the genome of the african trypanosome s trypanosoma …fieldlab.org/reprints_files/416.pdfal ivens,...

R E S E A R C H A R T I C L E

The Genome of the African TrypanosomeTrypanosoma brucei

Matthew Berriman,1* Elodie Ghedin,2,3 Christiane Hertz-Fowler,1 Gaëlle Blandin,2 Hubert Renauld,1

Daniella C. Bartholomeu,2 Nicola J. Lennard,1 Elisabet Caler,2 Nancy E. Hamlin,1 Brian Haas,2 Ulrike Böhme,1

Linda Hannick,2 Martin A. Aslett,1 Joshua Shallom,2 Lucio Marcello,4 Lihua Hou,2 Bill Wickstead,5

U. Cecilia M. Alsmark,6 Claire Arrowsmith,1 Rebecca J. Atkin,1 Andrew J. Barron,1 Frederic Bringaud,7 Karen Brooks,1

Mark Carrington,8 Inna Cherevach,1 Tracey-Jane Chillingworth,1 Carol Churcher,1 Louise N. Clark,1 Craig H. Corton,1

Ann Cronin,1 Rob M. Davies,1 Jonathon Doggett,1 Appolinaire Djikeng,2 Tamara Feldblyum,2 Mark C. Field,9

Audrey Fraser,1 Ian Goodhead,1 Zahra Hance,1 David Harper,1 Barbara R. Harris,1 Heidi Hauser,1 Jessica Hostetler,2

Al Ivens,1 Kay Jagels,1 David Johnson,1 Justin Johnson,2 Kristine Jones,2 Arnaud X. Kerhornou,1 Hean Koo,2

Natasha Larke,1 Scott Landfear,10 Christopher Larkin,2 Vanessa Leech,9 Alexandra Line,1 Angela Lord,1

Annette MacLeod,4 Paul J. Mooney,1 Sharon Moule,1 David M. A. Martin,11 Gareth W. Morgan,12 Karen Mungall,1

Halina Norbertczak,1 Doug Ormond,1 Grace Pai,2 Chris S. Peacock,1 Jeremy Peterson,2 Michael A. Quail,1

Ester Rabbinowitsch,1 Marie-Adele Rajandream,1 Chris Reitter,9 Steven L. Salzberg,2 Mandy Sanders,1 Seth Schobel,2

Sarah Sharp,1 Mark Simmonds,1 Anjana J. Simpson,2 Luke Tallon,2 C. Michael R. Turner,13 Andrew Tait,4

Adrian R. Tivey,1 Susan Van Aken,2 Danielle Walker,1 David Wanless,2 Shiliang Wang,2 Brian White,1 Owen White,2

Sally Whitehead,1 John Woodward,1 Jennifer Wortman,2 Mark D. Adams,14 T. Martin Embley,6 Keith Gull,5

Elisabetta Ullu,15 J. David Barry,4 Alan H. Fairlamb,11 Fred Opperdoes,16 Barclay G. Barrell,1 John E. Donelson,17

Neil Hall,1. Claire M. Fraser,2 Sara E. Melville,9 Najib M. El-Sayed2,3*

African trypanosomes cause human sleeping sickness and livestock trypanosomiasisin sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains9068 predicted genes, including È900 pseudogenes and È1700 T. brucei–specificgenes. Large subtelomeric arrays contain an archive of 806 variant surface gly-coprotein (VSG) genes used by the parasite to evade the mammalian immunesystem. Most VSG genes are pseudogenes, which may be used to generate expressedmosaic genes by ectopic recombination. Comparisons of the cytoskeleton andendocytic trafficking systems with those of humans and other eukaryotic organismsreveal major differences. A comparison of metabolic pathways encoded by thegenomes of T. brucei, T. cruzi, and Leishmania major reveals the least overallmetabolic capability in T. brucei and the greatest in L. major. Horizontal transfer ofgenes of bacterial origin has contributed to some of the metabolic differences inthese parasites, and a number of novel potential drug targets have been identified.

Human African trypanosomiasis, or sleepingsickness, primarily affects the poorest ruralpopulations in some of the least developedcountries of Central Africa (1). The incidence

may approach 300,000 to 500,000 cases peryear, and it is invariably fatal if untreated. Thedisease is caused by Trypanosoma brucei, anextracellular eukaryotic flagellate parasite, and

current treatments are inadequate: Drugs forlate-stage disease are highly toxic; there is noprophylactic chemotherapy and little or noprospect of a vaccine.

Livestock trypanosomiasis is caused byclosely related Trypanosoma species. It has thegreatest impact in sub-Saharan Africa, wherethe tsetse fly vector is common, and also occursin Asia and South America.

We present the sequence and analysis ofthe 11 megabase-sized chromosomes of the T.brucei genome (Table 1). The nuclear genomealso contains an unspecified number of smalland intermediate-sized chromosomes (30 to700 kb) (2, 3) known to encode sequences sim-ilar to subtelomeric regions of megabase-sizedchromosomes (4).

Genome structure and content. Thegenome data herein represent a haploid mosaic(5) of the diploid chromosomes and were de-termined by both whole chromosome shotgun(chromosomes 1 and 9 to 11) and bacterialartificial chromosome walking strategies (chro-mosomes 2 to 8). Most of the assembledchromosome sequences extend into the sub-telomeric regions (the sequence between thetelomere and the first housekeeping gene)(Plate 3 and table S1).

