Functional Genomics (EST) and Functional Genomics (EST) and

Genome Sequencing of the Genome Sequencing of the ProtistanProtistan

Parasite Parasite PerkinsusPerkinsus marinusmarinus

José A. F. Robledo1, Eric J. Schott1, Malcolm J. Gardner2, Jessica

Kissinger3, and Gerardo R. Vasta1

1COMB, UMBI, University of Maryland, Baltimore, MD 21202, USA;2Institute for Genomic Research, Rockville, MD 20850, USA;

3Center for Tropical & Emerging Global Diseases, University of Georgia, Athens, GA 30602, USADo not

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PerkinsusPerkinsus species describedspecies described

http://modis.gsfc.nasa.gov/ http://www.oie.int/

P. marinus 1950

P. qugwadi

1998

P. olseni (= P. atlanticus)

P. mediterraneous 2004

P. olseni 1981

P. chesapeaki

P. andrewsi

Perkinsus sp.

Perkinsus sp.Perkinsus sp.

Perkinsus sp.

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UltrastructureUltrastructure of of PerkinsusPerkinsus spsp

Trophozoite Zoospore

Coss,Robledo and Vasta, 2001Do n

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Debate on the taxonomic placement of Debate on the taxonomic placement of PerkinsusPerkinsus spsp

Dermocystidium sp.

Dermocystidium marinum (Mackin et al. 1950)

Fungus

Labyrinthomyxa marina (Mackin & Ray 1966)

Thraustochytrium thallus

Saprolegniales

Toxoplasma gondii

Perkinsus marinus (Levine 1978)

Apicomplexan

Perkinsus marinus (Siddall et al. 1997) rRNA and actin phylogeny

Pfiesteria piscicida

Dinozoan

Perkinsus marinus (Norén, et al. 1999) Ultrastructure and

rRNA phylogeny

Parviluciferainfectans

Cryptophagussubtilis

Perkinsozoan

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HSP90HSP90--based based phylogeny phylogeny

(Leander and Keeling 2003, Leander et al. 2004)

Alveolates

Perkinsus marinusPerkinsus marinus

OxyrrhisOxyrrhis marinamarina

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> 90%

50-89%

1-49%

http://modis.gsfc.nasa.gov/ (Based on Burreson et al. 1996)

DermoDermo prevalence in Chesapeake and Delaware Baysprevalence in Chesapeake and Delaware Bays

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• Detection of P. marinus prior to restocking

• Selective breeding of oysters surviving infections

• Treatment of oysters with anti-parasitic agents

• Introduction of exotic disease-resistant oyster species

• Identification of genetic markers for host resistance

and parasite targets for intervention (functional genomics)

Approaches for intervention in Approaches for intervention in DermoDermo diseasedisease

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Hypothesis Hypothesis

• Perkinsus marinus expresses genes involved in virulence

• Infection mechanisms (e.g. recognition, colonization, entry)

• Novel pathogenesis mechanisms

• Selected P. marinus genes and/or gene products may constitute suitable

candidates for intervention

• Homologues of genes used as targets in other parasites

• New target genes and/or gene productsDo not

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GenomicsGenomics

GENOME

GENE

EXPRESSION

STIMULUS

PHYSIOLOGICALHormonal, metabolic

RESPONSE

ENVIRONMENTPhysical, chemical, biological stress

Development,Differentiation

Infection orImmunity?

Stress ResponsePhysiology/Pathology

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GenomicsGenomics

GENOME

GENE

EXPRESSION

STIMULUS

PHYSIOLOGICALHormonal, metabolic

RESPONSE

ENVIRONMENTPhysical, chemical, biological stress

Development,Differentiation

Infection orImmunity?

