chains on these ligands allows the assemblies to extract anions from aqueous media into organic solvents. Phosphate can be removed from water efficiently and highly selectively, even in the presence of other anions.
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http://dx.doi.org/10.1002/anie.202009960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693201PMC
November 2020

Airyscan Superresolution Microscopy to Study Trypanosomatid Cell Biology.

Methods Mol Biol 2020 ;2116:449-461

School of Applied Sciences, University of Huddersfield, Huddersfield, UK.

The recent introduction by Carl Zeiss Ltd. of the Airyscan detector module for their LSM880 confocal laser-scanning microscope has enabled routine superresolution microscopy to be combined with the advantages of confocal-based fluorescence imaging. Resulting enhanced spatial resolution in X, Y, and Z provides tractable opportunity to derive new insight into protein localization(s), organelle dynamics, and thence protein function within trypanosomatids or other organisms. Here, we describe methods for preparing slides, cells, and basic microscope setup for fluorescence imaging of trypanosomatids using the LSM-880 with Airyscan platform.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_27DOI Listing
February 2021

Analysis of Base Excision and Single-Strand Break Repair Activities in Trypanosomatid Extracts.

Methods Mol Biol 2020 ;2116:353-364

Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK.

Cellular DNA is inherently unstable, subject to both spontaneous hydrolysis and attack by a range of exogenous and endogenous chemicals as well as physical agents such as ionizing and ultraviolet radiation. For parasitic protists, where an inoculum of infectious parasites is typically small and natural infections are often chronic with low parasitemia, they are also vulnerable to DNA damaging agents arising from innate immune defenses. The majority of DNA damage consists of relatively minor changes to the primary structure of the DNA, such as base deamination, oxidation, or alkylation and scission of the phosphodiester backbone. Yet these small changes can have serious consequences, often being mutagenic or cytotoxic. Cells have therefore evolved efficient mechanisms to repair such damage, with base excision and single strand break repair playing the primary role here. In this chapter we describe a method for analyzing the activity from cell extracts of various enzymes involved in the base excision and single strand break repair pathways of trypanosomatid parasites.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_22DOI Listing
February 2021

Transcriptome, proteome and draft genome of Euglena gracilis.

BMC Biol 2019 02 7;17(1):11. Epub 2019 Feb 7.

School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.

Background: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts.

Results: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids.

Conclusions: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.
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http://dx.doi.org/10.1186/s12915-019-0626-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366073PMC
February 2019

A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes.

Open Biol 2018 07;8(7)

Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, UK

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, FOPL and OFD1, in the evolutionarily divergent sleeping sickness parasite , showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, FOPL, affects neither axoneme nor flagellum elongation. Instead, FOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar FOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, FOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for FOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of FOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.
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http://dx.doi.org/10.1098/rsob.170218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070722PMC
July 2018

Farming, slaving and enslavement: histories of endosymbioses during kinetoplastid evolution.

Parasitology 2018 09 13;145(10):1311-1323. Epub 2018 Jun 13.

Department of Biological Sciences,School of Applied Sciences, University of Huddersfield,Huddersfield, HD1 3DH,UK.

Parasitic trypanosomatids diverged from free-living kinetoplastid ancestors several hundred million years ago. These parasites are relatively well known, due in part to several unusual cell biological and molecular traits and in part to the significance of a few - pathogenic Leishmania and Trypanosoma species - as aetiological agents of serious neglected tropical diseases. However, the majority of trypanosomatid biodiversity is represented by osmotrophic monoxenous parasites of insects. In two lineages, novymonads and strigomonads, osmotrophic lifestyles are supported by cytoplasmic endosymbionts, providing hosts with macromolecular precursors and vitamins. Here we discuss the two independent origins of endosymbiosis within trypanosomatids and subsequently different evolutionary trajectories that see entrainment vs tolerance of symbiont cell divisions cycles within those of the host. With the potential to inform on the transition to obligate parasitism in the trypanosomatids, interest in the biology and ecology of free-living, phagotrophic kinetoplastids is beginning to enjoy a renaissance. Thus, we take the opportunity to additionally consider the wider relevance of endosymbiosis during kinetoplastid evolution, including the indulged lifestyle and reductive evolution of basal kinetoplastid Perkinsela.
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http://dx.doi.org/10.1017/S0031182018000781DOI Listing
September 2018

Variation in Basal Body Localisation and Targeting of Trypanosome RP2 and FOR20 Proteins.