As previously observed on a smaller scale(6, 7), both strands of the megabase chromo-somes contain long, nonoverlapping geneclusters (Plate 3) that are probably transcribedas polycistrons and subsequently trans-splicedand polyadenylated. The unusual mechanismsof transcription and their implications for theparasite are discussed elsewhere (8). Just over20% of the genome encodes subtelomeric genes

1Wellcome Trust Sanger Institute, Wellcome TrustGenome Campus, Hinxton CB10 1SA, UK. 2The Institutefor Genomic Research, Rockville, MD 20850, USA. 3De-partment of Microbiology and Tropical Medicine, GeorgeWashington University, Washington, DC 20052, USA.4Wellcome Centre for Molecular Parasitology, Universityof Glasgow, 56 Dumbarton Road, Glasgow G11 6NU,UK. 5Sir William Dunn School of Pathology, University ofOxford, South Parks Road, Oxford OX1 3RE, UK. 6Schoolof Biology, Devonshire Building, University of Newcastleupon Tyne, Newcastle NE1 7RU, UK. 7Laboratoire deGénomique Fonctionnelle des Trypanosomatides, Uni-versité Victor Segalen Bordeaux II, UMR-5162 CNRS,33076 Bordeaux cedex, France. 8Department of Bio-chemistry, University of Cambridge, Cambridge CB21GA, UK. 9Department of Pathology, University of Cam-bridge, Cambridge CB2 1QP, UK. 10Oregon Health andScience University, 3181 SW Sam Jackson Park Road,Mail Code L474, Portland, OR 97239–3098, USA.11School of Life Sciences, Wellcome Trust Biocentre,

University of Dundee, Dundee DD1 5EH, UK. 12Depart-ment of Biological Sciences, Imperial College, LondonSW7 2AY, UK. 13Institute of Biomedical and LifeSciences, Joseph Black Building, University of Glasgow,Glasgow G12 8QQ, UK. 14Department of Genetics, CaseWestern Reserve University, 10900 Euclid Avenue,Cleveland, OH 44106, USA. 15Department of InternalMedicine, Yale University School of Medicine, 333 CedarStreet, Post Office Box 208022, New Haven, CT 06520–8022, USA. 16Christian de Duve Institute of CellularPathology and Catholic University of Louvain, AvenueHippocrate 74-75, B-1200 Brussels, Belgium. 17Depart-ment of Biochemistry, University of Iowa, Iowa City, IA52242, USA.

*To whom correspondence should be addressed.E-mail: [email protected] (M.B.); [email protected](N.M.E.-S.).Present address: Institute for Genomic Research, Rock-ville, MD 20850, USA.

T H E T R Y P A N O S O M A T I D G E N O M E ST H E T R Y P A N O S O M A T I D G E N O M E S

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Fig. 1. Distribution of VSGs and ESAGs on chromosomes 1 to 11 of T. brucei.The four different VSG categories are defined in the text: VSG, atypical VSG,VSG pseudogene (gene fragments and full-length pseudogenes), and VSG-related. ESAG family members are identified (e.g., E1 is ESAG1), and degen-

erate, frameshifted, and truncated ESAGs are represented by y. Solid circlesindicate arrays of telomeric-repeat hexamers and/or known terminal subtelo-meric repeats, whereas open circles depict arrays ofÈ70-bp repeats. The VSG andESAG array at the right-hand end of chromosome 9 is highlighted as an example.


www.sciencemag.org SCIENCE VOL 309 15 JULY 2005 417

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(Plate 3) (9), the majority of which are T.brucei–specific and relate to the parasite_scapacity to undergo antigenic variation in thebloodstream of its mammalian host. Expansionof gene families by tandem duplication is amechanism by which the parasites can increaseexpression levels to compensate for a generallack of transcriptional control (8). Analysis ofprotein families (table S2) gives a measure ofthe resources that the parasite commits to keycellular processes. Kinesins and kinesin-likeproteins, protein kinases (8), and adenylatecyclases are among the largest families, and twoas-yet uncharacterized families of more than 10members each appear to be expanded in the T.brucei genome relative to L. major and T. cruzigenomes.

Antigenic variation. Antigenic variationenables evasion of acquired immunity duringinfection or in immune host populations.Invariant antigens on the surface of T. bruceiare shielded by a dense coat of 107 copies of asingle variant surface glycoprotein (VSG) thatmust be replaced as antibodies against it arise(10, 11). VSGs differ significantly in thehypervariable N-terminal domain, which isexposed to the immune system, whereas theC-terminal domain is more conserved and isburied in the coat. The genome was previouslyknown to contain a large archive of an es-timated 1000 nonexpressed VSG genes (VSGs)that are activated clonally during infection at arate of up to one activation event per 100 celldoublings (12, 13). These VSGs have a basiccassette organization, with one or more È70–base pair (bp) repeats at their 5¶ flank andsequence homology at their 3¶ flank thatincludes parts of the coding sequence and the3¶ untranslated region (14). VSG activationrelies on residence in specific transcriptionunits known as bloodstream-form expressionsites (BESs), which are immediately subtelo-meric on megabase and intermediate chromo-somes and possess additional co-transcribedexpression site–associated genes (ESAGs).VSG switching normally involves duplicationof VSG cassettes into BESs.

