Systems-Based Analysis of the Sarcocystis neurona Genome IdentifiesPathways That Contribute to a Heteroxenous Life Cycle

Tomasz Blazejewskia Nirvana Nursimuluab Viviana Pszennyc Sriveny Dangoudoubiyamd Sivaranjani Namasivayame

Melissa A Chiassonc Kyle Chessmanab Michelle Tonkinf Lakshmipuram S Swapnaa Stacy S Hungag Joshua Bridgerse

Stacy M Ricklefsh Martin J Boulangerf Jitender P Dubeyh Stephen F Porcellai Jessica C Kissingerejk Daniel K Howed

Michael E Griggc John Parkinsonagl

Program in Molecular Structure and Function Hospital for Sick Children Toronto Ontario Canadaa Department of Computer Science University of Toronto TorontoOntario Canadab Molecular Parasitology Section Laboratory of Parasitic Diseases NIAID National Institutes of Health Bethesda Maryland USAc Gluck Equine ResearchCenter Department of Veterinary Science University of Kentucky Lexington Kentucky USAd Department of Genetics University of Georgia Athens Georgia USAeDepartment of Biochemistry and Microbiology University of Victoria Victoria British Columbia Canadaf Department of Molecular Genetics University of TorontoToronto Ontario Canadag US Department of Agriculture Animal Parasitic Diseases Laboratory Beltsville Agricultural Research Center Agricultural Research ServiceBeltsville Maryland USAh Genomics Unit Research Technologies Section Rocky Mountain Laboratories National Institute of Allergy and Infectious Diseases HamiltonMontana USAi Center for Tropical and Emerging Global Diseases University of Georgia Athens Georgia USAj Institute of Bioinformatics University of Georgia AthensGeorgia USAk Department of Biochemistry University of Toronto Toronto Ontario Canadal

ABSTRACT Sarcocystis neurona is a member of the coccidia a clade of single-celled parasites of medical and veterinary impor-tance including Eimeria Sarcocystis Neospora and Toxoplasma Unlike Eimeria a single-host enteric pathogen SarcocystisNeospora and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediatehosts As a genus Sarcocystis is one of the most successful protozoan parasites all vertebrates including birds reptiles fish andmammals are hosts to at least one Sarcocystis species Here we sequenced Sarcocystis neurona the causal agent of fatal equineprotozoal myeloencephalitis The S neurona genome is 127 Mbp more than twice the size of other sequenced coccidian ge-nomes Comparative analyses identified conservation of the invasion machinery among the coccidia However many dense-granule and rhoptry kinase genes responsible for altering host effector pathways in Toxoplasma and Neospora are absent fromS neurona Further S neurona has a divergent repertoire of SRS proteins previously implicated in tissue cyst formation inToxoplasma Systems-based analyses identified a series of metabolic innovations including the ability to exploit alternativesources of energy Finally we present an S neurona model detailing conserved molecular innovations that promote the transi-tion from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts

IMPORTANCE Sarcocystis neurona is a member of the coccidia a clade of single-celled apicomplexan parasites responsible formajor economic and health care burdens worldwide A cousin of Plasmodium Cryptosporidium Theileria and Eimeria Sarco-cystis is one of the most successful parasite genera it is capable of infecting all vertebrates (fish reptiles birds and mammalsmdashincluding humans) The past decade has witnessed an increasing number of human outbreaks of clinical significance associatedwith acute sarcocystosis Among Sarcocystis species S neurona has a wide host range and causes fatal encephalitis in horsesmarine mammals and several other mammals To provide insights into the transition from a purely enteric parasite (eg Eime-ria) to one that forms tissue cysts (Toxoplasma) we present the first genome sequence of S neurona Comparisons with othercoccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies

Received 7 December 2014 Accepted 12 December 2014 Published 10 February 2015

Citation Blazejewski T Nursimulu N Pszenny V Dangoudoubiyam S Namasivayam S Chiasson MA Chessman Tonkin M Swapna LS Hung SS Bridgers J Ricklefs SMBoulanger MJ Dubey JP Porcella SF Kissinger JC Howe DK Grigg ME Parkinson J 2015 Systems-based analysis of the Sarcocystis neurona genome identifies pathways thatcontribute to a heteroxenous life cycle mBio 6(1)e02445-14 doi101128mBio02445-14

Editor Louis M Weiss Albert Einstein College of Medicine

Copyright copy 2015 Blazejewski et al This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 30 Unportedlicense which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original author and source are credited

Address correspondence to John Parkinson johnparkinsonutorontoca or Michael E Grigg griggmniaidnihgov

This article is a direct contribution from a Fellow of the American Academy of Microbiology

The coccidia are a large clade of protozoan parasites within thephylum Apicomplexa In addition to a single definitive host

species in which the parasite undergoes its sexual cycle a subgroupof coccidia the members of the family Sarcocystidae (SarcocystisToxoplasma and Neospora) have evolved the ability to infect abroad range of intermediate hosts (1 2) To drive this transitionthe members of the family Sarcocystidae produce infectious tissue

cysts surrounded by glycosylated cyst walls Different species andeven strains exhibit distinct patterns of organ tropism withToxoplasma forming cysts in any organ whereas Sarcocystis cystsare largely restricted to muscle Ingestion of tissue cysts throughpredation or scavenging by the definitive host propagates the lifecycle (eg felids for Toxoplasma canids for Neospora and hu-mans for two Sarcocystis species) (3)

RESEARCH ARTICLE crossmark

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To survive and persist in their respective hosts apicomplexanparasites have evolved a variety of molecular strategies These in-clude a group of specialized proteins that facilitate parasite entryegress and colonization as well as molecular decoys that modu-late host immune signaling (4 5) The majority of these proteinslocalize to exocytic organelles (micronemes rhoptries and densegranules) that discharge in a highly coordinated program of inva-sion (5) For Toxoplasma gondii initial host recognition and at-tachment are performed by members of the SAG1-related se-quence (SRS) family This is followed by secretion of themicroneme (MIC) proteins that strengthen host cell attachmentand result in the formation of a ldquomoving junctionrdquo that providesthe motive force required to penetrate the host cell The movingjunction is further controlled by a set of proteins known as rhoptryneck (RON) proteins that facilitate invasion (for a review seereference 6) Subsequently rhoptry (ROP) proteins and dense-granule (GRA) proteins are secreted into the host cytosol to inter-act with cellular targets to protect the (now) intracellular parasitefrom clearance The parasite is further protected through encase-ment within a parasitophorous vacuole (PV) that is interestinglyabsent from the schizont form of Sarcocystis

Recent genomic comparisons of Toxoplasma Neospora andHammondia (the closest extant relative of T gondii) have identi-fied a series of ROP proteins whose expression targets host-specific immune signaling pathways ROP5 ROP16 and ROP18have all been shown to affect parasite virulence and contribute tohost specialization in the mouse model (4 5) Recently an ex-panded repertoire of SRS proteins previously implicated in hostrange expansion of T gondii were also identified in Neospora (7)Modeling of T gondii metabolism also identified strain-specificdifferences in growth potential establishing metabolism as anevolutionary factor capable of influencing host adaptation (8) Tocomplement the recently generated Eimeria genome sequencesand understand the transition from a purely enteric monoxenouslife cycle (eg Eimeria) to a heteroxenous one that includes theformation of tissue cysts we sequenced the genome of Sarcocystisneurona

Sarcocystosis caused by parasites within the genus Sarcocystisis typically asymptomatic but can be associated with myositis di-arrhea or infection of the central nervous system (CNS) Thegenus is ancient (relative age 246 to 500 million years based onsmall-subunit RNA sequences) diverse (more than 150 cata-logued species) highly successful (all vertebrates are susceptiblehosts including fish birds reptiles and mammals) and prevalent(cattle exhibit a 90 infection rate worldwide) (9) InterestinglySarcocystis species are not structurally similar for example S neu-rona sporozoites like T gondii lack the crystalloid body present inother coccidia including S cruzi of cattle (10) Sarcocystis speciestypically have a two-host predator-prey life cycle with one hostsupporting asexual multiplication while the other acts as the de-finitive host supporting a sexual cycle that results in sporocystshedding in feces Humans are definitive hosts of S suihominis andS hominis and can be infected by S nesbitti with associated se-quelae including muscular sarcocystosis Opossums are the de-finitive hosts of S neurona (11) a species that has a broadintermediate-host range including raccoons cats skunks andmore recently a variety of mustelids pinnipeds and cetaceans(12ndash15) S neurona produces tissue cysts typically in muscle andoccasionally in the CNS (16 17) Horses are considered aberranthosts in which the parasite typically multiplies as schizonts in the

