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Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi letters to nature ................................................................. The gradual increase in G + C content at the core centre described in chromosome I (chrI) exists in all the chromosomes (maximum Genome sequence and gene 51.0%). The 1,997 protein-coding DNA sequences (CDSs) repre- sent about 90% of the chromosome cores, as a result of generally compaction of the eukaryote short intergenic regions (see Supplementary Information). Gene density is slightly lower than that observed in the nucleomorph parasite Encephalitozoon cuniculi genome of the cryptomonad Guillardia theta . Only about 44% of the CDSs are assigned to functional categories and about 6% to Michae È l D. Katinka*, Simone Duprat*, Emmanuel Cornillot², conserved hypothetical proteins (Fig. 1). In contrast to the nucleo- Guy Me  te  nier², Fabienne Thomarat³,Ge  rard Prensier², Vale  rie Barbe*, morph genome , no overlapping of CDSs with predicted functions Eric Peyretaillade², Philippe Brottier*, Patrick Wincker*, Fre  de  ric Delbac², Hicham El Alaoui², Pierre Peyret², William Saurin*, was revealed. Structural or functional clusters are rare and never Manolo Gouy³, Jean Weissenbach* & Christian P. Vivare Á s² composed of more than two CDSs (for example, histones H3 and H4 on chrIX). Genome compaction can also be related to gene * Genoscope, UMR CNRS 8030, CP 5706, 91057 Evry cedex, France shortening, as indicated by the length distribution of all potential ² Parasitologie Mole Âculaire et Cellulaire, Laboratoire de Biologie des Protistes, proteins (Fig. 2a). The mean and median lengths of all potential UMR CNRS 6023, Universite  Blaise Pascal, 63177 Aubie Áre cedex, France E. cuniculi proteins are only 359 and 281 amino acids, respectively. Laboratoire de Biome Âtrie et Biologie Evolutive, UMR CNRS 5558, We compared the lengths of 350 proteins with Saccharomyces Universite  Lyon I, 69622 Villeurbanne cedex, France cerevisiae homologues (Fig. 2b). More than 85% of these proteins .............................................................................................................................................. are shorter than in yeast, with a mean relative size difference of Microsporidia are obligate intracellular parasites infesting many 14.6%. From the analysis of the protein size distributions derived animal groups . Lacking mitochondria and peroxysomes, these from sequenced genomes, it has been suggested that the lengthening unicellular eukaryotes were ®rst considered a deeply branching of proteins in eukaryotes (non-parasitic species) allows for more 2 14 protist lineage that diverged before the endosymbiotic event that complex regulation networks . Thus, protein shortening in E. led to mitochondria. The discovery of a gene for a mitochondrial- cuniculi may re¯ect reduced protein± protein interactions as a 3±5 6±9 type chaperone combined with molecular phylogenetic data result of various gene losses linked to the intracellular parasitic later implied that microsporidia are atypical fungi that lost nature (Fig. 2a, b). mitochondria during evolution. Here we report the DNA Perfect segmental duplications of 0.5 ± 10-kb coding and non- sequences of the 11 chromosomes of the ,2.9-megabase (Mb) coding sequences represent about 3.7% of the core region in average genome of Encephalitozoon cuniculi (1,997 potential protein- (from ,1% in chrII, III, IV and VI to ,7% in chrIX; see coding genes). Genome compaction is re¯ected by reduced inter- Supplementary Information). A segment carrying four enzyme- genic spacers and by the shortness of most putative proteins coding genes is perfectly duplicated in the extreme part of the chrI relative to their eukaryote orthologues. The strong host depen- core but partially duplicated near the end of chrVIII (truncated dence is illustrated by the lack of genes for some biosynthetic serine hydroxymethyltransferase gene). The genome core sequences pathways and for the tricarboxylic acid cycle. Phylogenetic analy- exhibit a very low base polymorphism, restricted to a few positions sis lends substantial credit to the fungal af®liation of microspor- in eight chromosomes. A duplicated gene homologous to CTP idia. Because the E. cuniculi genome contains genes related to synthases (chrXI) revealed a rare polymorphic tract of 189 base pairs some mitochondrial functions (for example, Fe ± S cluster assem- (bp) (116,847 ± 117,036). Hybridization experiments indicate that bly), we hypothesize that microsporidia have retained a mito- each polymorphic sequence is present in about 50% of the DNA chondrion-derived organelle. molecules (data not shown), suggesting that they are alleles and 10,12 Encephalitozoon cuniculi infects various mammals, including supporting the diploidy hypothesis . humans, and can cause digestive and nervous clinical syndromes A large proportion of CDSs is assigned to the conservation and in HIV-infected or cyclosporine-treated people . Its reproduction transmission of genetic information as well as to protein modi®ca- proceeds as a sequence of two major stages: merogony, involving the tion and intracellular transport processes (Fig. 1), but with a multiplication of large, wall-lacking cells (meronts); and sporogony, signi®cant degree of simpli®cation, mainly related to the lack of leading to small, thick-walled spores. The sporal invasive apparatus DNA-containing organelles. For example, subunits for DNA pol I is characterized by a long polar tube that can be quickly extruded (d), pol II (e) and pol III (a but not b) are present while there is no then used for transferring the sporoplasm into the target cell. candidate for the mitochondrial DNA polymerase g. Apart from the Consisting of 11 linear chromosomes ranging from 217 to 315 kb, telomerase catalytic subunit, no RNA-dependent reverse transcrip- the E. cuniculi genome is remarkably reduced (,2.9 Mb) . The tase was found. This and the lack of any retrotransposition