The first new insight of the genome analysisis thatmost sequenced silentVSGs are defective.It is not known what proportion of VSGs areintact among the intermediate chromosomes,minichromosomes, or the yet-to-be-sequencedsubtelomeric regions. Of 806 analyzed (Fig. 1and table S3), only 57 (7%) are fully functional(that is, encode all recognizable features ofknown functional VSGs), whereas 9% areatypical (complete genes possibly encodingproteins with inconsistent VSG folding or post-translational modification), 66% are full-lengthpseudogenes (with frameshifts and/or in-framestop codons), and 18% are gene fragments,most of which encode C-terminal domains. ThisVSG archive could be a repository of defunctmaterial, but it might also be an informationpool of an unprecedented size among parasites.

Antigen pseudogenes are known to exist inseveral bacterial and other protozoan patho-gens and to contribute to immune evasion bypartial gene conversions to become expressedmosaic genes (15). This combinatorial use ofinformation economizes on the genome and,theoretically, can greatly increase the potentialfor variation. In African trypanosomes, it haslong been known that VSGs of antigenic var-iants can be encoded by mosaics of pseudo-genes and also by hybrids, where the N- andC-terminal domains are derived from separategenes (16, 17). It now appears that more thanone-third of the N-terminal domains encoded byfull-length VSG are intact, whereas C-terminaldomains are more degenerate, further indicat-ing the likely importance of hybrid gene for-mation. It is also possible that a trypanosomemay be able to reinfect a reservoir host pre-viously exposed to trypanosomes by using theVSG archive to embark stochastically on adifferent pathway of mosaic formation.

The second new insight is that almost allVSGs form arrays, numbering 3 to È250(pseudo)genes, and that most of these aresubtelomeric (Fig. 1). In contrast to genes intelomeric BESs, the majority of VSGs areoriented away from the telomere. This isconsistent with other protozoa Eexcept Giar-dia (18)^, where antigen gene archives are atleast partly subtelomeric, probably due to theability of these chromosome domains torecombine with each other ectopically ratherthan merely between chromosome homologs(19). More than 90% of the VSGs have one ormoreÈ70-bp repeats upstream. In addition, thenon–long terminal repeat retransposons ingiand RIME are associated with the VSG arrays,in particular where coding strand switchesoccur, in keeping with the role of suchelements in genome restructuring (20). Wefound no obvious organization of the mostsimilar VSGs into subfamilies within arrays;VSGs are scattered apparently at randomwithin and among arrays (fig. S1) or betweenhomologous chromosomes (fig. S2).

VSGs can be subdivided into N- and C-terminal domains as defined by cysteinedistribution and sequence homology (21).Two previously unknown types of C-terminaldomain are encoded in the genome, anddiversification among the three known N-terminal domains has occurred. Also, weidentified a previously unknown set of 29VSG-related genes that cluster distinctly fromother VSGs in a multiple alignment and lackupstreamÈ70-bp repeats. Themajority of theseputative proteins do not contain cysteineresidues in their C-terminal regions. Theycould have evolved novel functions, as hashappened with the serum resistance-associatedprotein (22). This arrangement of genes resem-bles that of the VAR family in the malariaparasite Plasmodium falciparum; VARs en-code variant erythrocyte surface proteins and

most are subtelomeric, but some are internallylocated and differ from their subtelomericcounterparts (23).

Members of up to 11 families of ESAGs arenormally associated with VSGs in the poly-cistronic BESs. All BESs appear to harbour anESAG6 and ESAG7, and most have a total offive to 10 ESAGs and pseudo-ESAGs (24). Ofthe 11 known ESAG families (Fig. 1 and tableS4), ESAG3 and ESAG4 are remarkablyabundant, but ESAG3s are preferentially asso-ciated with VSG arrays, where it is present ex-clusively as pseudogenes. A single copy of thetandem ESAG6 and ESAG7 (normally found atthe BESs) resides outside BESs but not in theVSG arrays; it appears in tandem on chromo-some 7 at an interruption in the conservation ofsynteny found between the Tritryps (9).

Intracellular protein vesicle trafficking.Membrane transport functions are criticallyimportant for the Tritryps, both in terms oftheir interactions with insect vectors and mam-malian hosts as well as the recycling of sur-face membrane components, such as VSGs.The Tritryps have a polarized endomembranesystem and restrict both exocytosis and endo-cytosis, which occur exclusively via a clathrin-dependent mechanism, to the flagellar pocket(25, 26).

T. brucei has served as a paradigm for thestudy of transport processes in kinetoplastids,elucidating the role of cytoplasmic coat pro-teins (tables S5 and S6) and suggesting afundamental difference in the mechanisms of

Table 1. Summary of the T. brucei genome. Ge-nome size and chromosome numbers exclude in-termediate and mini-chromosomes. Details ofcontig coverage for each chromosome are de-scribed in table S1. Intergenic regions are regionsbetween protein-coding sequences (CDSs). Theexact number of spliced leader (sl) RNA copiescannot be resolved in the assembly.