Protist 2017 Aug 13;168(4):452-466. Epub 2017 Jul 13.

Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, UK. Electronic address:

TOF-LisH-PLL motifs define FOP family proteins; some members are involved in flagellum assembly. The critical role of FOP family protein FOR20 is poorly understood. Here, we report relative localisations of the four FOP family proteins in parasitic Trypanosoma brucei: TbRP2, TbOFD1 and TbFOP/FOP1-like are mature basal body proteins whereas TbFOR20 is present on pro- and mature basal bodies - on the latter it localises distal to TbRP2. We discuss how the data, together with published work for another protist Giardia intestinalis, informs on likely FOR20 function. Moreover, our localisation study provides convincing evidence that the antigen recognised by monoclonal antibody YL1/2 at trypanosome mature basal bodies is FOP family protein TbRP2, not tyrosinated α-tubulin as widely stated in the literature. Curiously, FOR20 proteins from T. brucei and closely related African trypanosomes possess short, negatively-charged N-terminal extensions absent from FOR20 in other trypanosomatids and other eukaryotes. The extension is necessary for protein targeting, but insufficient to re-direct TbRP2 to probasal bodies. Yet, FOR20 from the American trypanosome T. cruzi, which lacks any extension, localises to pro- and mature basal bodies when expressed in T. brucei. This identifies unexpected variation in FOR20 architecture that is presently unique to one clade of trypanosomatids.
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http://dx.doi.org/10.1016/j.protis.2017.07.002DOI Listing
August 2017

Peroxisomes in parasitic protists.

Mol Biochem Parasitol 2016 Sep - Oct;209(1-2):35-45. Epub 2016 Feb 16.

Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, UK; Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, UK; Laboratorio de Enzimología de Parásitos, Departamento de Biología, Universidad de Los Andes, Mérida, Venezuela. Electronic address:

Representatives of all major lineages of eukaryotes contain peroxisomes with similar morphology and mode of biogenesis, indicating a monophyletic origin of the organelles within the common ancestor of all eukaryotes. Peroxisomes originated from the endoplasmic reticulum, but despite a common origin and shared morphological features, peroxisomes from different organisms show a remarkable diversity of enzyme content and the metabolic processes present can vary dependent on nutritional or developmental conditions. A common characteristic and probable evolutionary driver for the origin of the organelle is an involvement in lipid metabolism, notably HO-dependent fatty-acid oxidation. Subsequent evolution of the organelle in different lineages involved multiple acquisitions of metabolic processes-often involving retargeting enzymes from other cell compartments-and losses. Information about peroxisomes in protists is still scarce, but available evidence, including new bioinformatics data reported here, indicate striking diversity amongst free-living and parasitic protists from different phylogenetic supergroups. Peroxisomes in only some protists show major involvement in HO-dependent metabolism, as in peroxisomes of mammalian, plant and fungal cells. Compartmentalization of glycolytic and gluconeogenic enzymes inside peroxisomes is characteristic of kinetoplastids and diplonemids, where the organelles are hence called glycosomes, whereas several other excavate parasites (Giardia, Trichomonas) have lost peroxisomes. Amongst alveolates and amoebozoans patterns of peroxisome loss are more complicated. Often, a link is apparent between the niches occupied by the parasitic protists, nutrient availability, and the absence of the organelles or their presence with a specific enzymatic content. In trypanosomatids, essentiality of peroxisomes may be considered for use in anti-parasite drug discovery.
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http://dx.doi.org/10.1016/j.molbiopara.2016.02.005DOI Listing
November 2017

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

Curr Biol 2016 Jan 24;26(2):161-172. Epub 2015 Dec 24.

Pathogen Genomics Group, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

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.
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http://dx.doi.org/10.1016/j.cub.2015.11.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728078PMC
January 2016

Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions.

J Cell Sci 2015 Aug 6;128(16):3117-30. Epub 2015 Jul 6.

