The transcriptome of Schistosoma mansoni – insight into the biology of a primitive metazoan

Emmanuel Dias Neto

Universidade de São Paulo, São Paulo, Brasil

emmanuel@usp.br

The transcriptome The transcriptome of Schistosoma mansoni:of Schistosoma mansoni:insights into the biology insights into the biology of a primitive metazoanof a primitive metazoan

UNESCOUNESCOFirst SouthFirst South--North Human Genome North Human Genome

Conference. Conference. 12 a 15 de maio de 1992.12 a 15 de maio de 1992.CaxambCaxambúú, MG , MG -- BRAZILBRAZIL

UNESCO UNESCO -- First NorthFirst North--South Human Genome South Human Genome

ConferenceConference

•• Is there a way to integrate the Is there a way to integrate the research performed in developing research performed in developing countries with the US/Europe countries with the US/Europe ‘Human Genome Project’ ?‘Human Genome Project’ ?

•• After the completion of the ‘Human After the completion of the ‘Human Genome sequencing’, how can we Genome sequencing’, how can we gain access or make use of the gain access or make use of the technology developed ?technology developed ?

•• InIn orderorder toto be ablebe able to exploreto explore the human the human genomegenome data,data, we should be ablewe should be able to do genomicsto do genomics

•• ThereThere is no timeis no time--spacespace, , nor technology nor technology availableavailable, for , for third world countries third world countries to to generategeneratesignificantsignificant data for data for the completion the completion of of the the human genome sequencinghuman genome sequencing

•• We should do gWe should do genomics using organisms enomics using organisms of of local local importanceimportance

How can we learn ?How can we learn ?

•• Initiate an EST sequencing project of a Initiate an EST sequencing project of a parasite of local importance parasite of local importance ((SchistosomaSchistosoma mansonimansoni))

•• cDNA libraries prepared with Marcelo cDNA libraries prepared with Marcelo Bento SoaresBento Soares

•• cDNA sequencing performed at TIGR cDNA sequencing performed at TIGR (Craig Venter)(Craig Venter)

•• Some 1,000 ESTs generated (Some 1,000 ESTs generated (GlóriaGlóriaFranco, UFMG)Franco, UFMG)

Strategies Strategies for EST for EST generation generation

•• Conventional random selection Conventional random selection of cDNA of cDNA clones fromclones from directionaldirectional cDNA librariescDNA libraries

•• Development Development of of an alternative technique an alternative technique based on the based on the use of PCR use of PCR using randomly using randomly selected oligonucleotides and low selected oligonucleotides and low stringency stringency (RAP(RAP--PCR PCR or or ORESTES).ORESTES).

dbEST: database of "Expressed Sequence Tags"

dbEST release 120800 Summary by Organism - December 8, 2000

29th) Schistosoma mansoni (blood fluke) 13,017

68th) Schistosoma japonicum (blood fluke) 1,954

July 13th, 2000

•• FAPESPFAPESP•• MinMin.. CiCiêênciancia e Tecnologia, MCTe Tecnologia, MCT

Project FundingProject Funding::

http://www.fapesp.br/

ObjectivesObjectives::

•• To To generate generate 120,000 120,000 partial cDNA partial cDNA sequencessequences, , derivedderived from different from different lifelife--cyclecycle stagesstages in 18 in 18 monthsmonths..

•• To explore geneTo explore gene discoverydiscovery in in different parasitedifferent parasite stages suchstages such asaseggseggs,, miracidiamiracidia,, cercariaecercariae,,schistosomula and adult wormsschistosomula and adult worms, , depositdepositinging thethe sequence data insequence data inpublicpublic databasesdatabases

Project OrganiProject Organissationation::

• General Coordination• Sergio Verjovski-Almeida (Inst.