elements nucleotide sequence of the smallest chromosome has been recently may explain the absence of pseudoretrogenes. The potential tran- reported . The full sequencing of this minimal genome among scription machinery includes RNA pol I, II and III, more than 70 eukaryotes was expected to provide insight into the metabolism and messenger RNA transcription-associated proteins and a virtually general biology of microsporidia and to help in the understanding of the evolutionary history of amitochondriate eukaryotes currently considered `curious fungi' (see Table 1). Table 1 General features of the E. cuniculi genome The chromosome sequences were determined through a plasmid Total sequenced length 2,507,519 bp library (,3 kb inserts) and a miniBAC library (,20 ± 25 kb inserts) G + C content Protein-coding regions 47.6% totalling 15 genome equivalents (,46 Mb). All chromosomes Intergenic regions 45.0% possess a `unique sequences' core region ¯anked by two 28-kb Telomeric and subtelomeric regions 52.9% divergently oriented regions, each including one ribosomal DNA No. of protein-coding sequences 1,997 11,12 Mean intergenic distance 129 bp unit . The subtelomeric repeats upstream from rDNA are mostly Gene density of chromosome cores 1 CDS per 1,025 bp degenerated minisatellites, whereas downstream repeats consist No. and sizes of spliceosomal introns 13 (23±52 bp) essentially of non-polymorphic microsatellite arrays. The chromo- No. of 16S±23S rRNA genes 22* No. of 5S rRNA genes 3 (on chrV, VII, IX) some cores lack simple sequence repeats, minisatellite arrays and No. of tRNA genes 44² known transposable elements. No imprint of retrogenes or pseudo- No. and sizes of tRNA introns 2 (16, 42 bp) ............................................................................................................................................................................. retrogenes of either polymerase (pol) II or pol III type was found. * Two per chromosome. General features of genome organization are indicated in Table 1. ² On all chromosomes. 450 NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature complete set of genes for splicing, 59 and 39 processing. An (A + T)- the E1 component of the mitochondrial pyruvate dehydrogenase rich consensus transcription initiation sequence is revealed in the complex. Pyruvate decarboxylation could be inferred, but, in the 120-bp region upstream from the initiation codon of numerous absence of evidence for E2 and E3 components, a subsequent genes, suggesting short 59 leaders. Seventy different eukaryotic-type production of acetyl coenzyme A cannot be concluded. ribosomal proteins (40 from the large subunit, 30 from the small Microsporidia have a presumably simpli®ed Golgi apparatus in subunit) are predicted, which is slightly lower than in the amito- which a cis ±trans polarity is not cytologically distinguishable but 15 1 chondriate protist Giardia lamblia (74) . Compared with the that is central in sporogony-speci®c secretion processes . The spore cytoplasmic ribosome of S. cerevisiae, the missing proteins are wall protein SWP1 (ref. 20) is encoded by a unique gene on chrX LP1, L14, L29, L38, L40, L41, S27 and S27A. Small putative whereas two genes for the polar tube proteins PTP1 and PTP2 are spliceosomal introns with usual GT ± AG boundaries were detected arranged in tandem on chrVI . The set of chaperones for protein in 11 ribosomal protein genes (S8, S17, S24, S26, L5, L19, L27A, L37, folding includes a complete oligomeric TCP-1 complex, four L37A and L39). They start either in the initiator ATG or the next members of the HSP70 system but no chaperonin CPN60. The 16 13 codon, as often observed in yeast or nucleomorph genomes. Two mitochondrial-type HSP70 has been previously characterized in 3±5 more internal introns create frame shifts within a CDP-diacyl- three different microsporidian species . Initial steps of N-glyco- glycerol serine phosphatidyltransferase gene (chrXI). Two of the sylation using UDP ±N-acetylglucosamine (UDP-GNAc) and GDP- Ile Tyr 44 transfer RNA genes (tDNA and tDNA ) also harbour a small mannose (GDP-Man) may occur, but further trimming by manno- intron. sidases associated with endoplasmic reticulum or Golgi apparatus Reduced metabolic capacities and low diversity of transporters and formation of a complex N-linked oligosaccharide are not can be inferred from the genome sequence, as illustrated in Fig. 3. supported. The major sugar used for O-linked glycosylations The repertoire for the biosynthesis of amino acids is restricted to would be mannose (two mannosyltransferases of the fungal PMT asparagine synthetase and serine hydroxymethyltransferase genes. family). The lack of genes for the two enzymes involved in Genes for de novo biosynthesis of purine and pyrimidine nucleo- phosphorylation of mannose residues argues for the absence of tides are absent but several nucleotide interconversions are pre- sorting of lysosomes. Membrane fusion and recognition of some dicted. Genes encoding a fatty acid synthase complex are lacking, target membrane processes are sustained by a restricted set of which supports the uptake of host-derived fatty acids . The potential proteins for Golgi and post-Golgi traf®cking. The con- E. cuniculi spore membrane contains cholesterol. This sterol stitutive secretion pathway leading to the plasma membrane may might also be of host origin, as no gene for the conversion of involve some characteristic Rab proteins. Several potential partners farnesyl-PP into cholesterol was detected. In contrast, E. cuniculi for trans-Golgi and endosome transport include b1-adaptin, Vps1- seems to be capable of synthesizing usual membrane phospholipids. like dynamin and vacuolar protein sorting-associated proteins, Genes for principal enzymes for the synthesis and degradation of con®rming that the Golgi-like apparatus is functionally polarized. trehalose con®rm that this disaccharide could be the major sugar Endocytosis of certain macromolecules was previously shown in 18 18 reserve in microsporidia , as in other fungi. A complete glycolytic Spraguea lophii sporoplasms , when maintained in vitro in a cell glucose-to-pyruvate pathway is predicted. In contrast, genes culture medium. Several genes are also suggestive of an endocytosis required for the tricarboxylic acid cycle, fatty acid b-oxidation, pathway in E. cuniculi. This might drive the internalization of respiratory electron-transport chain and the F F -ATPase complex macromolecular ligands representing sources of fatty acids, choles- 0 1 are absent. Thus, ATP production in microsporidia would be terol or iron (see Fig. 4). possible by substrate-level phosphorylation only. As proliferating The evolutionary origin of microsporidia has been much debated microsporidia recruit host mitochondria near their plasma mem- brane, it has been proposed that these parasites could use host- a 600 b derived ATP . This is reinforced by the ®nding of four genes encoding ADP/ATP carrier proteins that are homologous to ADP/ ATP translocases from chloroplasts and obligate intracellular bac- 500 60 teria (Rickettsia and Chlamydia) capable of importing host ATP . The fate of pyruvate remains dif®cult to predict because of the lack 50 of genes for lactate and ethanol fermentation as well as for glycolysis reversal. A potential cytosolic glycerol-3P dehydrogenase (GPDH) might serve to reoxidize the NADH produced during glycolysis. Surprisingly, two CDSs have signi®cant similarity to the subunits of Cellular communication/signal transduction Cell rescue, defence, cell death Cellular organization and ageing and biogenesis 0 0 2 4 6 8 10 10–35 –15 0 15 30 45 Hypotheticals Intracellular transport Protein length Protein size reduction (%), (no. of amino acids (× 10 )) relative to yeast Transport facilitation Protein destination Figure 2 Sizes of E. cuniculi (Ec) proteins and comparison with S. cerevisiae (Sc) Unknown homologues. a, Distribution of the lengths of all the potential E. cuniculi protein chains. Protein synthesis Only six have more than 2,000 amino acids (maximum 3,456 amino acids). b, Degrees of reduction in length of E. cuniculi proteins (n = 350) relative to those of S. cerevisiae, Transcription expressed as a percentage: 100(Sc protein length - Ec protein length)/(Sc protein length). The positive classes representative of shorter E. cuniculi proteins are in grey. The dynein Cell growth, cell division Metabolism heavy chain, the largest protein chain with a clearly predicted function (3,151 amino and DNA synthesis acids), is ,23% shorter than the yeast homologue (4,092 amino acids). Mean values Energy associated with major functional categories are 7.3% for `protein synthesis', 11.0% for Figure 1 Distribution of predicted E. cuniculi proteins among functional groups. The `protein destination', 11.4% for `metabolism/energy', 14.4% for `intracellular transport', corresponding gene list is given in the Supplementary Information. 15.3% for `transcription' and 20.1% for `cell growth, cell division and DNA synthesis'. NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com 451 © 2001 Macmillan Magazines Ltd No. of proteins No. of proteins CHITIN HOST CELL Other ABC Nicotinic Glucose Oligo- Folate Nucleosides ATP ATP Sugars Amino acids Met transporters acid Fructose peptides PV ADP ADP Spore Encephalitozoon TREHALOSE 6P-gluconate Glucose wall NUCLEOTIDE METABOLISM cuniculi INTERCONVERSIONS PENTOSES-P UDP-GNAc dATP, dGTP ATP, GTP G-6P G-1P UDPG PATHWAY L-Gln L-Glu (Sporogony) NADPH GN-6P GNAc-1P ADP, GDP dADP, dGDP F-6P CMP L-Serine Palmitoyl CoA GLYCOLYSIS CDP CTP Sphingosine L-Gln L-Glu GA-3P DHAP Glycerol-3P UTP NADH CTP dCTP Phospholipid Fatty acids anabolism ATP Acetyl CoA UDP dUDP dUTP dCMP Acetate dTDP dTMP Pyruvate dUMP H4F Dolichyl-PP Isopentenyl-PP Farnesyl-PP Acetyl CoA dTTP Thymidine GPI Glycine L-Serine 70S Protein ribosome UDP-GNAc Signal transduction modifications L-Asparagine L-Aspartate GDP-Man (kinase C) L-Gln L-Glu Endoplasmic reticulum IP3, DAG? Plasma membrane COP I V-type H -ATPase Rabs COP II Polaroplast Golgi (Sporogony) Endosomes Polar tube VESICULAR TRANSPORT Clathrin-associated proteins REGULATED + ENDOCYTOSIS Na EXOCYTOSIS P-type Antiporter V-type ATPase Water (MIP) Na Pi ATPase Cations letters to nature GMP HMG CoA PI PIP PIP2 Proteasome Figure 3 An overview of metabolism and transport in E. cuniculi, as deduced from with the plasma membrane are shown with indications on substrate speci®city. Question genome sequence analysis. Pathways for nucleotide biosynthesis, energy production and marks correspond to major uncertainties about the fate of pyruvate and the production of chitin biosynthesis are indicated. Endocytosis and vesicular transport involving a second messengers for signal transduction. The parasite is represented within a cis ± trans polarized Golgi apparatus are also illustrated. Potential transporters associated parasitophorous vacuole (PV) of the host cytoplasm. but strong evidence supporting a fungal origin of these organisms The presence of characteristic domains and key amino acids 6±9 has recently accumulated . The present genome sequence extends suggests that the potential mitochondrial-type proteins are func- this evidence: phylogenetic analyses of putative genes for seryl- tional. Moreover, PSORT analysis predicts amino-terminal prese- tRNA synthetase, transcription initiation factor IIB, subunit A of quences for the targeting of ®ve of these proteins (see vacuolar ATPase, and a GTP-binding protein place microsporidia as Supplementary Information). A common feature is an arginine a sister group of fungi with bootstrap supports ranging from 70% to residue at -2 relative to the cleavage site, similar to presequences of 92% (see Supplementary Information; a systematic phylogenetic mitochondrial and hydrogenosomal proteins . The amitochondri- analysis of the genome will be presented elsewhere). Genes of ate protozoan Entamoeba histolytica has recently been shown to 26 ± 28 putative mitochondrial evolutionary origin in the E. cuniculi contain a residual