Faculty of Sciences, University of South Bohemia, České Budějovice (Budweis) 37005, Czech Republic Institute of Parasitology, Biology Centre, Czech Academy of Sciences, České Budějovice (Budweis) 37005, Czech Republic Canadian Institute for Advanced Research, Toronto, Ontario, Canada M5G 1Z8

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.
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http://dx.doi.org/10.1242/jcs.171645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541047PMC
August 2015

Protein moonlighting in parasitic protists.

Authors:
Michael L Ginger

Biochem Soc Trans 2014 Dec;42(6):1734-9

*Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster LA1 4YG, U.K.

Reductive evolution during the adaptation to obligate parasitism and expansions of gene families encoding virulence factors are characteristics evident to greater or lesser degrees in all parasitic protists studied to date. Large evolutionary distances separate many parasitic protists from the yeast and animal models upon which classic views of eukaryotic biochemistry are often based. Thus a combination of evolutionary divergence, niche adaptation and reductive evolution means the biochemistry of parasitic protists is often very different from their hosts and to other eukaryotes generally, making parasites intriguing subjects for those interested in the phenomenon of moonlighting proteins. In common with other organisms, the contribution of protein moonlighting to parasite biology is only just emerging, and it is not without controversy. Here, an overview of recently identified moonlighting proteins in parasitic protists is provided, together with discussion of some of the controversies.
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http://dx.doi.org/10.1042/BST20140215DOI Listing
December 2014

TrypanoCyc: a community-led biochemical pathways database for Trypanosoma brucei.

Nucleic Acids Res 2015 Jan 9;43(Database issue):D637-44. Epub 2014 Oct 9.

University of Liverpool, Liverpool, Merseyside L69 3BX, UK.

The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite.
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http://dx.doi.org/10.1093/nar/gku944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384016PMC
January 2015

Modulation of a cytoskeletal calpain-like protein induces major transitions in trypanosome morphology.

J Cell Biol 2014 Aug;206(3):377-84

Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, England, UK

Individual eukaryotic microbes, such as the kinetoplastid parasite Trypanosoma brucei, have a defined size, shape, and form yet transition through life cycle stages, each having a distinct morphology. In questioning the structural processes involved in these transitions, we have identified a large calpain-like protein that contains numerous GM6 repeats (ClpGM6) involved in determining T. brucei cell shape, size, and form. ClpGM6 is a cytoskeletal protein located within the flagellum along the flagellar attachment zone (FAZ). Depletion of ClpGM6 in trypomastigote forms produces cells with long free flagella and a shorter FAZ, accompanied by repositioning of the basal body, the kinetoplast, Golgi, and flagellar pocket, reflecting an epimastigote-like morphology. Hence, major changes in microbial cell form can be achieved by simple modulation of one or a few proteins via coordinated association and positioning of membrane and cytoskeletal components.
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http://dx.doi.org/10.1083/jcb.201312067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4121973PMC
August 2014

Evidence for loss of a partial flagellar glycolytic pathway during trypanosomatid evolution.

PLoS One 2014 22;9(7):e103026. Epub 2014 Jul 22.

Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster, United Kingdom.

Classically viewed as a cytosolic pathway, glycolysis is increasingly recognized as a metabolic pathway exhibiting surprisingly wide-ranging variations in compartmentalization within eukaryotic cells. Trypanosomatid parasites provide an extreme view of glycolytic enzyme compartmentalization as several glycolytic enzymes are found exclusively in peroxisomes. Here, we characterize Trypanosoma brucei flagellar proteins resembling glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK): we show the latter associates with the axoneme and the former is a novel paraflagellar rod component. The paraflagellar rod is an essential extra-axonemal structure in trypanosomes and related protists, providing a platform into which metabolic activities can be built. Yet, bioinformatics interrogation and structural modelling indicate neither the trypanosome PGK-like nor the GAPDH-like protein is catalytically active. Orthologs are present in a free-living ancestor of the trypanosomatids, Bodo saltans: the PGK-like protein from B. saltans also lacks key catalytic residues, but its GAPDH-like protein is predicted to be catalytically competent. We discuss the likelihood that the trypanosome GAPDH-like and PGK-like proteins constitute molecular evidence for evolutionary loss of a flagellar glycolytic pathway, either as a consequence of niche adaptation or the re-localization of glycolytic enzymes to peroxisomes and the extensive changes to glycolytic flux regulation that accompanied this re-localization. Evidence indicating loss of localized ATP provision via glycolytic enzymes therefore provides a novel contribution to an emerging theme of hidden diversity with respect to compartmentalization of the ubiquitous glycolytic pathway in eukaryotes. A possibility that trypanosome GAPDH-like protein additionally represents a degenerate example of a moonlighting protein is also discussed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0103026PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4106842PMC
November 2015