CNS but fails to encyst Unabated destruction of neural tissue canbe fatal to horses and many other hosts and the disease was calledequine protozoal myeloencephalitis before the etiologic proto-zoan S neurona was identified and named in 1991 (2) With mi-gration of opossums to the west coast of North America during thelast century (14) the S neurona host range expanded to causeepizootics in sea otters harbor seals and harbor porpoises (18) Sneurona is now being monitored for its potential as an emergingdisease threat Here we sequenced and performed a systems-based analysis of the genome of type II S neurona strain SO SN1isolated from a southern sea otter that died of protozoal enceph-alitis (19) which represents the most common genotype infectinganimals throughout the United States

RESULTSThe S neurona SO SN1 genome is more than twice the size of theT gondii genome Combining 7020033 reads from the 454 LifeSciences sequencing platform with 529830690 reads from theIllumina Hi-Seq sequencing platform we generated 47722 Mbpof sequence data from S neurona SO SN1 DNA By integrating avariety of assembly algorithms (see Materials and Methods) thesedata were assembled into 116 genomic scaffolds with a combinedsize of 127 Mbp over twice the size of the Neospora caninum andT gondii genomes (Table 1 is a summary of the genome statisticsobtained) An additional 31 Mbp of sequence is encoded in 2950unscaffolded contigs (each greater than 500 bp in length) Theassembly N50 value was 3117290 bp with a maximum scaffoldlength of 9217112 bp To help annotation efforts we generatedan additional 59622019 reads from S neurona RNA From thesedata we predict a complement of 7093 genes 5853 of which aresupported by RNA-Seq evidence (see Table S1 in the supplemen-tal material) The number of genes predicted is comparable to thatof the related coccidia T gondii and Eimeria tenella Comparisonsof gene orderings reveal blocks of syntenic relationships betweenhomologous genes in S neurona and T gondii with the longestsuch block aligning 43 genes on scaffold 1 of S neurona and chro-mosome IX of T gondii (Fig 1A) Chromosome-wide synteny wasnot observed suggesting a significant level of genome rearrange-ment between S neurona and T gondii A more detailed view ofthe largest syntenic region reveals the extent of gene order preser-vation but also reveals differences in the structures of individualgenes (Fig 1B)

Given similarities in gene numbers and exon lengths we nextdetermined the source of the additional sequence associated with

TABLE 1 Genome statistics

Parameter Statistic

Genome size (bp) 130222184Genome GC 515

No of scaffolds 116Total scaffolded length (bp) 127077592No of contigs in scaffolds 11452Scaffold N50 (bp) 3117920

No of unscaffolded contigs 2950Contig N50 (bp) 20915

Total no of bp in gaps 12350913No of genes 7093

Mean gene length (bp) 9121Mean no of exons 55Mean coding size (no of amino acid residues) 856

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FIG 1 Architecture and syntenic relationships of the S neurona genome (A) Circos representations (50) of syntenic relationships between the genomes of Sneurona and T gondii ME49 The inner circle shows syntenic relationships among the 10 largest S neurona genomic scaffolds (maximum size 92 Mb minimumsize 35 Mb) and the 14 chromosomes of T gondii Bandwidths indicate alignment length and colors represent the S neurona scaffold of origin for the geneclusters Grey circles indicate the largest regions of genomic synteny between S neurona scaffold SO SN1 and T gondii chromosome 9 The outer circles show a

(Continued)

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the S neurona genome Comparisons of intron numbers andlengths show that S neurona possesses a similar number of exonsper gene (550 in T gondii 593 in N caninum and 544 in Sneurona) but that the average length of the introns in S neurona(14375 bp) is roughly triple that of T gondii and N caninum(4975 and 4652 bp respectively) Further comparisons of inter-genic regions reveal these to be larger in S neurona (8495 222 bp) than in T gondii and E tenella (2381 72 and 2934 268 bp respectively) To identify factors responsible for the in-creased intra- and intergenic region sizes we performed a system-atic analysis of repetitive regions across representative apicompl-exans with the software tool RepeatModeler (20) This analysisrevealed that the S neurona genome is rich in repeats largely asso-ciated with long interspersed nucleotide element (LINE) andDNA element sequences (class I and II transposons respectively)Mapping of the repeats to scaffolds revealed that many of therepeats are associated with genes (Fig 1B) Further comparisonsof introns exons and intergenic regions showed clear differencesin the repeat type based on the genomic context (Fig 1C) DNAelement-type repeats were enriched in intronic regions and virtu-ally absent from exons suggesting evolutionary pressure againstthe integration of DNA elements within coding regions Con-versely LINE-like repeats were equally distributed across exonsintrons and intergenic regions In total 31 Mbp of the S neuronagenome had repetitive sequences compared to 179 Mbp of theE tenella genome and 25 Mbp of the T gondii genome (Fig 2A)

S neurona displays a diverse set of repetitive elements Therepetitive sequences present in S neurona are extraordinarily di-verse with 203 families of repeats discovered with RepeatModelercompared to 101 families in E tenella and 5 in Plasmodium falci-parum The majority of simple repeats within the S neurona ge-nome belong to more diverse families unlike other apicomplexanparasites where simple repeats are largely composed of short re-peats (eg CAGn in E tenella [21]) For example 33 simple repeatfamilies in S neurona were composed of consensus sequences withan average length of 287 bp The average length of the simplerepeats was 105 bp in S neurona compared to 48 and 68 bp inP falciparum and E tenella respectively

Type II transposons or DNA elements with 64732 membersrepresent the largest family of repeats present in S neurona total-ing 146 Mbp (115) of the genome considerably more than inE tenella (Fig 2A) All of the DNA elements identified belong tothe ldquocut-and-pasterdquo families of transposons which propagatethrough genomes through excision and insertion of DNA inter-mediates The most abundant family of DNA elements belongedto the CACTA-Mirage-Chapaev family of transposons although aminority of Mutator-like elements was also identified ActiveDNA transposons contain transposase genes however we wereunable to detect any such gene within the S neurona genomesuggesting that these DNA elements are ancient and degradedSupporting this view we found that the ratio of transversions totransitions in alignments of repetitive sequences to DNA repeatfamilies was almost exactly 21 the statistically expected rate ofmutation in the absence of evolutionary pressure

Given the relative lack of repeats in the T gondii genome weexplored whether the repeats identified in S neurona are less activethan those identified in E tenella When repeats are active it ispossible to identify clades of repeats with significant sequence sim-ilarity Pairwise sequence alignments of members of five familiesof repeats (Fig 2B) were highly divergent indicating that theLINEs and DNA elements are no longer active in S neurona Fur-ther the LINEs in S neurona are more diverse and therefore likelyto be more ancient than those in E tenella Interestingly E tenellaT gondii and P falciparum all feature a bimodal distribution ofsimple repeats that is lacking in S neurona Finally all three of thecoccidian genomes analyzed here displayed similar distributionsof sequence divergence of DNA elements albeit with slightly dif-ferent means (225 281 and 278 for E tenella S neurona andS neurona-like DNA repeats in T gondii respectively) This sug-gests that while DNA elements are no longer active in these ge-nomes they did remain active for slightly longer within E tenellaFrom these analyses we conclude that the maintenance of largenumbers of LINEs and DNA elements in S neurona (andE tenella) even though they are inactive likely plays a functionalrole since T gondii has removed most of these elements from itsgenome

The S neurona apicoplast genome is well conserved withother Apicomplexa In addition to its nuclear genome the apico-plast genome of S neurona SO SN1 was studied by reference map-ping to the assembled S neurona SN3 apicoplast sequence (seeFig S1 in the supplemental material) Both organellar genomearchitectures are highly similar to those of Toxoplasma (GenBankaccession no U871452) and P falciparum (22) There are how-ever a few key differences As in Toxoplasma both Sarcocystisapicoplast sequences are missing open reading frame A (ORFA)However unlike Toxoplasma both S neurona sequences show aloss of rpl36 and a loss of one copy of tRNA-Met (from the tRNAcluster between rps4 and rpl4) S neurona also has a feature firstobserved in the Piroplasmida that is not seen in Toxoplasmanamely a division of the RNA polymerase C2 gene into two dis-tinct genes (23) Both S neurona apicoplast genome sequencesuniquely have the insertion of a fragment of rps4 between ORFGand one copy of the large-subunit rRNA in common (see Fig S1 inthe supplemental material) The insert was verified in S neuronaSN3 via PCR and sequencing across this region (see Fig S2 S3and S4 in the supplemental material) The rps4 fragment insertionappears to be very recent because the S neurona SN3 fragmentinsert is identical in sequence to the corresponding region in thefull-length rps4 gene Comparison of the S neurona SO SN1 andSN3 nucleotide sequences to each other reveals a few indels but nosingle-nucleotide polymorphisms (see Text S1 in the supplemen-tal material) Indels when present occur in up to one-third of thereads for the locus The dominant sequence is identical to thatdetermined for SN3 Each S neurona apicoplast genome was se-quenced to greater than 200 coverage