Parameter Number

The genomeSize (bp) 26,075,396GþC content (%) 46.4Chromosomes 11Sequence contigs 30Percent coding 50.5

Protein-coding genesGenes 9068Pseudogenes 904Mean CDS length (bp) 1592Median CDS length (bp) 1242GþC content (%) 50.9Gene density (genes per Mb) 317

Intergenic regionsMean length (bp) 1279GþC content (%) 41

RNA genestransfer RNA 65ribosomal RNA 56slRNA 928small nuclear RNA 5small nucleolar RNA 353



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endocytosis between the Tritryps (26). Analy-sis of the T. brucei Rab guanine triphosphatases(GTPases), regulators of membrane transport,indicates a complex, highly regulated vesiculartransport system (27). This complexity nowappears to be mirrored in the L. major and T.cruzi genomes (tables S5 and S7). A secondGTPase family includes adenosine diphosphateribosylation factors (ARFs), ARF-like pro-teins (ARLs), and Sar1, regulators of mem-brane traffic and the cytoskeleton (table S5).Phylogenetic reconstruction of the divergentTritryp ARF and ARL family indicated pre-viously unknown isoforms, some of which arenot found in metazoan organisms (fig. S3)(28). This likely acquisition of a large family oftrypanosomatid-specific GTPases may reflectthe extreme emphasis of the trypanosomatidcytoskeleton on tubulin- rather than actin-mediated mechanisms.

Cytoskeleton. Generally, the eukaryoticcytoskeleton is divided into three filamentclasses: microtubules, intermediate filaments,and actin microfilaments. Proteins of theintermediate filament network are very diverse

and poorly conserved in evolution. It appearsthat no homologs to known intermediatefilament genes (table S8) are encoded in theTritryp genome sequences. Homologs of com-ponents of actin- and tubulin-based cyto-skeleton, however, are readily identifiable(Fig. 2 and table S8). Compared to other eu-karyotic organisms, the Tritryps appear to havea reduced dependence on the acto-myosinnetwork balanced by an elaboration of thetubulin-based cytoskeleton.

Six members of the tubulin superfamily arepresent in the Tritryps: a-, b,- g-, d-, e-, and z-tubulin. The presence of d- and e-tubulin ischaracteristic of organisms with basal bodies andflagella. Five putative genes encoding centrins,elongation factor (EF)–hand proteins functioningat microtubule-organizing centers (MTOCs),occur in each genome. Yeasts, in contrast, havea single MTOC and a single centrin gene. Theexistence of a family of centrins in the Tritrypsmay reflect the diversity of dispersed MTOCsinvolved in cytoskeleton organization in theseparasites. Outside of the centrins, few homologsto the components of the mammalian centro-

some or yeast spindle-pole body were identifiedin the three genomes (Fig. 2).

The Tritryp dependence on diverse micro-tubule function is best exemplified by thekinesin superfamily. Each genome encodes940 putative kinesins (excluding near-identicalcopies) compared with only 31 in humans. Fur-thermore, these kinesins have a huge diversityof primary sequences (unlike, for example, thenumerous kinesins of Arabidopsis). Some ofthese kinesins fall into previously identifiedfunctional clades, but many belong to novelclades. There are no homologs for the kineto-chore motor, CENP-E, or the spindle motor,BimC. However, there has been an expansionand diversification of the mitotic centromere-associated kinesin family. Characteristic tri-laminar kinetochore-like plaques are seen inkinetoplastid mitotic nuclei attached to thespindle (29). Generally, the proteins of the mi-totic kinetochore are only poorly conserveddespite the near-ubiquity of the structure itself(Fig. 3). However, this appears to be particu-larly pronounced in the case of the Tritryps,where there are no homologs of CENP-C/Mif2,

+

Growth

+

ARP2/3 complexFormins

profilinG-actin binding

twinfilin

Activators of ARP2/3 complex

F-actin nucleation

F-actin cappingCapZ-α + CapZ-β

Dynactin complex

ADF/cofilinActin turnover

thymosin-β4CAP/Srv2p

WASP/ScarLas17pEPS15/Pan1p ActA

verprolinABP1

F-actin bundling / cross-linkingfimbrinvillin

filamin

α-actininplastinspectrin

ARP1dynactin1-6ARP11Cytoplasmic dynein

F-actin severinggelsolin

1

Tubulin Tyr-ligaseTubulin modification

Tubulin sequesteringstathmin

katanin p60MT severing

katanin p80

+

γ-tubulinMT nucleation

GCP2/Spc97p

GCP4-6GCP3/Spc98p

EB1/Bim1pMT capping

CLIP-170/Tip1p

MT minus-end organisation

Tea1pAPC

TOG/MOR1MT decoration

CLASPsMAP1-6tau

Actin cytoskeletonTubulin cytoskeleton

centrinspericentrinLIP1

Spc72pSpc42p

LIP8Kar1pSpc29p

+

-

-

-

-

Fig. 2. Core cytoskeletal components in T. brucei, T. cruzi, and L. major. Aschematic representation of the tubulin- and actin-based cytoskeleton.(Left) a- and b-tubulin subunits are depicted as light and dark bluecircles, either assembled into the microtubule lattice or as dimers. (Right)Actin filaments composed of actin momomers (yellow circles). Black textindicates processes involved in cytoskeleton organization. Components

for which the Tritryp genomes encode one or more homologs are shownin red text. Those that do not appear to be encoded by the three genomesare indicated in gray text. The CapZ proteins only present in T. cruzi arehighlighted in blue text, whereas orange text points to homologs onlyencoded by the T. brucei and T. cruzi genomes. The Arp2/3 complex is di-vergent in L. major.