An alternative model for the role of RP2 protein in flagellum assembly in the African trypanosome.

J Biol Chem 2014 Jan 20;289(1):464-75. Epub 2013 Nov 20.

From the Faculty of Health and Medicine, Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, United Kingdom.

The tubulin cofactor C domain-containing protein TbRP2 is a basal body (centriolar) protein essential for axoneme formation in the flagellate protist Trypanosoma brucei, the causal agent of African sleeping sickness. Here, we show how TbRP2 is targeted and tethered at mature basal bodies and provide novel insight into TbRP2 function. Regarding targeting, understanding how several hundred proteins combine to build a microtubule axoneme is a fundamental challenge in eukaryotic cell biology. We show that basal body localization of TbRP2 is mediated by twinned, N-terminal TOF (TON1, OFD1, and FOP) and LisH motifs, motifs that otherwise facilitate localization of only a few conserved proteins at microtubule-organizing centers in animals, plants, and flagellate protists. Regarding TbRP2 function, there is a debate as to whether the flagellar assembly function of specialized, centriolar tubulin cofactor C domain-containing proteins is processing tubulin, the major component of axonemes, or general vesicular trafficking in a flagellum assembly context. Here we report that TbRP2 is required for the recruitment of T. brucei orthologs of MKS1 and MKS6, proteins that, in animal cells, are part of a complex that assembles at the base of the flagellum to regulate protein composition and cilium function. We also identify that TbRP2 is detected by YL1/2, an antibody classically used to detect α-tubulin. Together, these data suggest a general processing role for TbRP2 in trypanosome flagellum assembly and challenge the notion that TbRP2 functions solely in assessing tubulin "quality" prior to tubulin incorporation into the elongating axoneme.
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http://dx.doi.org/10.1074/jbc.M113.509521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879569PMC
January 2014

The tubulin cofactor C family member TBCCD1 orchestrates cytoskeletal filament formation.

J Cell Sci 2013 Dec 7;126(Pt 23):5350-6. Epub 2013 Oct 7.

Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, UK.

TBCCD1 is an enigmatic member of the tubulin-binding cofactor C (TBCC) family of proteins required for mother-daughter centriole linkage in the green alga Chlamydomonas reinhardtii and nucleus-centrosome-Golgi linkage in mammalian cells. Loss of these linkages has severe morphogenetic consequences, but the mechanism(s) through which TBCCD1 contributes to cell organisation is unknown. In the African sleeping sickness parasite Trypanosoma brucei a microtubule-dominant cytoskeleton dictates cell shape, influencing strongly the positioning and inheritance patterns of key intracellular organelles. Here, we show the trypanosome orthologue of TBCCD1 is found at multiple locations: centrioles, the centriole-associated Golgi 'bi-lobe', and the anterior end of the cell body. Loss of Trypanosoma brucei TBCCD1 results in disorganisation of the structurally complex bi-lobe architecture and loss of centriole linkage to the single unit-copy mitochondrial genome (or kinetoplast) of the parasite. We therefore identify TBCCD1 as an essential protein associated with at least two filament-based structures in the trypanosome cytoskeleton. The last common ancestor of trypanosomes, animals and green algae was arguably the last common ancestor of all eukaryotes. On the basis of our observations, and interpretation of published data, we argue for an unexpected co-option of the TBCC domain for an essential non-tubulin-related function at an early point during evolution of the eukaryotic cytoskeleton.
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http://dx.doi.org/10.1242/jcs.136515DOI Listing
December 2013

Molecular paleontology and complexity in the last eukaryotic common ancestor.