The S neurona genome encodes many novel genes and iden-tifies many coccidian-specific innovations InParanoid predic-tions suggest that S neurona has more orthologs in common with

Figure Legend Continued

detailed view of synteny indicated by the grey circles Red and green bars indicate exons of S neurona and T gondii respectively and yellow and blue bars indicateintronic and repeat regions respectively (B) Detailed view of the synteny map shown in panel A revealing larger introns in S neurona relative to those in T gondiiand the relative positioning of repetitive elements (C) Incidence of repeats in different genomic regions as defined by RepeatModeler (20)

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FIG 2 Repeat incidence and diversity in selected alveolate genomes (A) Incidence of repeats as defined by RepeatModeler (20) in a selected group of alveolategenomes While E tenella is rich in LTR elements S neurona is rich in DNA elements Neither type of element is abundant in other alveolates (B) Diversity ofdifferent repeat families within coccidian genomes Bar graphs indicate the relative abundance of each repeat class as a function of Kimura divergence from theconsensus repeat sequence Note that DNA elements were not initially detected in the T gondii genome however subsequent searches revealed the presence ofDNA elements predicted from the S neurona genome (indicated in the inset bar chart of S neurona repeats)

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T gondii than with E tenella supporting a closer evolutionaryrelationship (3169 versus 1759 groups of orthologs respectively)(Fig 3A) Consistent with previous gene studies we identified1285 (18) S neurona genes with no detectable homology(BLAST score 50) to any known gene suggesting either a highdegree of gene innovation or significant sequence divergence fromremote homologs Among the conserved genes 715 (10) wereconserved (possessing orthologs) in both Cryptosporidium par-vum and either P falciparum or Theileria annulata identifying alarge collection of proteins that could be amenable to broad-spectrum drug development These include members of a varietyof ATPase genes heat shock proteins DEADDEAH helicasesproteins with EF-hand domains and protein kinases In additionwe identified 1285 (18) genes with homology only within thefamily Sarcocystidae representing potential drug targets againsttissue cyst-forming coccidia A majority (55) of these are anno-tated as ldquohypothetical proteinsrdquo in the ToxoDB resource (24) Ofthe proteins that are annotated AP2 domain transcription factorsrhoptry kinase (ROPK) and neck proteins and zinc fingers as wellas proteins with RNA recognition motifs are prevalent

The S neurona attachment and invasion machinery isbroadly conserved with T gondii The process of host cell inva-

sion by apicomplexan parasites is a rapid and complex processthat relies on a coordinated cascade of interactions between theinvading parasite and the host cell To orchestrate these processesapicomplexans have evolved families of invasion proteins that arebroadly conserved but nevertheless exhibit unique lineage-specific innovations (25) To identify S neurona gene models in-volved in invasion relative to T gondii we constructed an invasionprotein coexpression network (Fig 3B) in which pairs of T gondiiproteins are linked if they exhibit significant coexpression with Sneurona (Pearson correlation coefficient 08) as has been donefor other organisms (26ndash28) This network provides a scaffoldonto which conservation and expression data from S neurona aremapped to yield insights into evolutionary and functional rela-tionships Consistent with previous studies we found that con-served proteins (those that have an ortholog in common with Sneurona) tend to have more correlated expression (high Pearsoncorrelation coefficients) and more connections (high node de-gree) and are better connected within the network (shorter aver-age path lengths and higher betweenness) than their noncon-served counterparts (Fig 3C) These findings highlight thepotential importance of conserved proteins to the function of theinvasion machinery Within our T gondii invasion network we

FIG 3 Coexpression network for T gondii invasion-associated genes (A) Ortholog distribution of S neurona genes Orthologs were predicted by using theInParanoid pipeline (51) (B) Network of T gondii proteins involved in the invasion process Nodes indicate genes colored by family or location with sizeindicating the relative expression of the S neurona ortholog as determined through RNA-Seq expression Square nodes indicate the absence of an ortholog in Sneurona Links between nodes indicate significant coexpression (Pearson correlation coefficient [PCC] 08) Two main clusters of proteins are observed oneinvolving batteries of rhoptry proteins (ROPs and RONs) and one involving microneme proteins (MICs) (C) Network statistics associated with the invasionnetwork

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identified two main clusters of highly correlated genes associatedwith key invasion events The first involves proteins associatedwith the micronemes (MIC proteins) which strengthen host cellattachment and play a major role in the formation of the movingjunction that forms a specific interface facilitating invasion Thesecond involves proteins associated with the rhoptries (RON andROP proteins) an organelle that is absent from the merozoitestage of all Sarcocystis species including S neurona (29)

The genome analyses identified nine previously reported Sneurona orthologs of T gondii MICs (MIC7 MIC8 MIC10MIC12 MIC13 MIC14 MIC15 MIC16 and M2AP) (30) Wealso identified potential homologs of MIC2 MIC4 and MIC9 thathad not been annotated through the gene model prediction pipe-line MIC4 has previously been shown to form part of a hetero-complex with MIC1 and MIC6 (31) The absence of the latter twohomologs from S neurona suggests that MIC4 likely mediates themore important functional role MIC7 MIC8 MIC9 and MIC12are relatively unique in T gondii with the possession of epidermalgrowth factor-like domains suggesting a potential role in ligandbinding MIC10 together with MIC11 (absent from S neurona) isthought to be involved in the organization of organellar contentsAlso secreted by the microneme is apical membrane antigen 1(AMA1) which functions to link the inner membrane complex(IMC) to the host cell via interactions with RON proteins thattogether make up the moving junction (6 32) Our searches re-vealed two loci separated by approximately 80 kb on the S neu-rona assemblyrsquos largest scaffold homologous to T gondii AMA1protein TGME49_315730 Interestingly the reading frames of thetwo S neurona paralogs (SnAMA1a and SnAMA1b) occur in op-posite directions suggesting an inverted duplication Supportingthis two inverted repeats 100 bp in length and with 70 iden-tity were identified ~20000 bp apart separating the two paralogsWhile the region upstream of the paralogs appears repeat richcontaining simple repeats as well as LINEs and DNA elementsthe region downstream of the second paralog is uncharacteristi-cally repeat poor with no repetitive sequences in ~14000 bp ofsequence T gondii possesses additional paralogs of the AMA1protein including TGME49_300130 Again S neurona appears topossess these two additional AMA1 paralogs (SnAMA1c andSnAMA1d) but in this case they are present on two differentscaffolds AMA1 has been shown to interact remarkably stronglywith RON2 RON4 and RON5 (32)

In general RONs were well conserved in S neurona and T gon-dii with RON2 RON3 RON5 and RON8 orthologs displayingsignificant sequence similarity across their entire length Threeparalogs of T gondii RON3 (TgRON3) were identified on a singlescaffold suggesting a tandem duplication two of which appear tobe expressed as predicted by the RNA-Seq data Putative S neu-rona orthologs of RON4 and RON6 were identified through man-ual inspection of sequence alignments A pattern of conservationand divergence was observed for a putative ortholog of TgRON9In T gondii RON9 and RON10 form a stable complex distinctfrom the AMA1-RON2458 complex with disruption of eithergene leading to the retention of its partner in the endoplasmicreticulum followed by degradation This complex does not play arole in T gondii invasion and virulence but because of its conser-vation with C parvum has been linked to interactions involvingepithelial cells (33) While an S neurona protein could be alignedover ~25 of the TgRON9 sequence it was found to lack a singlecopy of the 22 copies of the PAEENAEEPKQAEEQANASQSSET

motif associated with the T gondii protein No homologs toRON1 or RON10 could be identified

Another critical organelle required for host invasion is theIMC which additionally confers stability and shape on the cell andis thought to mediate critical roles in cytokinesis and host cellegress (34) We identified 20 putative S neurona IMC orthologswith additional evidence of a further six (see Table S2A in thesupplemental material) Only TgGAP70 TgAlv6 and TgAlv7 ap-pear to lack homologs