Stress ResponsePhysiology/Pathology

EXPRESSED SEQUENCE TAGS (ESTs)

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Gene identification through EST analysis Gene identification through EST analysis

• Construction of two P. marinus bacteriophage cDNA libraries

- P. marinus grown in standard medium

- P. marinus grown in standard medium supplemented with oyster

serum

• Random single pass sequencing (5’ EST)

• Sequence analysis

• Identification of genes of interest

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Cryptosporidium parvumPlasmodium yoelliP. falciparumP. reichenowiP. knowlesiEimeria tenellaToxoplasma gondii

Tetrahymena thermophila

Tripanosoma cruzi

Trichomonas vaginalis

Genbank NRGenbank ESTJuly 4-5 2004 releases

Dino EST

Entamoeba histolytica

• ORF (≥75 aa)

• Analysis

- BLAST

- SignalP

- TargetP

- Pfam

- GO terms

Perkinsids

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P. marinus ESTs

Number Std medium Oyster serum Total

Independent clones 2.4 x 106 4.0 x 106

Av. insert size (bp) 806 1,057

ESTs number 1,228 1,285 2,513

Clusters 328

Singletons 1,306

Unigenes 1,634

EST summaryEST summary

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EST functional categoriesEST functional categories

Ribosomal Proteins

DNA/RNA Proteins

Metabolic Enzymes

Hydrolases

Stress Proteins

Signal and Transcription

Cytoskeleton

Membrane Proteins

EST Homologues

Unknown

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Perkinsus Perkinsus cDNAcDNA normalized librariesnormalized libraries

1,2% Agarose, EtBr, 1X TAE

1,5% Agarose, EtBr, 1X TAE

1 K

p

Pm

CB

5 D

4

Pan

dA

8-4

1 K

p

Pm

CB

5 D

4

Pan

dA

8-4

Pm CB5 D4

Pand A8-41

00

bp

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Perkinsus Perkinsus cDNAcDNA normalized librariesnormalized libraries

1,2% Agarose, EtBr, 1X TAE

1,5% Agarose, EtBr, 1X TAE

1 K

p

Pm

CB

5 D

4

Pan

dA

8-4

1 K

p

Pm

CB

5 D

4

Pan

dA

8-4

Pm CB5 D4

Pand A8-41

00

bp

From each normalized library 12,000 EST will be sequencedDo n

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GenomicsGenomics

GENOME

GENE

EXPRESSION

STIMULUS

PHYSIOLOGICALHormonal, metabolic

RESPONSE

ENVIRONMENTPhysical, chemical, biological stress

Development,Differentiation

Infection orImmunity?

Stress ResponsePhysiology/Pathology

SEQUENCE COMPLETE GENOME

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Genome sequencing strategyGenome sequencing strategy

• Whole genome shotgun plus closure

• Two genomic shotgun libraries prepared in pHOS2

� 2.3 kb average insert size

� 11 kb average insert size

• Sequence to 8X coverage

• Generate 10,000 ESTDo not

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Number Total Length Avg. Length

Contigs 18,156 32.8 Mb 1.8 kb

Scaffolds 5,753 19.8 Mb 3.4 kb

Singletons 22,104 NA NA

• 163,052 sequences; 861 nt/read @ 92% success rate

• Contigs match P. marinus GenBank sequences > 98% id

• Sequences from 3X assembled with Celera Assembler

• Sequencing to 6X coverage is underway

Genome sequencing: Current statusGenome sequencing: Current status

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Perkinsus Perkinsus sppspp.. trophozoitestrophozoites/Oyster /Oyster

hemocyteshemocytes

(Gauthier & Vasta 1998)

Host Host oxidaseoxidase

OO22--..

OO22

HH22 OO

22

SODSOD

HO

Cl

HO

Cl

MPOMPO

OHOH . . OHOH ..

FeFe22++

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Novel components of the Novel components of the P. marinusP. marinus

antioxidant pathwayantioxidant pathway

PmSOD1

PmSOD2

(Schott et al. 2003a,b, 2004)

GRX1 (PMAAF76TH)

GRX2 (PMBA504TH)

PRX1 (PMBAA58TH)

PRX2 (PMBAE15TH)

TRX1 (PMBA920TH)

TRX2 (PMBAA56TH)

APX

GR

Asc. MDA

GSH GSSG

(Asc. Red’se)

HH22OO22OO22--..

HH22O O

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P. marinusP. marinus virulence factorsvirulence factors

Transcription

factors

Cathepsin D (PMBA667TH)

Cathepsin Z (PMBAE46TH)

Cys protease (PMBAG12TH)

Ser protease (PmS+018)

Pepsinogen A (PMBAA90TH)

Protein Phosphatase(PMBA602TH)

Chitinase (BAA36TH)

HSP70 (PMAAG23TH)

H+Glucose

Nucleoside

Multidrug efflux pump

Aminoacid

ABCtransporter

H+

H+

Na+

PO43+

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P. marinus

CvNramp1 ?