Crit Rev Biochem Mol Biol 2013 Jul-Aug;48(4):373-96

Biomedical Research Foundation, Academy of Athens, Soranou Efesiou 4, Athens 115 27, Greece.

Eukaryogenesis, the origin of the eukaryotic cell, represents one of the fundamental evolutionary transitions in the history of life on earth. This event, which is estimated to have occurred over one billion years ago, remains rather poorly understood. While some well-validated examples of fossil microbial eukaryotes for this time frame have been described, these can provide only basic morphology and the molecular machinery present in these organisms has remained unknown. Complete and partial genomic information has begun to fill this gap, and is being used to trace proteins and cellular traits to their roots and to provide unprecedented levels of resolution of structures, metabolic pathways and capabilities of organisms at these earliest points within the eukaryotic lineage. This is essentially allowing a molecular paleontology. What has emerged from these studies is spectacular cellular complexity prior to expansion of the eukaryotic lineages. Multiple reconstructed cellular systems indicate a very sophisticated biology, which by implication arose following the initial eukaryogenesis event but prior to eukaryotic radiation and provides a challenge in terms of explaining how these early eukaryotes arose and in understanding how they lived. Here, we provide brief overviews of several cellular systems and the major emerging conclusions, together with predictions for subsequent directions in evolution leading to extant taxa. We also consider what these reconstructions suggest about the life styles and capabilities of these earliest eukaryotes and the period of evolution between the radiation of eukaryotes and the eukaryogenesis event itself.
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http://dx.doi.org/10.3109/10409238.2013.821444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3791482PMC
February 2014

Calmodulin is required for paraflagellar rod assembly and flagellum-cell body attachment in trypanosomes.

Protist 2013 Jul 19;164(4):528-40. Epub 2013 Jun 19.

Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, UK.

In the flagellum of the African sleeping sickness parasite Trypanosoma brucei calmodulin (CaM) is found within the paraflagellar rod (PFR), an elaborate extra-axonemal structure, and the axoneme. In dissecting mechanisms of motility regulation we analysed CaM function using RNAi. Unexpectedly CaM depletion resulted in total and catastrophic failure in PFR assembly; even connections linking axoneme to PFR failed to form following CaM depletion. This provides an intriguing parallel with the role in the green alga Chlamydomonas of a CaM-related protein in docking outer-dynein arms to axoneme outer-doublet microtubules. Absence of CaM had no discernible effect on axoneme assembly, but the failure in PFR assembly was further compounded by loss of the normal linkage between PFR and axoneme to the flagellum attachment zone of the cell body. Thus, flagellum detachment was a secondary, time-dependent consequence of CaM RNAi, and coincided with the loss of normal trypomastigote morphology, thereby linking the presence of PFR architecture with maintenance of cell form, as well as cell motility. Finally, wider comparison between the flagellum detachment phenotypes of RNAi mutants for CaM and the FLA1 glycoprotein potentially provides new perspective into the function of the latter into establishing and maintaining flagellum-cell body attachment.
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http://dx.doi.org/10.1016/j.protis.2013.05.002DOI Listing
July 2013

Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling.

Genome Biol 2013 Feb 1;14(2):R11. Epub 2013 Feb 1.

Background: The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan.

Results: Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms.

Conclusions: Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.
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http://dx.doi.org/10.1186/gb-2013-14-2-r11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053784PMC
February 2013

Divergence of Erv1-associated mitochondrial import and export pathways in trypanosomes and anaerobic protists.

Eukaryot Cell 2013 Feb 21;12(2):343-55. Epub 2012 Dec 21.

Biology Centre, Institute of Parasitology, Czech Academy of Sciences and Faculty of Sciences, University of South Bohemia, České Budějovice (Budweis), Czech Republic.

In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O(2), unexpectedly find O(2) and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O(2) by TbERV1 is not dependent upon a simple O(2) channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.
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http://dx.doi.org/10.1128/EC.00304-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3571301PMC
February 2013

Guidelines for the use and interpretation of assays for monitoring autophagy.