Molecular modeling reveals that SnAMA1a is capable of in-timately coordinating SnRON2 To examine if S neurona AMA1homologs can bind S neurona RON2 homologs we generatedstructural models of SnAMA1a and SnAMA1b which show thehighest sequence identity (49 and 44 respectively) withTgAMA1 Both models possess a PAN-like domain architecturefor DI and DII (SnAMA1a Fig 4A) consistent with homologsfrom other apicomplexans (35 36) A key feature of DII is anextended loop that packs into the groove of DI and regulatesRON2 binding in related AMA1 proteins (37) In the SnAMA1amodel a cysteine pair localized within the DII loop is a novelfeature of AMA1s and may serve as a hinge to regulate loop dis-placement and consequently RON2 binding (Fig 4A) Further-more the SnAMA1a DII loop appears to be loosely anchoredwithin the DI groove via a Val-Val pair surrounding a central Leu(Fig 4A) This is in contrast to TgAMA1 (Fig 4C) where a Trp-Trp pair surround a central Tyr and the SnAMA1b model(Fig 4B) where a Trp-Leu pair surround a central Tyr (Fig 4B)These models suggest that AMA1 paralogs in S neurona employdivergent strategies that control DII loop dynamics and governaccess to the ligand-binding groove Focusing on the interactionwith RON2 removal of the apical segment of the DII loop fromthe model of SnAMA1a (mimicking the mature binding surface)led to a pronounced groove similar to the RON2 binding surfaceobserved in TgAMA1 (Fig 4D) Indeed an energy-minimizeddocked model revealed that SnRON2 domain 3 was accommo-dated in a U-shaped conformation (SnRON2D3 Fig 4E) with anoverall topology conserved with respect to the TgAMA1 costruc-ture with a synthetic TgRON2D3 peptide (Fig 4D) (37) Key fea-tures of the TgAMA1-TgRON2sp interface appear to be conservedat the SnAMA1-SnRON2D3 interface including a RON2 prolineresidue that occupies an AMA1 pocket exposed by displacementof the DII loop (Fig 4D and E yellow arrow) and a reliance onhydrophobic interactions to engage AMA1

Overall modeling of apo SnAMA1a and SnAMA1b in combi-nation with the complex of SnAMA1a with SnRON2D3 supportsthe hypothesis that these two proteins can form an intimate binarycomplex as observed in related apicomplexan homologs (37 38)Of note both SnAMA1a and SnAMA1b exhibited relatively lowlevels of expression in the merozoite stage sampled (104 and 67fragments per kilobase of exon model per million mapped reads[FPKM] respectively) compared to SnAMA1c and SnAMA1d(631 and 839 FPKM respectively) perhaps reflecting a stage-specific role for each AMA1-RON2 pairing

Proteins involved in host regulation in T gondii are not wellconserved in S neurona In addition to RONs rhoptries alsosecrete a battery of ROP proteins the products of a group of genesdisplaying high levels of correlated expression (Fig 3B) ROP pro-teins are secreted into the host cytosol to interact with host celltargets manipulating pathways that protect the intracellular par-asite against clearance To identify putative S neurona ROP ho-

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mologs we used previously published hidden Markov models(HMMs) (39) In addition to the eight SnROPKs reported previ-ously (39) our phylogenetic analysis identified seven new ROPKorthologs including ROP20 ROP26 ROP33 ROP34 andROP45 as well as two SnROPKs that appear unique to S neurona(Fig 5A) RNA-Seq data support the expression of six of these(ROP14 ROP21 ROP27 ROP30 ROP35 and ROP37) duringthe merozoite stage despite this stagersquos lack of rhoptries and theability of schizonts to develop in host cell cytoplasm in the absenceof a PV (3)

Overall S neurona contains a smaller complement of ROPKs(n 15) than E tenella (n 27) and a considerably smaller setthan T gondii (n 55) and N caninum (n 44) both of whichfeature distinct lineage-specific expansions However despite itslower number of ROPKs S neurona was found to have moreROPKs in common with T gondii and N caninum than withE tenella only two of the ROPKs in the three tissue cyst-formingcoccidia are conserved with E tenella (ROP2127 and ROP35)Importantly we did not find S neurona homologs correspondingto T gondii ROPK proteins implicated in murine virulence (ROP5and ROP18) modulation of STAT3 and STAT6 signaling(ROP16) or mitogen-activated protein (MAP) kinase signaling(ROP38) which suggests that S neuronarsquos success and pathogen-esis are not dependent on the inactivation of these host-specificpathways and may explain in part why this parasite is not infec-tious in rodents No information is available regarding the func-tional role of S neurona ROPKs However six are likely to beactive kinases since they retain key ldquocatalytic triadrdquo residuescritical for protein kinase function (SnROP2127 SnROP30SnROP33 SnROP34 SnROP35) Further five are likely to bepseudokinases (SnROP20 SnROP22 SnROP26 SnROP36SnROP37) that have been shown to act as cofactors of the activekinases (eg SnROP5 to SnROP18)

Finally only two dense-granule (GRA) protein homologs ofT gondii GRA10 and GRA12 were identified The discovery ofthe latter is surprising given that it has not been annotated in theN caninum genome In addition like the ROPKs the majority ofthe GRA proteins encoded by T gondii that specifically target hostimmune signaling pathways to alter parasite pathogenesis are notencoded by S neurona These include GRA6 which regulates theactivation of the host transcription factor nuclear factor of acti-vated T cells (NFAT4) GRA15 which regulates NF-B activation(40) GRA24 which promotes nuclear translocation of host cellp38a MAP kinase (41) and the phosphoprotein GRA25 whichalters CXCL1 and CCL2 levels to regulate immune responses andcontrol parasite replication (42) These data indicate either that adifferent suite of GRA proteins facilitate Sarcocystis host and nichespecialization or that Sarcocystis does not require an expandedrepertoire of GRA proteins during merozoite replication since itreplicates in the host cytosol and is not contained within a PV likeT gondii or N caninum

S neurona encodes a distinct set of SRS proteins The SRSproteins exist as a developmentally regulated superfamily of par-asite surface adhesins within the tissue cyst-forming coccidia thatpromote host cell attachment and modulate host immunity toregulate parasite growth and virulence In previous work we iden-tified 109 and 246 SRS proteins in the T gondii and N caninumgenomes respectively (4 7) Applying our previously generatedHMMs we identified a more restricted set of only 23 SRS-encoding genes in the S neurona genome Twenty of the 23 SRS-encoding genes were distributed across 11 of the major scaffoldsbut unlike the SRS-encoding genes in T gondii and N caninumonly one genomic locus (SnSRS7 on scaffold 4) existed as a tan-dem array of duplicated paralogs The 23 SRS-encoding geneswere made up of 75 putative SRS domains (Fig 5B) Of note 63(84) of these 75 domains were associated with family 2 (fam2)domains including SRS7A which contained 26 fam2 domains Ingeneral each SRS protein possessed either one or two SRS do-mains with individual domains classifiable into one of the eightpreviously defined families although no fam5 domains were iden-tified The 26-fam2-domain SRS7A protein genomic locus alsocontained several gaps bordered by nucleic acids with which ahigh number of reads could be aligned This might indicate repet-itive elements that could promote domain expansion within thislocus through ectopic recombination Interestingly the SRS7Aprotein fam2 domains possessed the highest sequence similarity tothe 13 fam2 domains encoded by TgSRS44 a protein previouslyimplicated as an integral structural constituent of the T gondii cystwall (43) TgSRS44 also possesses a mucin domain which has beenshown to be highly glycosylated and is thought to protect the cystfrom immune recognition andor dehydration However we didnot identify any mucin domains in our S neurona homolog Onlyfour SRS proteins possessed either fam7 or fam8 domains (oneand three copies respectively) in contrast to T gondii and N cani-num where the majority of SRS proteins possess one or the otherof these two fam domains Previous work suggested that the rela-tive expansion of fam7 and fam8 domains in T gondii and N cani-num is linked to their role in host specificity (7) Other noteworthyfeatures include unique combinations of a fam1 domain with afam8 domain (SnSRS1) and a fam3 domain with a fam6 domain(SnSRS16) which likely promote specific cell recognition eventsfor S neurona

RNA-Seq data identified at least seven SRS proteins express-ed in merozoites which was confirmed by TaqMan reversetranscription-PCR (Fig 5C) The three most abundantly ex-pressed SnSRS proteins were SnSRS12 (SnSAG3) SnSRS8(SnSAG2) and SnSRS4 (SnSAG4) as has been observed previ-ously (44) Importantly the SO SN1 strain did not expressSnSRS10 (SnSAG1) SnSRS10 is a highly immunogenic proteinand is the major surface antigen expressed on the SN3 strain (45)which explains the high number of SN3-derived expressed se-

FIG 4 SnAMA1a and SnAMA1b accessorize the canonical AMA1 DI and DII domains with unique features but maintain an apical surface capable ofcoordinating SnRON2D3 (A) Secondary-structure (left) and surface representations of SnAMA1a DI (purple) and DII (orange) five conserved disulfides andtwo extra cysteines in the DII loop are highlighted as ball-and-stick structures Two cysteines predicted to form a disulfide at the DII loop hinge are shown as aball-and-stick structure (black arrow) Residues anchoring the DII loop are labeled and surround a central Leu residue colored yellow (B) Surface representationsof SnAMA1b colored and labeled as for SnAMA1a (C) Surface representation of TgAMA1 (Protein Data Bank [PDB] accession no 2x2z) DI light grey DII darkgrey (D) Complementary views of the TgAMA1-TgRON2sp costructure (PDB accession no 2y8t) with TgAMA1 colored light grey (DI) or dark grey (DII) andTgRON2sp colored cyan (E) Complementary views of the SnAMA1a-SnRON2D3 costructure model with SnAMA1 colored as in panel A and SnRON2D3 ingreen Residues making up the RON2 cystine loop tip are shown as ball-and-stick structures to highlight shape complementarity