Química/USP)

• Parasite Life Cycle• Cybele Gargioni and Toshie Kawano

(Inst. Adolfo Lutz and Inst. Butantã)

• RNA Coordination• Sergio Verjovski-Almeida (IQ/USP)

• cDNA Coordination• Emmanuel Dias Neto (IPq - HCFMUSP)

Project OrganiProject Organissationation::

• Sequencing groups• Sergio Verjovski Almeida (IQ/USP)• Emmanuel Dias Neto (IPq HCFMUSP)• Alda Maria B.N. Madeira (Vet. School/USP)• Carlos Menck (ICB/USP)• Vanderlei Rodrigues (USP – Rib. Preto)• Luciana C. C. Leite (Inst. Butantã)

Project OrganiProject Organissationation::

• Bioinformatics:• João Carlos Setubal (UNICAMP)• João Paulo Kitajima (UNICAMP)• Milton Nishiyama Jr (IQ/USP)• Apuã Paquola (IQ/USP)

Project OrganiProject Organissationation::

ORESTES -a new concept

5’untranslated Coding 3’ untranslated

4kb

5’ ESTs 3’ ESTsORESTES

Consensus sequence

Reasons for sequencing the Reasons for sequencing the S. S. mansonimansoni transcriptometranscriptome

•• Early Early bilaterianbilaterian•• First First acoelomateacoelomate to be sequencedto be sequenced

Schistosoma are platyhelminths, invertebrates that diverged early at the stem of bilaterians. Thus, the transcriptome data disclose sequences that may represent ancient orthologs of known vertebrate genes, providing hints on how certain metabolic pathways have evolved and helping to draw the history of metazoans.

In fact, comparative genomics revealed that in S. mansoni transcriptome there are sequences, corresponding to approximately 645 genes, specific to metazoans.

Reasons for sequencing the Reasons for sequencing the S. S. mansonimansoni transcriptometranscriptome

•• Among the first organisms to present sexual Among the first organisms to present sexual dimorphismdimorphism

•• Close relationship with its hosts (has the Close relationship with its hosts (has the receptors but not their receptors but not their ligandsligands))

•• LongLong--lived parasite (40 years, maybe more)lived parasite (40 years, maybe more)

FindingsFindings

• The parasite has some 14,000 genes• 7,200 are expressed in adult worms• 92% of the transcriptome has been

sampled• 77% represent new Sm genes• 55% with unknown function

8,000 clusters

mostmost, if, if not all the cellular and not all the cellular and physiological systemsphysiological systems ofof higher higher

organisms were established before organisms were established before the divergencethe divergence ofof the platyhelminthsthe platyhelminths

The metazoan-specific sequences were predominantly for genes involved in signal transduction and genes involved in cell-cell interactions, developmental processes and response to external stimuli. Examples: -orthologs of MEDEA (SmAE 602187.1), member of the TGF-beta receptor-signaling pathway.-Na+/Cl– dependent neurotransmitter transporter-like protein (SmAE 714725.1) -ryanodine receptor (SmAE 600004.1).

In contrast, among eukarya-conserved sequences (~1400 genes), those relating to metabolism were the most numerous. Protein kinases were the single, most abundant protein family found among the S. mansonitranscripts. A complete list of such genes is available at the project website.

Tissue formation in metazoans

Genes which control the transport of material into and out of the parasite body.

Components of the receptor-mediated endocytosis pathway.

Neurological and hormonal signaling, for example the nicotinic and muscarinic acetylcholine receptors and the glutamate receptor, and for primitive neuroendocrine processes, for example those from the nuclear receptor family, thyroid and steroid hormones

Adaptation to parasitismProlonged association of the parasite with the host seems to have generated specific adaptations to parasitism.

Living in the blood streamrequires dealing with homeostasis, thromboregulation and platelet activation; genes coding for proteins involved in these processes were identified such as the echicetin alpha-subunit, a snake C-type lectin from Echis carinatus that inhibits binding of von Willebrand factor to platelets, and the ATP-diphosphohydrolase, an ecto-ATPase that is involved in platelet aggregation and thromboregulation.