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HEC1/Ndc80, or the centromeric checkpointprotein BUB1, each of which is highly con-served among yeast, plants, and mammals (Fig.3). Indeed, it appears that the sole variant his-tone H3 in these genomes does not evenfunction at the centromere (30).

The proteins of the flagellar axonemeappeared to be extremely well conserved. Withthe exception of tektin, there are homologs inthe three genomes for all previously identifiedstructural components as well as a full comple-ment of flagellar motors and both complex Aand complex B of the intraflagellar transportsystem (Fig. 4). Thus, the 9þ2 axoneme,which arose very early in eukaryotic evolution,appears to be constructed around a core set ofproteins that are conserved in organisms pos-sessing flagella and cilia. The major diver-gence apparent in flagellar organization inKinetoplastida is the augmentation of theaxoneme by a paracrystalline extra-axonemalstructure, the paraflagellar rod, that is uniqueto the Kinetoplastida and Euglenida.

No actin-based motility has been describedin trypanosomatids; they do not exhibit theruffles or pseudopodia seen in amoebae andmetazoa or themyosin-based glidingmotility ofthe Apicomplexa. Acto-myosin may not evenbe necessary for cytokinesis in these organisms.Actin is, however, involved in endocytosis(31). The genome sequences revealed putativehomologs for proteins involved in monomeric-actin binding and actin filament nucleation (Fig.2 and table S8) and for two myosin families(32). Surprisingly, the cohort of highly con-served proteins involved in severing, bundling,cross-linking, and capping of filamentous actinare missing Ethe latter is not the case for T. cruzi(32)^. Moreover, the dynactin complex is ab-sent, indicating a major loss in the ability forcross-talk between the actin- and tubulin-filament networks (Fig. 2).

Metabolism and transport. Understand-ing the parasite_s metabolism will underpinnew drug discovery. To date, biochemical

studies of trypanosomatids have been restrictedto specific life-cycle stages that can be readilycultured in vitro or in vivo. In contrast, ge-nome analysis provides a global view of theparasite_s metabolic potential (Plate 2). Interms of overall capabilities, T. brucei hasthe most restrictive metabolic repertoire, fol-lowed by T. cruzi. This may reflect that,unlike T. cruzi or L. major, the parasite doesnot have an intracellular life cycle stage andtherefore has greater access to nutrients inthe plasma. Horizontal gene transfer mayhave provided additional metabolic versa-tility for the parasites. In almost 50 cases,there was strong evidence of putative hori-zontal transfer from bacteria into the Tritryplineage (5) (table S11). Moreover, these pro-vide us with candidate enzymes for new drugintervention points.

Across the three species, 633 genes (tableS9) are annotated as transporters or channels,and 8113 are annotated as enzymes. Of the latter,more than 2000 have been classified withEnzyme Commission (EC) numbers (table S10).

Carbohydrate metabolism. The Tritrypspossess a full complement of candidate genesnecessary for uptake and degradation of glucosevia glycolysis or the pentose phosphate shuntand the tricarboxylic acid (TCA) cycle (Plate 2).A limited number of hexose transporters arepresent in all species, but only T. cruzi pos-sesses hexose phosphate transporters as seenin bacteria. This may reflect the fact thatonly T. cruzi amastigotes reside in the cyto-sol of the host cell with ready access to sugarphosphates. In contrast, only L. major appearscapable of hydrolyzing disaccharides. Thisis consistent with the biology of the insectvectors: Tsetse and triatomines are obligateblood feeders, whereas sandflies also feed onnectar and aphid honeydew. T. brucei lacksseveral sugar kinases present in L. major orT. cruzi, possibly an adaptation to the re-stricted range of sugars available in plasmaand in tsetse blood meals.

Despite having potential pathways for com-plete oxidation, the Tritryps partially degradeglucose to succinate, ethanol, acetate, alanine,pyruvate, and glycerol (33) (Plate 2). L-lactateis not an end product because lactate dehy-drogenase is absent from all three species,although D-lactate can be formed from methyl-glyoxal in L. major. A major route for formationof succinate from glucose in the insect stagesis via an unusual nicotinamide adenine dinu-cleotide (NADH)–dependent fumarate reductase(34). In mitochondria, pyruvate is principallyconverted to acetate via an acetate:succinatecoenzyme A (CoA)–transferase (35).

In many insects, including tsetse andsandflies, proline is a highly abundant energysource used in flight muscles. Proline is also apreferred energy source for the insect stagesof the parasites. However, proline is only par-tially degraded to succinate (procyclic stage,T. brucei) or alanine, succinate, and otherTCA intermediates (promastigote stage, L.major). In the Tritryps, neither an isocitratelyase nor malate synthase could be identi-fied, making a glyoxalate bypass in the TCAcycle unlikely.