Autophagy 2012 Apr;8(4):445-544

Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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http://dx.doi.org/10.4161/auto.19496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883PMC
April 2012

Probing why trypanosomes assemble atypical cytochrome c with an AxxCH haem-binding motif instead of CxxCH.

Biochem J 2012 Dec;448(2):253-60

Faculty of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, UK.

Mitochondrial cytochromes c and c1 are core components of the respiratory chain of all oxygen-respiring eukaryotes. These proteins contain haem, covalently bound to the polypeptide in a catalysed post-translational modification. In all eukaryotes, except members of the protist phylum Euglenozoa, haem attachment is to the cysteine residues of a CxxCH haem-binding motif. In the Euglenozoa, which include medically relevant trypanosomatid parasites, haem attachment is to a single cysteine residue in an AxxCH haem-binding motif. Moreover, genes encoding known c-type cytochrome biogenesis machineries are all absent from trypanosomatid genomes, indicating the presence of a novel biosynthetic apparatus. In the present study, we investigate expression and maturation of cytochrome c with a typical CxxCH haem-binding motif in the trypanosomatids Crithidia fasciculata and Trypanosoma brucei. Haem became attached to both cysteine residues of the haem-binding motif, indicating that, in contrast with previous hypotheses, nothing prevents formation of a CxxCH cytochrome c in euglenozoan mitochondria. The cytochrome variant was also able to replace the function of wild-type cytochrome c in T. brucei. However, the haem attachment to protein was not via the stereospecifically conserved linkage universally observed in natural c-type cytochromes, suggesting that the trypanosome cytochrome c biogenesis machinery recognized and processed only the wild-type single-cysteine haem-binding motif. Moreover, the presence of the CxxCH cytochrome c resulted in a fitness cost in respiration. The level of cytochrome c biogenesis in trypanosomatids was also found to be limited, with the cells operating at close to maximum capacity.
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http://dx.doi.org/10.1042/BJ20120757DOI Listing
December 2012

Proteomic insights into parasite biology.

Parasitology 2012 Aug;139(9):1101-2

Division of Biomedical and Life Sciences, Lancaster University, LA1 4YK, UK.

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http://dx.doi.org/10.1017/S0031182012000947DOI Listing
August 2012

Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species.

Proc Natl Acad Sci U S A 2012 Feb 13;109(9):3416-21. Epub 2012 Feb 13.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

Antigenic variation enables pathogens to avoid the host immune response by continual switching of surface proteins. The protozoan blood parasite Trypanosoma brucei causes human African trypanosomiasis ("sleeping sickness") across sub-Saharan Africa and is a model system for antigenic variation, surviving by periodically replacing a monolayer of variant surface glycoproteins (VSG) that covers its cell surface. We compared the genome of Trypanosoma brucei with two closely related parasites Trypanosoma congolense and Trypanosoma vivax, to reveal how the variant antigen repertoire has evolved and how it might affect contemporary antigenic diversity. We reconstruct VSG diversification showing that Trypanosoma congolense uses variant antigens derived from multiple ancestral VSG lineages, whereas in Trypanosoma brucei VSG have recent origins, and ancestral gene lineages have been repeatedly co-opted to novel functions. These historical differences are reflected in fundamental differences between species in the scale and mechanism of recombination. Using phylogenetic incompatibility as a metric for genetic exchange, we show that the frequency of recombination is comparable between Trypanosoma congolense and Trypanosoma brucei but is much lower in Trypanosoma vivax. Furthermore, in showing that the C-terminal domain of Trypanosoma brucei VSG plays a crucial role in facilitating exchange, we reveal substantial species differences in the mechanism of VSG diversification. Our results demonstrate how past VSG evolution indirectly determines the ability of contemporary parasites to generate novel variant antigens through recombination and suggest that the current model for antigenic variation in Trypanosoma brucei is only one means by which these parasites maintain chronic infections.
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http://dx.doi.org/10.1073/pnas.1117313109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295286PMC
February 2012

The Naegleria genome: a free-living microbial eukaryote lends unique insights into core eukaryotic cell biology.

Res Microbiol 2011 Jul-Aug;162(6):607-18. Epub 2011 Mar 21.