mitochondrion-derived organelle that some genome were systematically sought by comparison with the 423 authors have called mitosome . Considering the set of potential recently surveyed yeast mitochondrial proteins encoded by the E. cuniculi proteins usually associated with mitochondria, we nuclear and the mitochondrial genomes . Twenty-two genes with propose that a cryptic organelle is also present in microsporidia signi®cant similarity to the yeast genes were identi®ed and phylo- (Fig. 4). This mitosome would be signi®cantly different from genetic analysis showed that six of them are closely related to hydrogenosomes found in several anaerobic unicellular eukaryotes homologues from a-proteobacteria, the bacterial group from (type II anaerobes) . Hydrogen production through pyruvate which mitochondria are believed to derive . The yeast homologues catabolism seems unlikely in microsporidia (because of a lack of a of these six proteins are ATM1 (ABC transporter), ISU1/ISU2 hydrogenase gene). The development of microsporidia in various (NIFU-like protein), NFS1 (unique homologue of bacterial ISC-S aerobic host cells is suggestive of a rather high O tolerance and and NIF-S), SSQ1 (heat-shock protein of relative molecular mass therefore of an ef®cient protection against oxidative stress. No 70,000), YAH1 (ferredoxin) and PDB1 (b-subunit of pyruvate catalase gene is identi®ed, in agreement with the lack of peroxi- dehydrogenase component E1). The ®rst ®ve proteins are typically somes. Thus, in addition to glutathione and thioredoxin-based involved in the Fe ±S cluster assembly machinery, an essential systems, E. cuniculi might use its unique manganese superoxide function of mitochondria . dismutase as an antioxidant. 452 NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature the fraction of invariant sites estimated once from the original data. Sequences of Glycolysis individual chromosomes were submitted to EMBL under the accession numbers AL39173 for chrI and AL590442 ± AL590451 for chrII ± chrXI, respectively. Acetyl CoA NADH NAD Pyruvate Received 23 April; accepted 31 August 2001. ACS1 CYTOSOL GPDH-C 1. Wittner, M. & Weiss, L. M. The Microsporidia and Microsporidiosis (American Society of Acetate Microbiology, Washington DC, 1999). 2. Vossbrinck, C. R., Maddox, J. V., Friedman, S., Debrunner-Vossbrinck, B. A. & Woese, C. R. 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Microsporidia are related to fungi: evidence from the largest subunit of RNA MITOSOME polymerase II and other proteins. Proc. Natl Acad. Sci. USA 96, 580 ± 585 (1999). Fe–S-cluster 7. Baldauf, S. L., Roger, A. J., Wenk-Siefert, I. & Doolittle, W. F. A kingdom-level phylogeny of eukaryotes assembly and transport based on combined protein data. Science 290, 972 ± 977 (2000). 8. Keeling, P. J., Luker, M. A. & Palmer, J. D. Evidence from b-tubulin phylogeny that microsporidia NifU-like/IscU HSP70-M Protein translocation NFS1, frataxin evolved from within the Fungi. Mol. Biol. Evol. 17, 23 ± 31 (2000). and folding TIM22 ATM1 9. Van de Peer, Y., Ben Ali, A. & Meyer, A. Microsporidia: accumulating evidence that a group of amitochondriate and suspectedly primitive eukaryotes are just curious fungi. Gene 246, 1 ± 8 (2000). 10. Biderre, C., Page Á s, M., Me  te  nier, G., Canning, E. U. & Vivare Á s, C. P. Evidence for the smallest nuclear genome (2.9 Mb) in the microsporidian Encephalitozoon cuniculi. Mol. Biochem. Parasitol. 74, 229 ± TOM70 231 (1995). 11. Peyret, P. et al. Sequence and analysis of chromosome I of the amitochondriate intracellular parasite Matrix preproteins Iron Cytosolic Fe–S Encephalitozoon cuniculi (Microspora). Genome Res. 11, 198 ± 207 (2001). proteins 12. Bruge Á re, J. F., Cornillot, E., Me  te  nier, G., Bensimon, A. & Vivare Á s, C. P. Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all Ferritin chromosomes. Nucleic Acids Res. 28, 2026 ± 2033 (2000). Figure 4 Conceptual scheme of a mitochondrion-derived organelle (`mitosome') in 13. Douglas, S. et al. The highly reduced genome of an enslaved algal nucleus. Nature 410, 1091 ± 1096 (2001). E. cuniculi suggested by the detection of several homologues of mitochondrial proteins. 14. Zhang, I. Protein-length distributions for the three domains of life. 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The predicted manganese superoxide dismutase glycine- and serine-rich spore wall protein in the microsporidian pathogen Encephalitozoon cuniculi. Infect. Immun. 68, 2268 ± 2275 (2000). (Mn-SOD) would ensure protection against oxygen radicals. A homologue of yeast ERV1 (a   Á 21. Delbac, F., Peuvel, I., Metenier, G., Peyretaillade, E. & Vivares, C. P. Microsporidian invasion small protein required for mitochondrial biogenesis) is indicated. In the lower part of the apparatus: identi®cation of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 scheme are depicted some of the major potential factors required for targeting of proteins genes in three Encephalitozoon species. Infect. Immun. 69, 1016± 1024 (2001). 22. Karlberg, O., Canback, B., Kurland, C. G. & Andersson, S. G. The dual origin of the yeast to the organelle and for biosynthesis of Fe± S clusters and transport of Fe ±S proteins mitochondrial proteome. Yeast 17, 170 ± 187 (2000). towards the cytosol. 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Hsp60 is targeted to a cryptic mitochondrion-derived organelle (`crypton') in the test the mitosome hypothesis. In addition, we expect the E. cuniculi microaerophilic protozoan parasite Entamoeba histolytica. Mol. Cell. Biol. 19, 2198± 2205 (1999). genome to provide a useful reference for comparative genomics of 28. Tovar, J., Fischer, A. & Clark, C. G. The mitosome, a novel organelle related to mitochondria in the microbial eukaryotes, particularly to identify the relative impor- amitochondrial parasite Entamoeba histolytica. Mol. Microbiol. 32, 1013 ± 1021 (1999). 29. Martin, W. & Mu È ller, M. The hydrogen hypothesis for the ®rst eukaryote. Nature 392, 37± 41 (1998). tance of shared genes among various evolutionarily distant intra- 30. Artiguenave, F. et al. Genomic exploration of the hemiascomycetous yeasts: 2. Data generation and cellular parasites, including major human-infecting parasites such processing. FEBS Lett. 487, 13± 16 (2000). as Plasmodium and Leishmania. M Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Methods The reference mouse isolate of Encephalitozoon cuniculi (GB-M1), cloning and library construction, nucleotide sequencing, sequence validation and sequence analysis have been Acknowledgements described in detail previously (see Supplementary Information). Further information on We are grateful to everyone who provided computer resources and system management: recombinant DNA preparation, sequencing and sequence analysis can be found D. Ferney and P. Bernard (LMA, UMR CNRS 6620), P. L. Reichstadt and B. Michel (LPC, elsewhere . Annotation was manually performed with the help of AceDB and Artemis IN2P3, UMR CNRS 6533). We thank B. Chebance and R. Guerry for technical assistance. graphic interfaces. Genes were characterized by Glimmer prediction of coding DNA L. Pereira, B. Duperrier, R. Me  te  ry and E. Delorme are acknowledged for their sequences, combined with BLAST all-homology results (BLAST X, BLAST N against `nr', contribution to the development of the annotation environment in the Universite  Blaise BLAST P against SwissProt, PSI-BLAST). Transfer RNA genes were detected with the Pascal. We also express gratitude to C. White for critical reading of the manuscript. F.D., tRNA Scan program. Spliceosomal-type introns were manually detected. Phylogenetic H.E.A and E.P were supported by a grant from the Ministe Á re de la Recherche. analyses were done as in Keeling et al. . Gamma-corrected ML distances with eight rate categories and invariant sites were computed with TREE-PUZZLE version 5.0 and trees Correspondence and requests for materials should be addressed to C.P.V. were derived with BioNJ. Bootstrapping was on 500 replicates with alpha parameters and (e-mail: [email protected]). NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com 453 © 2001 Macmillan Magazines Ltd http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Springer Journals

Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi

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Springer Journals
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Copyright © 2001 by The Author(s)
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Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
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Abstract

letters to nature ................................................................. The gradual increase in G + C content at the core centre described in chromosome I (chrI) exists in all the chromosomes (maximum Genome sequence and gene 51.0%). The 1,997 protein-coding DNA sequences (CDSs) repre- sent about 90% of the chromosome cores, as a result of generally compaction of the eukaryote short intergenic regions (see Supplementary Information). Gene density is slightly lower than that observed in the nucleomorph parasite Encephalitozoon cuniculi genome of the cryptomonad Guillardia theta . Only about 44% of the CDSs are assigned to functional categories and about 6% to Michae È l D. Katinka*, Simone Duprat*, Emmanuel Cornillot², conserved hypothetical proteins (Fig. 1). In contrast to the nucleo- Guy Me  te  nier², Fabienne Thomarat³,Ge  rard Prensier², Vale  rie Barbe*, morph genome , no overlapping of CDSs with predicted functions Eric Peyretaillade², Philippe Brottier*, Patrick Wincker*, Fre  de  ric Delbac², Hicham El Alaoui², Pierre Peyret², William Saurin*, was revealed. Structural or functional clusters are rare and never Manolo Gouy³, Jean Weissenbach* & Christian P. Vivare Á s² composed of more than two CDSs (for example, histones H3 and H4 on chrIX). Genome compaction can also be related to gene * Genoscope, UMR CNRS 8030, CP 5706, 91057 Evry cedex, France shortening, as indicated by the length distribution of all potential ² Parasitologie Mole Âculaire et Cellulaire, Laboratoire de Biologie des Protistes, proteins (Fig. 2a). The mean and median lengths of all potential UMR CNRS 6023, Universite  Blaise Pascal, 63177 Aubie Áre cedex, France E. cuniculi proteins are only 359 and 281 amino acids, respectively. Laboratoire de Biome Âtrie et Biologie Evolutive, UMR CNRS 5558, We compared the lengths of 350 proteins with Saccharomyces Universite  Lyon I, 69622 Villeurbanne cedex, France cerevisiae homologues (Fig. 2b). More than 85% of these proteins .............................................................................................................................................. are shorter than in yeast, with a mean relative size difference of Microsporidia are obligate intracellular parasites infesting many 14.6%. From the analysis of the protein size distributions derived animal groups . Lacking mitochondria and peroxysomes, these from sequenced genomes, it has been suggested that the lengthening unicellular eukaryotes were ®rst considered a deeply branching of proteins in eukaryotes (non-parasitic species) allows for more 2 14 protist lineage that diverged before the endosymbiotic event that complex regulation networks . Thus, protein shortening in E. led to mitochondria. The discovery of a gene for a mitochondrial- cuniculi may re¯ect reduced protein± protein interactions as a 3±5 6±9 type chaperone combined with molecular phylogenetic data result of various gene losses linked to the intracellular parasitic later implied that microsporidia are atypical fungi that lost nature (Fig. 2a, b). mitochondria during evolution. Here we report the DNA Perfect segmental duplications of 0.5 ± 10-kb coding and non- sequences of the 11 chromosomes of the ,2.9-megabase (Mb) coding sequences represent about 3.7% of the core region in average genome of Encephalitozoon cuniculi (1,997 potential protein- (from ,1% in chrII, III, IV and VI to ,7% in chrIX; see coding genes). Genome compaction is re¯ected by reduced inter- Supplementary Information). A segment carrying four enzyme- genic spacers and by the shortness of most putative proteins coding genes is perfectly duplicated in the extreme part of the chrI relative to their eukaryote orthologues. The strong host depen- core but partially duplicated near the end of chrVIII (truncated dence is illustrated by the lack of genes for some biosynthetic serine hydroxymethyltransferase gene). The genome core sequences pathways and for the tricarboxylic acid cycle. Phylogenetic analy- exhibit a very low base polymorphism, restricted to a few positions sis lends substantial credit to the fungal af®liation of microspor- in eight chromosomes. A duplicated gene homologous to CTP idia. Because the E. cuniculi genome contains genes related to synthases (chrXI) revealed a rare polymorphic tract of 189 base pairs some mitochondrial functions (for example, Fe ± S cluster assem- (bp) (116,847 ± 117,036). Hybridization experiments indicate that bly), we hypothesize that microsporidia have retained a mito- each polymorphic sequence is present in about 50% of the DNA chondrion-derived organelle. molecules (data not shown), suggesting that they are alleles and 10,12 Encephalitozoon cuniculi infects various mammals, including supporting the diploidy hypothesis . humans, and can cause digestive and nervous clinical syndromes A large proportion of CDSs is assigned to the conservation and in HIV-infected or cyclosporine-treated people . Its reproduction transmission of genetic information as well as to protein modi®ca- proceeds as a sequence of two major stages: merogony, involving the tion and intracellular transport processes (Fig. 1), but with a multiplication of large, wall-lacking cells (meronts); and sporogony, signi®cant degree of simpli®cation, mainly related to the lack of leading to small, thick-walled spores. The sporal invasive apparatus DNA-containing organelles. For example, subunits for DNA pol I is characterized by a long polar tube that can be quickly extruded (d), pol II (e) and pol III (a but not b) are present while there is no then used for transferring the sporoplasm into the target cell. candidate for the mitochondrial DNA polymerase g. Apart from the Consisting of 11 linear chromosomes ranging from 217 to 315 kb, telomerase catalytic subunit, no RNA-dependent reverse transcrip- the E. cuniculi genome is remarkably reduced (,2.9 Mb) . The tase was found. This and the lack of any retrotransposition elements nucleotide sequence of the smallest chromosome has been recently may explain the absence of pseudoretrogenes. The potential tran- reported . The full sequencing of this minimal genome among scription machinery includes RNA pol I, II and III, more than 70 eukaryotes was expected to provide insight into the metabolism and messenger RNA transcription-associated proteins and a virtually general biology of microsporidia and to help in the understanding of the evolutionary history of amitochondriate eukaryotes currently considered `curious fungi' (see Table 1). Table 1 General features of the E. cuniculi genome The chromosome sequences were determined through a plasmid Total sequenced length 2,507,519 bp library (,3 kb inserts) and a miniBAC library (,20 ± 25 kb inserts) G + C content Protein-coding regions 47.6% totalling 15 genome equivalents (,46 Mb). All chromosomes Intergenic regions 45.0% possess a `unique sequences' core region ¯anked by two 28-kb Telomeric and subtelomeric regions 52.9% divergently oriented regions, each including one ribosomal DNA No. of protein-coding sequences 1,997 11,12 Mean intergenic distance 129 bp unit . The subtelomeric repeats upstream from rDNA are mostly Gene density of chromosome cores 1 CDS per 1,025 bp degenerated minisatellites, whereas downstream repeats consist No. and sizes of spliceosomal introns 13 (23±52 bp) essentially of non-polymorphic microsatellite arrays. The chromo- No. of 16S±23S rRNA genes 22* No. of 5S rRNA genes 3 (on chrV, VII, IX) some cores lack simple sequence repeats, minisatellite arrays and No. of tRNA genes 44² known transposable elements. No imprint of retrogenes or pseudo- No. and sizes of tRNA introns 2 (16, 42 bp) ............................................................................................................................................................................. retrogenes of either polymerase (pol) II or pol III type was found. * Two per chromosome. General features of genome organization are indicated in Table 1. ² On all chromosomes. 450 NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com © 2001 Macmillan Magazines Ltd letters to nature complete set of genes for splicing, 59 and 39 processing. An (A + T)- the E1 component of the mitochondrial pyruvate dehydrogenase rich consensus transcription initiation sequence is revealed in the complex. Pyruvate decarboxylation could be inferred, but, in the 120-bp region upstream from the initiation codon of numerous absence of evidence for E2 and E3 components, a subsequent genes, suggesting short 59 leaders. Seventy different eukaryotic-type production of acetyl coenzyme A cannot be concluded. ribosomal proteins (40 from the large subunit, 30 from the small Microsporidia have a presumably simpli®ed Golgi apparatus in subunit) are predicted, which is slightly lower than in the amito- which a cis ±trans polarity is not cytologically distinguishable but 15 1 chondriate protist Giardia lamblia (74) . Compared with the that is central in sporogony-speci®c secretion processes . The spore cytoplasmic ribosome of S. cerevisiae, the missing proteins are wall protein SWP1 (ref. 20) is encoded by a unique gene on chrX LP1, L14, L29, L38, L40, L41, S27 and S27A. Small putative whereas two genes for the polar tube proteins PTP1 and PTP2 are spliceosomal introns with usual GT ± AG boundaries were detected arranged in tandem on chrVI . The set of chaperones for protein in 11 ribosomal protein genes (S8, S17, S24, S26, L5, L19, L27A, L37, folding includes a complete oligomeric TCP-1 complex, four L37A and L39). They start either in the initiator ATG or the next members of the HSP70 system but no chaperonin CPN60. The 16 13 codon, as often observed in yeast or nucleomorph genomes. Two mitochondrial-type HSP70 has been previously characterized in 3±5 more internal introns create frame shifts within a CDP-diacyl- three different microsporidian species . Initial steps of N-glyco- glycerol serine phosphatidyltransferase gene (chrXI). Two of the sylation using UDP ±N-acetylglucosamine (UDP-GNAc) and GDP- Ile Tyr 44 transfer RNA genes (tDNA and tDNA ) also harbour a small mannose (GDP-Man) may occur, but further trimming by manno- intron. sidases associated with endoplasmic reticulum or Golgi apparatus Reduced metabolic capacities and low diversity of transporters and formation of a complex N-linked oligosaccharide are not can be inferred from the genome sequence, as illustrated in Fig. 3. supported. The major sugar used for O-linked glycosylations The repertoire for the biosynthesis of amino acids is restricted to would be mannose (two mannosyltransferases of the fungal PMT asparagine synthetase and serine hydroxymethyltransferase genes. family). The lack of genes for the two enzymes involved in Genes for de novo biosynthesis of purine and pyrimidine nucleo- phosphorylation of mannose residues argues for the absence of tides are absent but several nucleotide interconversions are pre- sorting of lysosomes. Membrane fusion and recognition of some dicted. Genes encoding a fatty acid synthase complex are lacking, target membrane processes are sustained by a restricted set of which supports the uptake of host-derived fatty acids . The potential proteins for Golgi and post-Golgi traf®cking. The con- E. cuniculi spore membrane contains cholesterol. This sterol stitutive secretion pathway leading to the plasma membrane may might also be of host origin, as no gene for the conversion of involve some characteristic Rab proteins. Several potential partners farnesyl-PP into cholesterol was detected. In contrast, E. cuniculi for trans-Golgi and endosome transport include b1-adaptin, Vps1- seems to be capable of synthesizing usual membrane phospholipids. like dynamin and vacuolar protein sorting-associated proteins, Genes for principal enzymes for the synthesis and degradation of con®rming that the Golgi-like apparatus is functionally polarized. trehalose con®rm that this disaccharide could be the major sugar Endocytosis of certain macromolecules was previously shown in 18 18 reserve in microsporidia , as in other fungi. A complete glycolytic Spraguea lophii sporoplasms , when maintained in vitro in a cell glucose-to-pyruvate pathway is predicted. In contrast, genes culture medium. Several genes are also suggestive of an endocytosis required for the tricarboxylic acid cycle, fatty acid b-oxidation, pathway in E. cuniculi. This might drive the internalization of respiratory electron-transport chain and the F F -ATPase complex macromolecular ligands representing sources of fatty acids, choles- 0 1 are absent. Thus, ATP production in microsporidia would be terol or iron (see Fig. 4). possible by substrate-level phosphorylation only. As proliferating The evolutionary origin of microsporidia has been much debated microsporidia recruit host mitochondria near their plasma mem- brane, it has been proposed that these parasites could use host- a 600 b derived ATP . This is reinforced by the ®nding of four genes encoding ADP/ATP carrier proteins that are homologous to ADP/ ATP translocases from chloroplasts and obligate intracellular bac- 500 60 teria (Rickettsia and Chlamydia) capable of importing host ATP . The fate of pyruvate remains dif®cult to predict because of the lack 50 of genes for lactate and ethanol fermentation as well as for glycolysis reversal. A potential cytosolic glycerol-3P dehydrogenase (GPDH) might serve to reoxidize the NADH produced during glycolysis. Surprisingly, two CDSs have signi®cant similarity to the subunits of Cellular communication/signal transduction Cell rescue, defence, cell death Cellular organization and ageing and biogenesis 0 0 2 4 6 8 10 10–35 –15 0 15 30 45 Hypotheticals Intracellular transport Protein length Protein size reduction (%), (no. of amino acids (× 10 )) relative to yeast Transport facilitation Protein destination Figure 2 Sizes of E. cuniculi (Ec) proteins and comparison with S. cerevisiae (Sc) Unknown homologues. a, Distribution of the lengths of all the potential E. cuniculi protein chains. Protein synthesis Only six have more than 2,000 amino acids (maximum 3,456 amino acids). b, Degrees of reduction in length of E. cuniculi proteins (n = 350) relative to those of S. cerevisiae, Transcription expressed as a percentage: 100(Sc protein length - Ec protein length)/(Sc protein length). The positive classes representative of shorter E. cuniculi proteins are in grey. The dynein Cell growth, cell division Metabolism heavy chain, the largest protein chain with a clearly predicted function (3,151 amino and DNA synthesis acids), is ,23% shorter than the yeast homologue (4,092 amino acids). Mean values Energy associated with major functional categories are 7.3% for `protein synthesis', 11.0% for Figure 1 Distribution of predicted E. cuniculi proteins among functional groups. The `protein destination', 11.4% for `metabolism/energy', 14.4% for `intracellular transport', corresponding gene list is given in the Supplementary Information. 