Electron transport and oxidative phos-phorylation. A fully functional mitochondrialelectron transport system and adenosine tri-phosphate (ATP) synthase is present in theTritryps. Several ubiquinone-linked dehydro-genases are present, including a flavin adeninedinucleotide (FAD)–dependent glycerol-3-phosphate dehydrogenase, which in T. bruceialone is linked to an alternative oxidase thatrepresents a potential drug target (36).

In many organisms, heme is synthesizedfrom glycine and succinyl-CoA and incor-porated into hemoproteins, such as cytochromes.However, all trypanosomatids (except thosewith bacterial endosymbionts such as Crithidiaoncopelti) are heme auxotrophs. Thus, theabsence of the first two enzymes in hemebiosynthesis (aminolevulinate synthase andaminolevulinate dehydratase) is not unexpected.It was surprising, however, to discover thatgenes for the last three enzymes (copropor-phyrinogen III oxidase, protoporphyrinogen IXoxidase, and ferrochelatase) of heme bio-synthesis are apparently present in L. major.Moreover, there is strong evidence (table S11and fig. S4, A to C) that these genes may havebeen horizontally acquired from bacteria.

Glycosylphosphatidylinositol anchorbiosynthesis. The surface of trypanosomatidplasma membranes contains a variety of gly-coproteins (e.g., VSGs), phosphosaccharides(e.g., lipophosphoglycan), and glycolipids thatare involved in immune evasion, attachment, orinvasion. The most abundant of these areattached via glycosylphosphatidylinositol (GPI)anchors. GPI anchors are essential for parasitesurvival and are therefore a potential drug target(37). The GPI backbone contains mannose,glucosamine, and myo-inositol-phospholipid,

Fig. 3. Conservation of proteins ofthe kinetochore in T. brucei, T.cruzi, and L. major. A schematicrepresentation of conserved com-ponents of a typical kinetochoreis shown. The pair of plate-likekinetochores are present on thesurface of the chromosome in thecentromeric region, with the out-er plate acting as microtubuleattachment points radiating fromspindle pole bodies. Kinetochorecomponents for which the Tri-tryps encode one or more homo-logs are indicated in red text.Those that do not appear to beencoded by the three genomesare indicated in gray text. Thekinetoplastid histone H3 variantdoes not appear to be centromer-ic (30).

DNA bindingCENP-ACENP-B

Peri-centromericHP1

Checkpoint proteinsBUB1BUB3

MAD1MAD2

ZW10

inner plate

outer plate

spindle

1

Inner kinetochoreCENP-CCENP-G

Mis12MCAK

Skp1p Nde10pCep3p Ctf13p

Outer kinetochoreCENP-ECENP-F

HEC1/Ndc80Nuf2

TOG/MOR1



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plus additional sugar modifications such asgalactose (Plate 2). Pathways to the appropri-ate sugar nucleotide precursors are completeexcept for phosphoglucomutase, which wasnot identified in T. brucei despite amplebiochemical evidence for its presence. More-over, uridine diphosphate (UDP)–galactose canonly be formed from glucose-6-phosphate viaUDP-glucose (38), consistent with the absenceof genes for conversion of galactose to thissugar in T. brucei. The T. brucei genome en-codes a large family of UDP-galactose– orUDP-N-acetylglucosomine–dependent glyco-syltransferases (table S2), nine of which appearto be pseudogenes associated with arrays ofVSG pseudogenes. Myo-inositol can either besynthesized de novo from glucose-6-phosphateor salvaged via an inositol transporter (39) forthe synthesis of phosphatidyl inositol, the firststep in the GPI biosynthetic pathway.

Surface glycoproteins are attached to theirGPI anchors via ethanolamine phosphate. In T.cruzi mucins, this linkage can optionally bereplaced by aminoethylphosphonate (AEP).Enzymes for the complete synthesis of AEPfrom phosphoenol pyruvate are present only inT. cruzi (Plate 2) and could represent noveldrug targets because they are absent fromhumans. The third (and last) step of thepathway (AEP transaminase) is also found inL. major and T. cruzi, and strong tree-basedevidence supports its presence via horizontalacquisition from bacteria (table S11 and fig.S5). The second step of the pathway (phos-phonopyruvate decarboxylase) is found in allthree parasites, and homologs in other eukary-otes could not be found.

Amino acid metabolism. The Tritrypshave some striking differences in amino acidmetabolism (Plate 2, boxed). Amino acid trans-porters constitute one of the largest families ofpermeases in these parasites, with 29 predictedmembers in L. major, 38 in T. brucei, and 42 inT. cruzi (table S9). This is consistent with theTritryps lacking biosynthetic pathways for theessential amino acids of humans and requiringexogenous proline as an energy source (exceptbloodstream T. brucei), glutamine for severalbiosynthetic pathways, cysteine as an additionalsulfur source, and tyrosine for protein synthesis.

Most of the enzymes of the classical path-ways for aromatic amino acid oxidation aremissing. A putative biopterin-dependentphenylalanine-4-hydroxylase capable of con-verting phenylalanine to tyrosine could only beidentified in L. major. However, Leishmaniaspp. are auxotrophic for tyrosine (40), so thisgene (LmjF28.1280) may have another func-tion. Candidate genes for transamination andreduction to the corresponding aromatic lactatederivative have been identified in all species.The role of this pathway is unclear, althoughsecretion of these aromatic acids is found in T.brucei infections (41) and their presence in thecentral nervous system may result in the neuro-

behavioral disturbances (42) typically associ-ated with human sleeping sickness.