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FIG 5 Coccidian-specific protein families implicated in virulence and host range determination (A) Maximum-likelihood-based phylogenetic tree of theROPK family Values indicate the bootstrap support (of 1000 replicates) S neurona members are red T gondii members are dark blue N caninum membersare cyan T gondii and N caninum clades are blue E tenella members and clades are yellow (B) Summary of the 23 SRS family members identified in the Sneurona genome Relative expression in the S neurona merozoite stage are provided as FPKM values and domain architectures are indicated ESTs number ofexpressed sequence tags (C) The expression of the SnSRS-encoding genes was assessed by TaqMan qPCR Genes were sorted in descending order by theirexpression levels

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quence tags that mapped to SnSRS10 which was transcriptionallysilent in this study (Fig 5)

Reconstruction and analysis of S neurona metabolism re-veal the potential to exploit alternative sources of energy S neu-rona has 372 metabolic enzymes (unique enzyme classification[EC] numbers excluding those involved in nonmetabolic reac-tions) in common with T gondii but is missing 42 enzymes andhas an additional 13 enzymes that are expressed by RNA-Seq inthe merozoite stage (Fig 6A see Text S1 in the supplemental ma-terial) Our analyses predict putative T gondii orthologs for 12 ofthese enzymes including the fatty acid elongation genes very-long-chain 3-oxoacyl coenzyme A synthase (EC 231199TGME49_205350) and very-long-chain (3R)-3-hydroxyacyl acylcarrier protein dehydratase (EC 421134 TGME49_311290)Only threonine ammonia-lyase (EC 43119) is unique and addsfunctionality to S neurona

We incorporated these differences into our previously pub-lished metabolic reconstruction of T gondii named iCS382 (8)and performed flux balance analyses of both iCS382 and the mod-ified S neurona reconstruction Scaling the iCS382 model to pro-duce a doubling time of 118 h with glucose as the sole energysource (see Text S1 in the supplemental material) we show that Sneurona has a slightly longer doubling time of 138 h Single-reaction knockouts identified 22 reactions whose deletion resultedin a significantly greater impact on S neurona than on T gondii(20 maximal growth rate difference) (Fig 6B see Table S2 inthe supplemental material) Critical reactions include members ofthe pentose phosphate and glycolysis pathways the tricarboxylicacid (TCA) cycle and two members of the pyrimidine biosyn-thetic pathway nucleoside-diphosphate kinase (EC 2746) andcytidylate kinase (EC 27414) Conversely we identified only asingle reaction catalyzed by pyruvate dehydrogenase whose de-letion had a significantly greater impact on T gondii than on Sneurona (20 maximal growth rate difference)

The S neurona annotation effort predicted a gene for alpha-glucosidase (EC 32120) (see Text S1 and Fig S4 in the supple-mental material) Since conversion of sucrose to fructose and glu-cose by alpha-glucosidase would add functionality to themetabolic reconstruction we tested in silico for its potential im-pact on growth S neurona was predicted to grow faster in thepresence of sucrose and the absence of glucose than in the pres-ence of glucose and the absence of sucrose (doubling time of 114 hversus 138 h) This is due in part to an increase in the concen-tration of fructose-6-phosphate caused by the action of hexoki-nase (EC 2711 Fig 6C) Consequently under conditions of su-crose uptake knockout of enzymes involved in glycolysis has agreater impact on the growth rate than conditions of glucose up-take (see Table S2 in the supplemental material) When we exam-ined the impact of combining access to different carbohydratesour simulations suggested that S neurona has the capacity to sig-nificantly enhance its growth by utilizing fructose with an evengreater effect when sucrose is used as an additional energy source(Fig 6D) For example while fructose supplementation alters par-asite growth to 120 supplementation with sucrose extends par-asite growth to 180 of its original rate Interestingly glucose-6-phosphate isomerase the enzyme responsible for the conversionof glucose-6-phosphate to fructose-6-phosphate operates in thereverse direction under glucose or sucrose uptake conditionsWhen only sucrose is available more glucose-6-phosphate is pro-duced from the conversion of fructose-6-phosphate which is pre-

dicted to feed into other pathways (eg the pentose phosphatepathway) resulting in the elevated production of NADPH and anincreased growth rate Importantly glycolysis is utilized morewhen sucrose is available so there is less reliance on the TCA cycleFurthermore the breakdown of sucrose makes fructose availablefor the synthesis of other key metabolites (eg branched-chainamino acids) decreasing the parasitersquos dependency on the TCAcycle for their production Hence the deletion of individual TCAcycle reactions has a greater impact on the growth rate in thepresence of glucose than in the presence of sucrose (Fig 6C)

DISCUSSION

Coccidian parasites represent a major clade within the phylumApicomplexa and the genomes of three species E tenella T gon-dii and N caninum have already been sequenced (7 21) S neu-rona is the first genome in the genus Sarcocystis to be sequencedThe 127-Mbp genome is more than twice the size of other se-quenced coccidian genomes largely because of a high proportionof repetitive LINEs and DNA elements The organization of the Sneurona genome into 116 genomic scaffolds produces the firstmolecular karyotype or physical linkage map which shouldgreatly facilitate future genetic and comparative genomic studiesof this important genus Sarcocystis chromosomes do not con-dense nor have they been resolved by pulse-field gel electropho-resis Our comparative genomic transcriptomic and metabolicflux data analyses show that the invasion machinery is largely con-served among the coccidia but that the tissue cyst-forming coc-cidia have evolved families of dense-granule (GRA) ROPK andsurface-associated SRS adhesins that promote their ability to per-sist chronically in cyst-like structures or disrupt the induction ofsterilizing immunity representing novel molecular strategies thatfacilitate their transition from largely enteric pathogens within asingle host (Eimeria) to heteroxenous pathogens that cycle be-tween a definitive host and an intermediate host(s) (Sarcocystis)

Genome comparisons reveal that S neurona has more or-thologs in common with T gondii than with E tenella (3169 ver-sus 1759 orthologs respectively) supporting the notion that theEimeria lineage is more divergent However S neurona is alsoquite distinct from T gondii it possesses only limited genomicsynteny restricted to only dozens of genes and additionally en-codes 1285 (18) genes with no detectable homology to anyother species As in E tenella LINEs and DNA elements are pres-ent in S neurona but the DNA elements are significantly ex-panded in S neurona partially accounting for its increased ge-nome size The presence of the LINEs and DNA elementshowever is not associated with gene model misannotation sinceLINEs are as frequently associated with T gondii orthologs(133) as they are with unique genes (116) indicating thatthey may drive genome innovations within S neurona (46) Wedid not find any examples of the coronavirus-like long terminalrepeat (LTR) element previously associated with the E tenella ge-nome (21) strengthening the suggestion that this element wasacquired by horizontal gene transfer within that lineage

The Sarcocystis invasion machinery was largely conservedwithin the coccidia and the construction of the S neurona inter-action network based on gene expression data identified two mainclusters of conservation one composed largely of MIC AMA1and RON proteins required for the mechanics of cell attachmentand invasion and another composed of a limited set of ROP andGRA proteins thought to alter host immune effector function

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FIG 6 Metabolic reconstruction and analysis of S neurona based on iCS382 (A) Overlap in enzyme predictions for genes from S neurona T gondii andP falciparum (B) Species-specific differences in growth rates of single-reaction knockouts Only reactions that show a growth rate difference of 20 betweenT gondii and S neurona are shown (C) Impact of deletion of reactions involved in glycolysis and the TCA cycle on S neurona growth under conditions ofexclusive glucose or sucrose uptake D-glc-6-P D-glucose-6-phosphate G6PI glucose-6-phosphate isomerase D-frc-6-P D-fructose-6-phosphate DF6P1P

(Continued)

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However the complement of the latter ROP and GRA proteins isgreatly reduced compared to that of other tissue cyst-forming coc-cidia such as Toxoplasma or Neospora While mouse models haveshown ROP5 and ROP18 which are absent from S neurona toimpact virulence in Toxoplasma the lack of a suitable such modelie immunocompetent mice for S neurona means that little isknown about its strain virulence determinants Additionally allstrains induce fatal encephalitis in immunodeficient mice irre-spective of the dose Only two ROPKs were conserved withE tenella implying specialization in the ROPK machinery re-quired for the different life cycles Hence the reduced comple-ment of ROPKs within the S neurona genome likely underscoresthe important role the expanded repertoire of ROPKs plays inpromoting Toxoplasma and Neospora host and niche adaptationamong the susceptible hosts in which these parasites establishtransmissible infections Likewise the distinctive set of ROPKspreviously reported for E tenella and thought to map to the sporo-zoite rhoptry (47) might suggest a specialized role for these pro-teins during the initial establishment of infection

Consistent with a transition from a strictly enteric coccidianpathogen to a tissue-invasive one capable of establishing long-term chronic infection by encystment within host cells S neu-rona expresses a distinct surface antigen coat of SRS proteins thatpromote parasite recognition attachment and long-term encyst-ment within host cells to promote transmissibility of infection Incomparison to T gondii and N caninum however the S neuronaSRS protein repertoire is surprisingly small and less divergent(25) there is a dramatic reduction in the number of SRS proteinscomposed of fam7 and fam8 domains with the vast majority ofthe 23 SnSRS-encoding genes composed of fam2 domains Previ-ous studies of T gondii suggest that proteins composed of theformer domains modulate host immune responses and mediatecritical roles in parasite virulence Our data suggest that with onlya single copy of a fam7- and fam8-containing SRS protein S neu-rona has evolved other mechanisms for control of immune acti-vation andor that such control is not required for the successfultransmission of this highly prevalent protozoan pathogen Thelatter point is consistent with observed differences between the Sneurona and ToxoplasmaNeospora life cycles Sarcocystis spponce encysted undergo a terminal commitment to their gamontstage requiring access to their definitive host to complete their lifecycle In contrast both Toxoplasma and Neospora are capable ofrecrudescing their infection after encystation and expansion offam7 and fam8 domain SRS proteins capable of altering host pro-tective immunity may function to increase the cyst burden or alterintermediate-host behavior promoting transmission of the para-site to the definitive host to complete its life cycle AlternativelySarcocystis spp are exclusively restricted to sexual developmentwithin the intestine of the definitive host whereas Toxoplasmainfection of its felid definitive host results in both sexual develop-ment and asexual expansion of infection so the expanded reper-toire of fam7 and fam8 domain SRS proteins may promote dis-semination of infection to a wide range of tissue and cell types andvaccinate the definitive host against reinfection

The sheer dominance of the fam2 domain proteins among thelimited repertoire of SnSRS proteins suggests that they play a crit-ical functional role in the life cycle A recent study (43) found thatTgSRS44 (CST1) a T gondii SRS protein with 13 tandemly re-peated fam2 domains is an important structural constituent ofthe cyst wall suggesting that the emergence of fam2 SnSRSdomain-containing proteins in the common ancestor of S neu-rona and T gondii likely provided the parasite with the ability toform cysts thereby extending its host range and promoting thetransition to a heteroxenous (two-host) life cycle Strains of Sneurona are known to exhibit important differences in the immu-nodominant SnSRS-encoding genes that they possess SnSRS10(SnSAG1) is encoded by an immunodominant gene present andexpressed abundantly in some S neurona isolates but absent fromothers (45) While the type II SO SN1 strain sequenced encodesSnSRS10 it does not express it during merozoite growth (Fig 5)whereas SN3 another type II isolate highly expresses this proteinWhile the mechanism of gene regulation within the SnSRS familyhas yet to be elucidated it may influence the host range the ca-pacity to promote coinfection andor pathogenicity among thebroad intermediate-host range of S neurona much the same waydifferential expression of TgSRS2 alters the parasite load and thepathology of T gondii infection in mice (4) Importantly a highprevalence of coinfection with different genetic types of S neuronawithin intermediate hosts would promote outcrossing during sex-ual reproduction Outcrossing in Toxoplasma has previously beenshown to produce progeny possessing altered biological poten-tials including virulence and a capacity to cause outbreaks (48)which has recently also been established for S neurona (15)

Finally regulation of energy production has likewise evolved asa strategy for parasites to extend their host range by tuninggrowth in relation to the host burden or carrying capacity (8) Wefound only a limited number of differences between the enzymecomplements of T gondii and S neurona Notably S neuronapossesses 13 enzymes not present in T gondii and a homolog of analpha-glucosidase (EC 32120) that preferentially gives S neu-rona the potential to use alternative carbon sources to help drivegrowth Hence our flux balance analysis showed that S neurona isless reliant on the TCA cycle when it is grown in the presence ofsucrose and that sucrose supplementation can increase parasitegrowth to 180 of its original rate a capability that may be im-portant for allowing the parasite to exploit new host niches Thesefindings serve to highlight subtle differences in pathway utiliza-tion that the two parasites may have adopted to optimize theirdistinct life cycle strategies

Together our data support a model in which following the splitwith the Eimeria lineage the ancestor of Sarcocystis andToxoplasma gained the ability to invade intermediate hosts and formtissue cysts This transition required the evolution of SRS family pro-teins as structural constituents of the cyst wall as well as immuneevasion molecules protecting the parasite from sterilizing immunitySubsequently while the Sarcocystis lineage abandoned the use of thePV during its schizont stage in the intermediate host committing theparasite to its sexual cycle after encystation the Toxoplasma lineage

Figure Legend Continued

diphosphate-fructose-6-phosphate 1-phosphotransferase FBA fructose-bisphosphate aldolase PK pyruvate kinase Cyt cytosol Mito mitochondrion PCpyruvate carboxylase CS citrate synthase AH aconitate hydratase ID isocitrate dehydrogenase (NADP) OD oxoglutarate dehydrogenase (succinyl-transferring) DS dihydrolipoyllysine-residue succinyltransferase SL succinate-CoA ligase (ADP-forming) SD succinate dehydrogenase (ubiquinone) FHfumarate hydratase MD malate dehydrogenase frc fructose (D) Relationship between fructose glucose and sucrose import and growth

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maintained the use of a PV during intermediate-host infection Theuse of the PV could conceivably shield the parasite from the hostdeveloping an effector memory CD8 T cell response that is naturallyinduced by the presence of parasite antigens in the cytosol of infectedhost cells This in turn allows Toxoplasma to recrudesce postencys-tation and aided by an expanded repertoire of ROPK GRA and SRSproteins provides further opportunities to increase the cyst burdenand extend its host range In addition to addressing questions of hostrange and specificity we expect that the availability of this resourcewill help drive the development of novel therapeutics that are ur-gently required for these devastating pathogens Further referencegenome mapping will facilitate genus-wide and population studiesthat focus on questions of host specialization and virulence mecha-nisms The latter for example might be expected to inform on thespate of fatal infections in marine mammals to resolve at the genomelevel the genetic basis of the emergence of these disease-producingstrains Key to these studies will be the generation of robust expres-sion data sets that allow the identification of critical proteins associ-ated with distinct stages of the parasitersquos life cycle

MATERIALS AND METHODSCulturing of parasites extraction of DNARNA and sequencing S neu-rona strain SO SN1 was isolated from a southern sea otter (19) and ob-tained from Patricia Conrad University of California Davis CA S neu-rona parasites were maintained in MA-104 cells as described previously(49) Genomic DNA was extracted from frozen pellets of S neurona SOSN1 by proteinase K digestion and subsequence phenol-chloroform extrac-tion Five libraries were prepared two Roche 454 Shotgun GS-Titanium li-braries prepared in accordance with the Rapid Library Preparation MethodManual (Roche) a Roche 454 8-kb paired-end GS-Titanium Library pre-pared in accordance with the Paired-End Library Preparation Method Man-ual with the modification of setting up four circularization reactions to in-crease the final library yield an Illumina 2- to 3-kb mate pair librarysynthesized with the TruSeq DNA sample prep kit (Illumina) and run on anIllumina GA IIx and a Nextera 8- to 15-kb mate pair library prepared inaccordance with the manufacturerrsquos recommendations and run on an Illu-mina HiSeq 2000 These sequencing efforts generated 7020033 shotgunreads 5919255 shotgun reads 1100788 paired-end reads 128614194mate pair reads and 136301151 mate pair reads respectively S neurona SOSN1 RNA was isolated from merozoites with the RNeasy minikit (Qiagen)snap-frozen and stored at 80degC A single TruSeq v2 RNA library (mRNAenriched) was prepared for Illumina sequencing by the standard Illuminaprotocol and used to generate 59622019 reads For further details of genomeassembly and annotation as well as bioinformatics and experimental analy-ses see Text S1 in the supplemental material

Nucleotide sequence accession number Further information on theS neurona genome project including sequence files is available throughthe bioproject repository at the National Center for Biotechnology Infor-mation (httpwwwncbinlmnihgovbioproject252030) using the ac-cession number SRP052925

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at httpmbioasmorglookupsuppldoi101128mBio02445-14-DCSupplemental

Text S1 PDF file 03 MBFigure S1 PDF file 06 MBFigure S2 PDF file 03 MBFigure S3 PDF file 03 MBFigure S4 PDF file 02 MBFigure S5 PDF file 13 MBTable S1 XLS file 18 MBTable S2 PDF file 03 MBTable S3 XLSX file 004 MBTable S4 PDF file 02 MB

ACKNOWLEDGMENTS

This study was financially supported by the Canadian Institute for HealthResearch (CIHR-MOP 84556 to JP and MEG) and the Intramural Re-search Program of the NIH and NIAID (MEG) MJB was supported bya discovery grant from the Natural Sciences and Engineering ResearchCouncil of Canada (NSERC) MEG is a scholar of the Canadian Institutefor Advanced Research (CIFAR) Integrated Microbial Biodiversity Pro-gram DKH and JCK were supported by a grant from the USDA NIFA(2009-65109-05918) DKH was additionally supported by the Amer-man Family Equine Research Fund

We gratefully acknowledge the assistance of J Wendte M QuinonesA Kennard A Khan D Bruno S Anzick C Martens and E Dahlstromin DNA sequencing and assembly We also thank Patricia Conrad for theSO SN1 strain and thank the University of Toronto SciNet facility for theuse of its computing resources

REFERENCES1 Cowper B Matthews S Tomley F 2012 The molecular basis for the

distinct host and tissue tropisms of coccidian parasites Mol BiochemParasitol 1861ndash10 httpdxdoiorg101016jmolbiopara201208007

2 Dubey JP Lindsay DS Saville WJ Reed SM Granstrom DE Speer CA2001 A review of Sarcocystis neurona and equine protozoal myeloenceph-alitis (EPM) Vet Parasitol 9589 ndash131 httpdxdoiorg101016S0304-4017(00)00384-8

3 Dubey JP Speer CA Fayer R 1988 Sarcocystosis of animals and manCRC Press Boca Raton FL

4 Wasmuth JD Pszenny V Haile S Jansen EM Gast AT Sher A BoyleJP Boulanger MJ Parkinson J Grigg ME 2012 Integrated bioinfor-matic and targeted deletion analyses of the SRS gene superfamily identifySRS29C as a negative regulator of Toxoplasma virulence mBio 3(6)e00321-12 httpdxdoiorg101128mBio00321-12

5 Sibley LD 2011 Invasion and intracellular survival by protozoan para-sites Immunol Rev 24072ndash91 httpdxdoiorg101111j1600-065X201000990x

6 Besteiro S Dubremetz JF Lebrun M 2011 The moving junction ofapicomplexan parasites a key structure for invasion Cell Microbiol 13797ndash 805 httpdxdoiorg101111j1462-5822201101597x

7 Reid AJ Vermont SJ Cotton JA Harris D Hill-Cawthorne GA Koumlnen-Waisman S Latham SM Mourier T Norton R Quail MA Sanders MShanmugam D Sohal A Wasmuth JD Brunk B Grigg ME Howard JCParkinson J Roos DS Trees AJ Berriman M Pain A Wastling JM2012 Comparative genomics of the apicomplexan parasites Toxoplasmagondii and Neospora caninum coccidia differing in host range and trans-mission strategy PLoS Pathog 8e1002567 httpdxdoiorg101371journalppat1002567

8 Song C Chiasson MA Nursimulu N Hung SS Wasmuth J Grigg MEParkinson J 2013 Metabolic reconstruction identifies strain-specific reg-ulation of virulence in Toxoplasma gondii Mol Syst Biol 9708 httpdxdoiorg101038msb201362

9 Levine ND 1986 The taxonomy of Sarcocystis (Protozoa Apicomplexa)species J Parasitol 72372ndash382 httpdxdoiorg1023073281676

10 Lindsay DS Mitchell SM Vianna MC Dubey JP 2004 Sarcocystisneurona (Protozoa Apicomplexa) description of oocysts sporocystssporozoites excystation and early development J Parasitol 90461ndash 465httpdxdoiorg101645GE-230R

11 Fenger CK Granstrom DE Langemeier JL Stamper S Donahue JMPatterson JS Gajadhar AA Marteniuk JV Xiaomin Z Dubey JP 1995Identification of opossums (Didelphis virginiana) as the putative definitivehost of Sarcocystis neurona J Parasitol 81916 ndash919 httpdxdoiorg1023073284040

12 Dubey JP Chapman JL Rosenthal BM Mense M Schueler RL 2006Clinical Sarcocystis neurona Sarcocystis canis Toxoplasma gondii andNeospora caninum infections in dogs Vet Parasitol 13736 ndash 49 httpdxdoiorg101016jvetpar200512017

13 Dubey JP Saville WJ Stanek JF Lindsay DS Rosenthal BM OglesbeeMJ Rosypal AC Njoku CJ Stich RW Kwok OC Shen SK Hamir ANReed SM 2001 Sarcocystis neurona infections in raccoons (Procyon lotor)evidence for natural infection with sarcocysts transmission of infection toopossums (Didelphis virginiana) and experimental induction of neuro-logic disease in raccoons Vet Parasitol 100117ndash129 httpdxdoiorg101016S0304-4017(01)00500-3

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14 Miller MA Conrad PA Harris M Hatfield B Langlois G Jessup DAMagargal SL Packham AE Toy-Choutka S Melli AC Murray MAGulland FM Grigg ME 2010 A protozoal-associated epizootic impact-ing marine wildlife mass-mortality of southern sea otters (Enhydra lutrisnereis) due to Sarcocystis neurona infection Vet Parasitol 172183ndash194httpdxdoiorg101016jvetpar201005019

15 Wendte JM Miller MA Lambourn DM Magargal SL Jessup DA GriggME 2010 Self-mating in the definitive host potentiates clonal outbreaksof the apicomplexan parasites Sarcocystis neurona and Toxoplasmagondii PLoS Genet 6e1001261 httpdxdoi org101371journalpgen1001261

16 Dubey JP Saville WJ Lindsay DS Stich RW Stanek JF Speert CARosenthal BM Njoku CJ Kwok OC Shen SK Reed SM 2000 Completionof the life cycle of Sarcocystis neurona J Parasitol 861276ndash1280 httpdxdoiorg1016450022-3395(2000)086[1276COTLCO]20CO2

17 Miller MA Barr BC Nordhausen R James ER Magargal SL MurrayM Conrad PA Toy-Choutka S Jessup DA Grigg ME 2009 Ultrastruc-tural and molecular confirmation of the development of Sarcocystis neu-rona tissue cysts in the central nervous system of southern sea otters (En-hydra lutris nereis) Int J Parasitol 391363ndash1372 httpdxdoiorg101016jijpara200904014

18 Gibson AK Raverty S Lambourn DM Huggins J Magargal SL GriggME 2011 Polyparasitism is associated with increased disease severity inToxoplasma gondii-infected marine sentinel species PLoS Negl Trop Dis5e1142 httpdxdoiorg101371journalpntd0001142

19 Miller MA Crosbie PR Sverlow K Hanni K Barr BC Kock N MurrayMJ Lowenstine LJ Conrad PA 2001 Isolation and characterization ofSarcocystis from brain tissue of a free-living southern sea otter (Enhydralutris nereis) with fatal meningoencephalitis Parasitol Res 87252ndash257httpdxdoiorg101007s004360000340

20 Smit AFA Hubley R 2008 ndash2010 posting date RepeatModeler Open-10 Inst i tute for Systems Biology Seat t le WA http wwwrepeatmaskerorg

21 Reid AJ Blake DP Ansari HR Billington K Browne HP Dunn MHung SS Kawahara F DM-S Malas TB Mourier TB Nagra H Nair MOtto TD Rawlings ND Rivailler P Sanchez-Flores A Sanders MSubramaniam C Tay Y-LL Wu X Dear PH Doerig C Gruber A IvensAC Parkinson J Shirley MW Wan K-L Berriman M Tomley FM PainA 2014 Genomic analysis of the causative agents of coccidiosis in domes-tic chickens Genome Res 241676 ndash1685 httpdxdoiorg101101gr168955113

22 Wilson RJ Denny PW Preiser PR Rangachari K Roberts K Roy AWhyte A Strath M Moore DJ Moore PW Williamson DH 1996Complete gene map of the plastid-like DNA of the malaria parasite Plas-modium falciparum J Mol Biol 261155ndash172 httpdxdoiorg101006jmbi19960449

23 Brayton KA Lau AO Herndon DR Hannick L Kappmeyer LS BerensSJ Bidwell SL Brown WC Crabtree J Fadrosh D Feldblum TForberger HA Haas BJ Howell JM Khouri H Koo H Mann DJBrayton KA Lau AO Herndon DR Hannick L Kappmeyer LS BerensSJ Bidwell SL Brown WC Crabtree J Fadrosh D Feldblum TForberger HA Haas BJ Howell JM Khouri H Koo H Mann DJNorimine J Paulsen IT Radune D Ren Q Smith RK Jr Suarez CEWhite O Wortman JR Knowles DP Jr McElwain TF Nene VM 2007Genome sequence of Babesia bovis and comparative analysis of apicom-plexan hemoprotozoa PLoS Pathog 3-1401ndash1413 httpdxdoiorg101371journalppat0030148