PmNramp

Fe2 + X+

Fe2+ X+

Fe2+ X+Type I ‘P’ ATPase ?

Fe2+

Fe2+ X+

Fe2+ X+

CvNramp2 ?Fe2+ X+

Fe2+ X+

Recycling

endosome

Cytoplasm

Oyster

Hemocyte

Proposed Role(s) of Proposed Role(s) of NrampNramp in in P. P. marinusmarinus and the Oysterand the Oyster

Fe2+ X+

Phagosome

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Perkinsus marinus databases

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Perkinsus marinus databases

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P. marinus genomics summary

• Functional genomics

- Constructed several P. marinus cDNA libraries

- Sequenced 2,513 EST (1,634 unigenes)

- Additional 12,000 EST underway

- Identified genes

- involved in basic functions of P. marinus

- whose products may be candidates for intervention

- unique to P. marinus

- Database under construction

• Perkinsus genome

- Established and cloned a new P. marinus strain

- Constructed two genomic shotgun libraries

- Generated 163,052 sequences

- 6X coverage underway

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GenomicsGenomics

GENOME

GENE

EXPRESSION

STIMULUS

PHYSIOLOGICALHormonal, metabolic

RESPONSE

ENVIRONMENTPhysical, chemical, biological stress

Development,Differentiation

Infection orImmunity?

Stress ResponsePhysiology/Pathology

PROTEOMICSPROTEOMICSDo n

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Integrated genomeIntegrated genome--/proteome/proteome--wide analysis wide analysis

of target/model organisms (of target/model organisms (P. P. marinusmarinus//C. C. virginicavirginica))

EST collection

Whole-genome sequence

Gene knockdown

Phenotype identification

Candidate genes

Microarray analysis

Proteomic analysis

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• Generate more EST and identify gene products

� Associated with effects of high temperature/salinity

� Involved in Fe uptake/detoxification

� Associated with exposure to oxidative stress

� Related to life cycle stages

� Effect(s) of long-term culture

• Expand/annotate PerkDB and TIGR databases

• Mine Perkinsus genome

� Microarrays

� Proteomics

• Extend the studies to other protozoan parasites of mollusks

Current researchCurrent research

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Collaborations and acknowledgements Collaborations and acknowledgements

Mario L. Mario L. AmzelAmzel

Mario A. Mario A. BianchetBianchet

AbelardoAbelardo M. SilvaM. Silva

ToyinToyin AsojoAsojo

Structural biologyStructural biology

Gregory W. Gregory W. WarrWarr

Robert ChapmanRobert Chapman

Matthew J. JennyMatthew J. Jenny

CrassostreaCrassostrea//PerkinsusPerkinsus microarraymicroarray

M. M. LeonorLeonor CancelaCancela

Manuela AlmeidaManuela Almeida

Patrícia A. Patrícia A. NunesNunes

Rita Rita AscensoAscenso

Perkinsus Perkinsus olseniolseni functional genomicsfunctional genomics

Funding AgenciesFunding Agencies

Ramón F. Ramón F. ConchasConchas

Jaime MontesJaime Montes

Other protozoan parasitesOther protozoan parasites

UGA

Boris Boris StriepenStriepen

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AcknowledgmentsAcknowledgmentsLaboratory participantsLaboratory participants

Julie D. Gauthier, Ph.D.

Adam G. Marsh, Ph.D.

Anita C. Wright, Ph.D.

Hafiz Ahmed, Ph.D.

Cathleen A. Coss, Ph.D.

Wolf Pecher, M.Sc.

CollaboratorsCollaborators

Abelardo M. Silva, Ph.D. (NCI, Frederick, MD)

Gregory M. Ruiz, Ph.D. (SERC, Edgewater, MD)

Jan Pohl, Ph.D. (Emory University, GA)

Valerie Culotta (Johns Hopkins University, MD)

Vern Carruthers (Johns Hopkins University, MD)

Grant SupportGrant Support

National Science Foundation (ICB)

NSF/USDA (Microbial Genomics)

MD Sea Grant/NOOA (ODRP)

USDA (NRAC)

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