The branched-chain amino acids can beconverted to acyl-CoA derivatives in mitochon-drion. However, no branched chain amino-transferase catalyzing the first step could beidentified in T. cruzi. Leucine is converted tohydroxymethylglutaryl-CoA (HMG-CoA),which then can be incorporated directly intosterols via the isoprenoid synthetic pathway(43). Alternatively, HMG-CoA can be cleavedinto acetyl-CoA and acetoacetate in T. bruceiand T. cruzi, but the lyase is apparently absentfrom L. major. Isoleucine and valine use asimilar pathway to form propionyl-CoA. How-ever, the mitochondrial enzymes for furthermetabolism of propionyl-CoA to succinyl-CoAseem to be absent from both T. brucei and T.cruzi. Similarly, methionine can be convertedto oxobutyrate, the precursor to propionyl-CoA, but no further. This could explain whythreonine is not oxidized via the 2-oxobutyratepathway in T. brucei: because the necessarythreonine hydratase is missing. Instead, threo-nine is degraded to acetyl-CoA and glycine viathe aminoacetone pathway by using a mito-chondrial threonine dehydrogenase (44) and anaminoacetone synthase. The latter two genesare present in T. brucei and T. cruzi but absentfrom L. major.

Histidine catabolism appears to be absentfrom T. brucei. However, T. cruzi does have apathway that looks typically eukaryotic, exceptfor the last enzyme (glutamate formiminoaminotransferase, Tc00.1047053507963.20),which only appears to be present in T. cruziand Tetrahymena. Histidine is the precursor forovothiol A, an antioxidant (45), which is in L.major and may help to protect the invadingparasite from macrophage-derived hydrogenperoxide and nitric oxide. The biosyntheticpathway is thought to involve two intermediatesteps requiring cysteine and adenosylmethio-nine. However, the sequences of these enzymesare not known in any organism and thereforecannot be identified.

A functional urea cycle (Plate 2) is missingfrom all three organisms. Although carbamoyl-phosphate synthetase is present in all three, thisenzyme is also essential for pyrimidine bio-synthesis. Argininosuccinate synthase and abona fide glycosomal arginase were onlyidentified in L. major. A second member ofthe arginase/agmatinase/formiminoglutamasegene family was found in all three parasites.However, deletion of the former gene in L.major renders it auxotrophic for ornithine,indicating that this second gene is probablynot an arginase (46). Arginine kinase, presentonly in T. brucei and T. cruzi, has been pro-posed as a possible chemotherapeutic target(47). Phosphoarginine may play a role as atransient source of energy for renewal of ATP,similar to phosphocreatine in vertebrates.

Trypanothione metabolism. Arginineand ornithine are precursors for polyaminebiosynthesis, essential for cell growth anddifferentiation and for the synthesis of the drugtarget, trypanothione (48). The first step inpolyamine biosynthesis (ornithine decarbox-ylase) is the target for the human sleepingsickness drug difluoromethylornithine (Plate 2boxed) (48) and was found in T. brucei and L.major but not T. cruzi. Arginine decarboxylase,catalyzing the proposed target for difluorometh-ylarginine in T. cruzi and an alternative routefor putrescine synthesis, could not be identifiedeither. Two candidate aminopropyltransferaseswere found in T. cruzi, in contrast to one in L.major and in T. brucei. The mammalianretroconversion pathway of polyamines appearsto be absent.

Cysteine can be produced from homo-cysteine by the trans-sulfuration pathway pres-ent in all three organisms. De novo synthesisfrom serine appears possible only in L. majorand T. cruzi. These parasites can also inter-convert glycine and serine via serine hydroxy-methyl transferase, which is apparently absentfrom T. brucei. Cysteine, glutamate, glycine, andmethionine (as decarboxylated AdoMet) are theprecursor amino acids for glutathione (GSH).

Fig. 4. Conservation of proteinsof the flagellum in T. brucei, T.cruzi, and L. major. The figureshows the kinetoplastid flagellumwith the axoneme (blue) and thelattice-like paraflagellar rod (PFR),unique to Kinetoplastida andEuglenida (yellow). Flagellarmotors are seen attached to theaxoneme. Components for whichthe Tritryps encode one or morehomologs are indicated in redtext. Those that do not appearto be encoded by the threegenomes are indicated in graytext. (IFT, intraflagellar transport).

IFT

PFR

CHE3/cytoplasmic dynein IIIntraflagellar transport

heterodimeric kinesin IIIFT172IFT140IFT122aIFT88IFT80

IFT71IFT57/55IFT52IFT20

axonemal dyneinsAxonemal components

PF16/SPAG6DIP13RIB43RSP4MBO2

PF20LF4RIB72ODA1tektin

axoneme



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Trypanothione is synthesized from spermi-dine and glutathione and subsumes many of theroles of the latter in defending against chemicaland oxidant stress. T. cruzi can also synthesizeor salvage spermine to form the spermine-containing analog of trypanothione. Trivalentarsenical and antimonial drugs form conjugateswith trypanothione and/or glutathione (48, 49),and the synthesis of GSH, polyamines, andtrypanothione are essential for survival in T.brucei (50–52).