15.3% for `transcription' and 20.1% for `cell growth, cell division and DNA synthesis'. NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com 451 © 2001 Macmillan Magazines Ltd No. of proteins No. of proteins CHITIN HOST CELL Other ABC Nicotinic Glucose Oligo- Folate Nucleosides ATP ATP Sugars Amino acids Met transporters acid Fructose peptides PV ADP ADP Spore Encephalitozoon TREHALOSE 6P-gluconate Glucose wall NUCLEOTIDE METABOLISM cuniculi INTERCONVERSIONS PENTOSES-P UDP-GNAc dATP, dGTP ATP, GTP G-6P G-1P UDPG PATHWAY L-Gln L-Glu (Sporogony) NADPH GN-6P GNAc-1P ADP, GDP dADP, dGDP F-6P CMP L-Serine Palmitoyl CoA GLYCOLYSIS CDP CTP Sphingosine L-Gln L-Glu GA-3P DHAP Glycerol-3P UTP NADH CTP dCTP Phospholipid Fatty acids anabolism ATP Acetyl CoA UDP dUDP dUTP dCMP Acetate dTDP dTMP Pyruvate dUMP H4F Dolichyl-PP Isopentenyl-PP Farnesyl-PP Acetyl CoA dTTP Thymidine GPI Glycine L-Serine 70S Protein ribosome UDP-GNAc Signal transduction modifications L-Asparagine L-Aspartate GDP-Man (kinase C) L-Gln L-Glu Endoplasmic reticulum IP3, DAG? Plasma membrane COP I V-type H -ATPase Rabs COP II Polaroplast Golgi (Sporogony) Endosomes Polar tube VESICULAR TRANSPORT Clathrin-associated proteins REGULATED + ENDOCYTOSIS Na EXOCYTOSIS P-type Antiporter V-type ATPase Water (MIP) Na Pi ATPase Cations letters to nature GMP HMG CoA PI PIP PIP2 Proteasome Figure 3 An overview of metabolism and transport in E. cuniculi, as deduced from with the plasma membrane are shown with indications on substrate speci®city. Question genome sequence analysis. Pathways for nucleotide biosynthesis, energy production and marks correspond to major uncertainties about the fate of pyruvate and the production of chitin biosynthesis are indicated. Endocytosis and vesicular transport involving a second messengers for signal transduction. The parasite is represented within a cis ± trans polarized Golgi apparatus are also illustrated. Potential transporters associated parasitophorous vacuole (PV) of the host cytoplasm. but strong evidence supporting a fungal origin of these organisms The presence of characteristic domains and key amino acids 6±9 has recently accumulated . The present genome sequence extends suggests that the potential mitochondrial-type proteins are func- this evidence: phylogenetic analyses of putative genes for seryl- tional. Moreover, PSORT analysis predicts amino-terminal prese- tRNA synthetase, transcription initiation factor IIB, subunit A of quences for the targeting of ®ve of these proteins (see vacuolar ATPase, and a GTP-binding protein place microsporidia as Supplementary Information). A common feature is an arginine a sister group of fungi with bootstrap supports ranging from 70% to residue at -2 relative to the cleavage site, similar to presequences of 92% (see Supplementary Information; a systematic phylogenetic mitochondrial and hydrogenosomal proteins . The amitochondri- analysis of the genome will be presented elsewhere). Genes of ate protozoan Entamoeba histolytica has recently been shown to 26 ± 28 putative mitochondrial evolutionary origin in the E. cuniculi contain a residual mitochondrion-derived organelle that some genome were systematically sought by comparison with the 423 authors have called mitosome . Considering the set of potential recently surveyed yeast mitochondrial proteins encoded by the E. cuniculi proteins usually associated with mitochondria, we nuclear and the mitochondrial genomes . Twenty-two genes with propose that a cryptic organelle is also present in microsporidia signi®cant similarity to the yeast genes were identi®ed and phylo- (Fig. 4). This mitosome would be signi®cantly different from genetic analysis showed that six of them are closely related to hydrogenosomes found in several anaerobic unicellular eukaryotes homologues from a-proteobacteria, the bacterial group from (type II anaerobes) . Hydrogen production through pyruvate which mitochondria are believed to derive . The yeast homologues catabolism seems unlikely in microsporidia (because of a lack of a of these six proteins are ATM1 (ABC transporter), ISU1/ISU2 hydrogenase gene). The development of microsporidia in various (NIFU-like protein), NFS1 (unique homologue of bacterial ISC-S aerobic host cells is suggestive of a rather high O tolerance and and NIF-S), SSQ1 (heat-shock protein of relative molecular mass therefore of an ef®cient protection against oxidative stress. No 70,000), YAH1 (ferredoxin) and PDB1 (b-subunit of pyruvate catalase gene is identi®ed, in agreement with the lack of peroxi- dehydrogenase component E1). The ®rst ®ve proteins are typically somes. 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M Supplementary Information accompanies the paper on Nature's website (http://www.nature.com). Methods The reference mouse isolate of Encephalitozoon cuniculi (GB-M1), cloning and library construction, nucleotide sequencing, sequence validation and sequence analysis have been Acknowledgements described in detail previously (see Supplementary Information). Further information on We are grateful to everyone who provided computer resources and system management: recombinant DNA preparation, sequencing and sequence analysis can be found D. Ferney and P. Bernard (LMA, UMR CNRS 6620), P. L. Reichstadt and B. Michel (LPC, elsewhere . Annotation was manually performed with the help of AceDB and Artemis IN2P3, UMR CNRS 6533). We thank B. Chebance and R. Guerry for technical assistance. graphic interfaces. Genes were characterized by Glimmer prediction of coding DNA L. Pereira, B. Duperrier, R. Me  te  ry and E. Delorme are acknowledged for their sequences, combined with BLAST all-homology results (BLAST X, BLAST N against `nr', contribution to the development of the annotation environment in the Universite  Blaise BLAST P against SwissProt, PSI-BLAST). Transfer RNA genes were detected with the Pascal. We also express gratitude to C. White for critical reading of the manuscript. F.D., tRNA Scan program. Spliceosomal-type introns were manually detected. Phylogenetic H.E.A and E.P were supported by a grant from the Ministe Á re de la Recherche. analyses were done as in Keeling et al. . Gamma-corrected ML distances with eight rate categories and invariant sites were computed with TREE-PUZZLE version 5.0 and trees Correspondence and requests for materials should be addressed to C.P.V. were derived with BioNJ. Bootstrapping was on 500 replicates with alpha parameters and (e-mail: [email protected]). NATURE | VOL 414 | 22 NOVEMBER 2001 | www.nature.com 453 © 2001 Macmillan Magazines Ltd

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