Department of Cellular and Molecular Pharmacology, UCSF Medical School, San Francisco, CA 94107, USA.

Naegleria gruberi, a free-living protist, has long been treasured as a model for basal body and flagellar assembly due to its ability to differentiate from crawling amoebae into swimming flagellates. The full genome sequence of Naegleria gruberi has recently been used to estimate gene families ancestral to all eukaryotes and to identify novel aspects of Naegleria biology, including likely facultative anaerobic metabolism, extensive signaling cascades, and evidence for sexuality. Distinctive features of the Naegleria genome and nuclear biology provide unique perspectives for comparative cell biology, including cell division, RNA processing and nucleolar assembly. We highlight here exciting new and novel aspects of Naegleria biology identified through genomic analysis.
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http://dx.doi.org/10.1016/j.resmic.2011.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929615PMC
December 2011

Autophagy in parasitic protists: unique features and drug targets.

Mol Biochem Parasitol 2011 Jun 21;177(2):83-99. Epub 2011 Feb 21.

Research Unit for Tropical Diseases, de Duve Institute, Brussels, Belgium.

Eukaryotic cells can degrade their own components, cytosolic proteins and organelles, using dedicated hydrolases contained within the acidic interior of their lysosomes. This degradative process, called autophagy, is used under starvation conditions to recycle redundant or less important macromolecules, facilitates metabolic re-modeling in response to environmental cues, and is also often important during cell differentiation. In this review, we discuss the role played by autophagy during the life cycles of the major parasitic protists. To provide context, we also provide an overview of the different forms of autophagy and the successive steps in the autophagic processes, including the proteins involved, as revealed in recent decades by studies using the model organism Saccharomyces cerevisiae, methylotrophic yeasts and mammalian cells. We describe for trypanosomatid parasites how autophagy plays a role in the differentiation from one life cycle stage to the next one and, in the case of the intracellular parasites, for virulence. For malarial parasites, although only a limited repertoire of canonical autophagy-related proteins can be detected, autophagy seems to play a role in the removal of redundant organelles important for cell invasion, when sporozoites develop into intracellular trophozoites inside the hepatocytes. The complete absence of a canonical autophagy pathway from the microaerophile Giardia lamblia is also discussed. Finally, the essential role of autophagy for differentiation and pathogenicity of some pathogenic protists suggests that the proteins involved in this process may represent new targets for drug development. Opportunities and strategies for drug design targeting autophagy proteins are discussed.
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http://dx.doi.org/10.1016/j.molbiopara.2011.02.003DOI Listing
June 2011

Intermediary metabolism in protists: a sequence-based view of facultative anaerobic metabolism in evolutionarily diverse eukaryotes.

Protist 2010 Dec 30;161(5):642-71. Epub 2010 Oct 30.

School of Health and Medicine, Division of Biomedical and Life Sciences, Lancaster University, Lancaster LA1 4YQ, UK.

Protists account for the bulk of eukaryotic diversity. Through studies of gene and especially genome sequences the molecular basis for this diversity can be determined. Evident from genome sequencing are examples of versatile metabolism that go far beyond the canonical pathways described for eukaryotes in textbooks. In the last 2-3 years, genome sequencing and transcript profiling has unveiled several examples of heterotrophic and phototrophic protists that are unexpectedly well-equipped for ATP production using a facultative anaerobic metabolism, including some protists that can (Chlamydomonas reinhardtii) or are predicted (Naegleria gruberi, Acanthamoeba castellanii, Amoebidium parasiticum) to produce H(2) in their metabolism. It is possible that some enzymes of anaerobic metabolism were acquired and distributed among eukaryotes by lateral transfer, but it is also likely that the common ancestor of eukaryotes already had far more metabolic versatility than was widely thought a few years ago. The discussion of core energy metabolism in unicellular eukaryotes is the subject of this review. Since genomic sequencing has so far only touched the surface of protist diversity, it is anticipated that sequences of additional protists may reveal an even wider range of metabolic capabilities, while simultaneously enriching our understanding of the early evolution of eukaryotes.
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http://dx.doi.org/10.1016/j.protis.2010.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021972PMC
December 2010
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