The reactive by-product of metabolism, meth-ylglyoxal, can be converted via the trypanothione-dependent glyoxalase pathway (53) to D-lactate.Homologs of glyoxalase I and II were foundin T. cruzi, whereas T. brucei apparently lacksglyoxalase I but does have glyoxalase II (54).Unlike many bacteria, none of the parasitescontain methylglyoxal synthase.

Trypanosomatids lack catalase and selenium-dependent peroxidases and are unusually depen-dent on trypanothione-dependent peroxidasesfor removal of peroxides. Tryparedoxins arehomologs of thioredoxins, and all species con-tain both redox proteins. Thioredoxin reduc-tase is absent, and all Tritryp peroxidases arecoupled to trypanothione and trypanothionereductase, a validated drug target.

Purine salvage and pyrimidine synthesis.Several studies have shown that the trypanoso-matids are incapable of de novo purine synthesis.This is now confirmed by the absence of 9 of the10 genes required to make inosine monophos-phate (IMP) fromphosphoribosyl pyrophosphate(PRPP). Adenylosuccinate lyase is present,but this plays a role in purine salvage convertingIMP to AMP. Present also are a large numberof nucleobase and nucleoside transporters (55),(table S9) numerous enzymes for the inter-conversion of purine bases, and nucleosidesand enzymes to synthesize pyrimidines denovo.

Lipid and sterol metabolism. Sterolmetabolism has attracted considerable inter-est as a drug target in T. cruzi and L. major(56). Except for bloodstream T. brucei, whichobtains cholesterol from the host, these para-sites synthesize ergosterol and are susceptibleto antifungal agents that inhibit this pathway.Candidate genes for most of ergosterol bio-synthesis are present in all three parasites.Intermediates of this pathway, namely farnesyl-and geranyl-pyrophoshosphate, are also pre-cursors for dolichols and the side chain ofubiquinone. L. major is capable of synthe-sizing the aromatic ring of ubiquinone fromacetate, parahydroxybenzoate being an im-portant intermediate. In this behavior it re-sembles prokaryotes.

The Tritryps seem to be capable ofoxidizing fatty acids via b-oxidation in twoseparate celular compartments: glycosomesand mitochondria. This contrasts with yeastsand plants, where b-oxidation of fatty acidstakes place exclusively in peroxisomes. In

addition, the three trypanosomatids are capa-ble of fatty acid biosynthesis.

Summary and concluding remarks.After more than a century of research aimed atunderstanding, controlling, and treating Africantrypanosomiasis, the genome sequence rep-resents a major step in this process. It will notprovide immediate relief to those suffering fromtrypanosomiasis. Instead, it will acceleratetrypanosomiasis research throughout the sci-entific community. Through early data re-lease before completion of the sequencing,the Tritryp genome projects have alreadyallowed scientists to identify many new drugtargets and pathways (e.g., GPI biosynthesisand isoprenoid metabolism) and have pro-vided a framework for functional studies. Be-cause about 50% of the genes of all threeparasites have no known function, many morebiochemical pathways and structural func-tions await discovery.

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(2003).57. We thank our colleagues at the Wellcome Trust Sanger

Institute and the Institute for Genomic Research,colleagues in the T. brucei and Tritryp genome networks,and the Trypanosomatid research community for theirsupport. We also thank K. Stuart, P. Myler, E. Worthey[Seattle Biomedical Research Institute (SBRI)], and B.Andersson (Karolinska Institutet) for helpful discussionsof the manuscript and P. Myler and G. Aggarwal (SBRI)for preparation of Plate 3. Funding for this work wasprovided by the Wellcome Trust and a grant from theNational Institute for Allergy and Infectious Diseases(NIAID) to N.M.E.-S. (AI43062). Funding for provision ofDNA resources was provided by Wellcome Trust grantsto S.E.M. (062513) and C.M.R.T. (062513). Funding forseveral T. brucei genome network and Tritryp genomemeetings over the past decade was provided by theBurroughs Wellcome Fund, NIAID, the Wellcome Trust,and the World Health Organisation Special Programmefor Research and Training in Tropical Diseases. Corre-spondence and requests for sequence data should beaddressed to M.B. ([email protected]) or N.M.E.-S.([email protected]). Requests for DNA resources shouldbe addressed to S.E.M. ([email protected]), and requestsfor updates or amendments to genome annotationshould be directed to C.H.-F. ([email protected]). Alldata sets and the genome sequence and annotationare available through GeneDB at www.genedb.org.Sequence data have been deposited at DNA Data Bankof Japan/European Molecular Biology Laboratory/GenBank with consecutive accession numbers CP000066to CP000071 for chromosomes 3 to 8 and project ac-cession numbers AAGZ00000000, AAHA00000000, andAAHB0000000 for the whole-chromosome shotgunprojects of chromosomes 9 to 11. The versions of chro-mosomes 9 to 11 described in this paper are the firstversions, AAGZ01000000, AAHA01000000, andAAHB01000000, and unassembled contigs (over-lapping contiguous sequences) have accession num-ber CR940345.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/309/5733/416/DC1Materials and MethodsFigs. S1 to S5Tables S1 to S11References and Notes

23 March 2005; accepted 22 June 200510.1126/science.1112642



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