24 Aurrecoechea C Barreto A Brestelli J Brunk BP Cade S Doherty RFischer S Gajria B Gao X Gingle A Grant G Harb OS Heiges M HuS Iodice J Kissinger JC Kraemer ET Li W Pinney DF Pitts B RoosDS Srinivasamoorthy G Stoeckert CJ Jr Wang H Warrenfeltz S2013 EuPathDB the eukaryotic pathogen database Nucleic Acids Res41D684 ndashD691 httpdxdoiorg101093nargks1113

25 Wasmuth J Daub J Peregriacuten-Alvarez JM Finney CA Parkinson J2009 The origins of apicomplexan sequence innovation Genome Res191202ndash1213 httpdxdoiorg101101gr083386108

26 Jordan IK Katz LS Denver DR Streelman JT 2008 Natural selectiongoverns local but not global evolutionary gene coexpression networks inCaenorhabditis elegans BMC Syst Biol 296 httpdxdoiorg1011861752-0509-2-96

27 Peregriacuten-Alvarez JM Xiong X Su C Parkinson J 2009 The modularorganization of protein interactions in Escherichia coli PLOS Comput Biol5e1000523 httpdxdoiorg101371journalpcbi1000523

28 Hu G Cabrera A Kono M Mok S Chaal BK Haase S Engelberg KCheemadan S Spielmann T Preiser PR Gilberger TW Bozdech Z2010 Transcriptional profiling of growth perturbations of the humanmalaria parasite Plasmodium falciparum Nat Biotechnol 2891ndash98httpdxdoiorg101038nbt1597

29 Speer CA Dubey JP 2001 Ultrastructure of schizonts and merozoites ofSarcocystis neurona Vet Parasitol 95263ndash271 httpdxdoiorg101016S0304-4017(00)00392-7

30 Hoane JS Carruthers VB Striepen B Morrison DP Entzeroth R HoweDK 2003 Analysis of the Sarcocystis neurona microneme proteinSnMIC10 protein characteristics and expression during intracellular de-velopment Int J Parasitol 33671ndash 679 httpdxdoiorg101016S0020-7519(03)00031-6

31 Reiss M Viebig N Brecht S Fourmaux M-N Soete M Di Cristina MDubremetz JF Soldati D 2001 Identification and characterization of anEscorter for two secretory adhesins in Toxoplasma gondii J Cell Biol 152563ndash578 httpdxdoiorg101083jcb1523563

32 Boothroyd JC Dubremetz JF 2008 Kiss and spit the dual roles ofToxoplasma rhoptries Nat Rev Microbiol 679 ndash 88 httpdxdoiorg101038nrmicro1800

33 Lamarque MH Papoin J Finizio AL Lentini G Pfaff AW Candolfi EDubremetz JF Lebrun M 2012 Identification of a new rhoptry neckcomplex RON9RON10 in the Apicomplexa parasite Toxoplasma gondiiPLoS One 7e32457 httpdxdoiorg101371journalpone0032457

34 Kono M Herrmann S Loughran NB Cabrera A Engelberg K Leh-mann C Sinha D Prinz B Ruch U Heussler V Spielmann T Parkin-son J Gilberger TW 2012 Evolution and architecture of the inner mem-brane complex in asexual and sexual stages of the malaria parasite MolBiol Evol 292113ndash2132 httpdxdoiorg101093molbevmss081

35 Crawford J Tonkin ML Grujic O Boulanger MJ 2010 Structuralcharacterization of apical membrane antigen 1 (AMA1) from Toxoplasmagondii J Biol Chem 28515644 ndash15652 httpdxdoiorg101074jbcM109092619

36 Tonkin ML Crawford J Lebrun ML Boulanger MJ 2013 Babesiadivergens and Neospora caninum apical membrane antigen 1 structuresreveal selectivity and plasticity in apicomplexan parasite host cell invasionProtein Sci 22114 ndash127 httpdxdoiorg101002pro2193

37 Tonkin ML Roques M Lamarque MH Pugniegravere M Douguet DCrawford J Lebrun M Boulanger MJ 2011 Host cell invasion by api-complexan parasites insights from the co-structure of AMA1 with aRON2 peptide Science 333463ndash 467 httpdxdoiorg101126science1204988

38 Vulliez-Le Normand B Tonkin ML Lamarque MH Langer S Hoos SRoques M Saul FA Faber BW Bentley GA Boulanger MJ Lebrun M2012 Structural and functional insights into the malaria parasite movingjunction complex PLoS Pathog 8e1002755 httpdxdoiorg101371journalppat1002755

39 Talevich E Kannan N 2013 Structural and evolutionary adaptationof rhoptry kinases and pseudokinases a family of coccidian virulencefactors BMC Evol Biol 13117 httpdxdoiorg1011861471-2148-13-117

40 Rosowski EE Lu D Julien L Rodda L Gaiser RA Jensen KD Saeij JP2011 Strain-specific activation of the NF-kappaB pathway by GRA15 anovel Toxoplasma gondii dense granule protein J Exp Med 208195ndash212httpdxdoiorg101084jem20100717

41 Braun L Brenier-Pinchart MP Yogavel M Curt-Varesano A Curt-Bertini RL Hussain T Kieffer-Jaquinod S Coute Y Pelloux H Tar-dieux I Sharma A Belrhali H Bougdour A Hakimi MA 2013 AToxoplasma dense granule protein GRA24 modulates the early immuneresponse to infection by promoting a direct and sustained host p38 MAPKactivation J Exp Med 2102071ndash2086 httpdxdoiorg101084jem20130103

42 Shastri AJ Marino ND Franco M Lodoen MB Boothroyd JC 2014Gra25 is a novel virulence factor of Toxoplasma gondii and influences thehost immune response Infect Immun 822595ndash2605 httpdxdoiorg101128IAI01339-13

43 Tomita T Bzik DJ Ma YF Fox BA Markillie LM Taylor RC Kim KWeiss LM 2013 The Toxoplasma gondii cyst wall protein CST1 is criticalfor cyst wall integrity and promotes bradyzoite persistence PLoS Pathog9e1003823 httpdxdoiorg101371journalppat1003823

44 Howe DK Gaji RY Mroz-Barrett M Gubbels MJ Striepen B StamperS 2005 Sarcocystis neurona merozoites express a family of immunogenic

Genome Sequence of Sarcocystis neurona

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surface antigens that are orthologues of the Toxoplasma gondii surfaceantigens (SAGs) and SAG-related sequences Infect Immun 731023ndash1033 httpdxdoiorg101128IAI7321023-10332005

45 Howe DK Gaji RY Marsh AE Patil BA Saville WJ Lindsay DS DubeyJP Granstrom DE 2008 Strains of Sarcocystis neurona exhibit differencesin their surface antigens including the absence of the major surface anti-gen SnSAG1 Int J Parasitol 38623ndash 631 httpdxdoiorg101016jijpara200709007

46 Toll-Riera M Radoacute-Trilla N Martys F Albagrave MM 2012 Role oflow-complexity sequences in the formation of novel protein codingsequences Mol Biol Evol 29883ndash 886 httpdxdoiorg101093molbevmsr263

47 Oakes RD Kurian D Bromley E Ward C Lal K Blake DP Reid AJPain A Sinden RE Wastling JM Tomley FM 2013 The rhoptry pro-teome of Eimeria tenella sporozoites Int J Parasitol 43181ndash188 httpdxdoiorg101016jijpara201210024

48 Grigg ME Bonnefoy S Hehl AB Suzuki Y Boothroyd JC 2001 Successand virulence in Toxoplasma as the result of sexual recombination be-tween two distinct ancestries Science 294161ndash165 httpdxdoiorg101126science1061888

49 Miller MA Sverlow K Crosbie PR Barr BC Lowenstine LJ GullandFM Packham A Conrad PA 2001 Isolation and characterization oftwo parasitic protozoa from a Pacific harbor seal (Phoca vitulina rich-ardsi) with meningoencephalomyelitis J Parasitol 87816 ndash 822 httpdxdoiorg1016450022-3395(2001)087[0816IACOTP]20CO2

50 Krzywinski M Schein J Birol I Connors J Gascoyne R Horsman DJones SJ Marra MA 2009 Circos an information aesthetic for compar-ative genomics Genome Res 191639 ndash1645 httpdxdoiorg101101gr092759109

51 Berglund AC Sjoumllund E Ostlund G Sonnhammer EL 2008 InParanoid6 eukaryotic ortholog clusters with inparalogs Nucleic Acids Res 36D263ndashD266 httpdxdoiorg101093nargkm1020

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