Long lifespan

Sir-2, encoding a histone deacetylase whose upregulation lengthens life-span by chromatin silencing under conditions of nutrient scarcity in Saccharomyces and C. elegans

Daf-2, an insulin/insulin-like growth factor receptor gene that when mutated confers oxidative stress resistance as well as life span extension in C. elegans, as well as a large repertoire of stress-related genes; these may help to explain why the parasite can live up to 40 years in some instances.

DNA polymorphisms in vaccine candidate genes

Triose Phosphate Isomerase A>G

Lysine Arginine

Schistosoma mansoni isolates

GlutatGlutathhionionee--SS--transferasetransferase G>A G>A

46,8%/34,3%29,1%

50%

6,6%

Serial Analysis of Gene ExpressionSerial Analysis of Gene Expression(SAGE) (SAGE)

in in SchistosomaSchistosoma mansonimansoni

The most abundant Schistosome genes

(Top 10)• GENE TAGS

• EGGSHELL PROTEIN PRECURSOR (CHORION PROTEIN) 1454 • Heat shock protein 86 - fluke 640• Hypothetical 588• Ribosomal protein L23a 513• HEAT SHOCK 70 KD HOMOLOG PROTEIN (HSP70) 464• Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 438• Fructose-bisphosphate aldolase 398• No hits to our database or complete Schisto genes >gtagtgcttg 364 • No hits to our database or complete Schisto genes >cttttctaaa 349• Hypothetical 340

Some quite abundant adult Schistosome transcripts were not sequenced yet

(3’ end at least...)

• TAG Sequences Clusters• >gtagtgcttg 364 0 • >cttttctaaa 349 0 • >tatcgttcta 302 0• >ccttcggtac 225 0• >gtaaccaatg 193 0

Using SAGE to evaluate alternative

polyadenylation

Polyadenylation in Schistosome genes

• Alternative polyadenylation (AP) is a frequent event in Schistosoma mansoni.

• It was found in 10% (using a cuttoff of 4 SAGE tags) to 50% (at least 2 SAGE tags) of the known genes.

• 24% of the human genes use AP

New drugs ??

Trans R Soc Trop Med Hyg. 2002 Sep-Oct;96(5):465-9.

New Drug targets identified

Vaccines ??

Approximately 450 novel paralogs of known S. mansoni genes were found, including 12 paralogs of six previously investigated S. mansoni vaccine candidates.

Schisto ESTs in Public databases

020000400006000080000

100000120000140000160000

2000 2003

Year

ESTs

S. japonicum S. mansoni

• Sequencing groups• Sergio Verjovski Almeida (IQ/USP)• Alda Maria B.N. Madeira (Veterinary

School/USP)• Carlos Menck (ICB/USP)• Vanderlei Rodrigues (USP – Ribeirão Preto)• Luciana C. C. Leite (Inst. Butantã)

AcknowledgmentsAcknowledgments::

• Bioinformatics:• João Carlos Setubal (UNICAMP)• João Paulo Kitajima (UNICAMP)• Milton Yutaka Nishiyama Junior (IQ/USP)• Apuã César de Miranda Paquola (IQ/USP)

AcknowledgmentsAcknowledgments::

Élida PB Ojopi, PhDCíntia Fridman, PhDPedro EM GuimarãesSheila GregórioCarolina A RodriguesGaëlle Chopin StukartFabio Borges MuryDaniel Martins

emmanuel@usp.br

UNESCO - First North-South Human Genome ConferenceHow can we learn ?Strategies for EST generation Project Funding:Objectives:Project Organisation:Project Organisation:Project Organisation:Project Organisation:Reasons for sequencing the �S. mansoni transcriptomeReasons for sequencing the �S. mansoni transcriptomeFindingsThe most abundant Schistosome genes �(Top 10)Some quite abundant adult Schistosome transcripts were not sequenced yet �(3’ end at least...)Using SAGE to evaluate �alternative polyadenylationPolyadenylation in Schistosome genesNew drugs ??Vaccines ??Acknowledgments:Acknowledgments: