Yeast as a surrogate genetic system for gene discovery in the human and animal pathogen Toxoplasma g

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Yeast as a surrogate genetic system for gene
discovery in the human and animal pathogen
Toxoplasma gondii
Christopher Kvaal Department of Veterinary
Molecular Biology
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Why study Toxoplasma gondii?

• Clinical Importance
• Life-long chronic disease
• Food safety
• Congenital Birth defects
• Central Nervous System Disease in
  immuno- compromised individuals-AIDS- transplant
  patients- elderly
• Abortions in livestock-- sheep/swine.

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Toxoplasma gondii is endemic

• Seroprevalence for Toxoplasma in the U.S. is
  5-30, although this is dropping.
• Source of Toxoplasma
• tissue cysts (bradyzoites) in meat products
• oocysts (sporozoites) in the environment
• Major problems in treating toxoplasmosis are
• drug intolerance/toxicity
• recrudescence (no current treatment is effective
  against the tissue cyst stage).

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Toxoplasmosis and HIV

• In the U.S., 30 of AIDS patients suffer from
  encephalitis.
• Toxoplasma gondii is the most common cause of
  central focal encephalitis.
• Toxoplasmosis is the reported cause of 7 of
  US/AIDS deaths.
• With 21 million HIV infected individuals world
  wide, in the coming decade over a million people
  will potentially suffer Toxoplasma encephalitis.

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Toxoplasma gondii cell division
Time-lapse microscopy of tachyzoites in Nuclear
Division (endodyogeny)
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T. gondii tachyzoites in culture
GFP Marked Nucleus
Phase Contrast Microscopy
Apical Complex Marker
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Toxoplasma induced brain lesions
Lesions
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Brain of a Patient Suffering Chronic Toxoplasmosis
Lesion
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The Tachyzoite to Bradyzoite Switch

• A Developmental Timer System operating at the
  level of the tachyzoite to bradyzoite switch
  event
• The molecular event(s) controlling switching is
  directly related to virulence in T. gondii
• Elucidation of the molecules effecting the switch
  event will provide novel target for drug
  development

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Cyclins and the Cell Cycle

• The eukaryotic cell cycle is based on a
  succession of active cyclin-cyclin dependent
  kinase complexes

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The T. gondii tachyzoite cell cycle
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Project Goals

• To identify protein interactors of the T. gondii
  homolog of the yeast cell cycle regulator CRK2
• To identify T. gondii cyclin-like proteins
  employing trans-complementation in yeast
• To identify T. gondii cyclins based on sequence
  homology from ongoing protozoan sequencing
  projects

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Approach -- Use yeast as a tool

• Experience the Power of Yeast Genetics
• Easy to grow and manipulate
• Successfully been used before in
  trans-complementation and heterologous gene
  expression experiments

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Saccharomyces cerevisiae

• Most notable in human history as
• The bug that rises your bread
• And ferments your beer
• Today it is the model eukaryotic organism for
  studies of all aspects of cell biology

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Phenotypic Characteristics
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Growth Characteristics

• Genome size of 14 Mb containing 6000 genes
• Arranged on 16 chromosomes.
• The genome is completely sequenced and is
  accessible, with associated links,
• at http//genome-www.stanford.edu/Saccharomyces/.

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What yeast really look like...
Clonal maintenance
Mass maintenance
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In yeast we can...

• Yeast Two-Hybrid System
• Functional Complementation

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Gal4 based YTHS

• Exploits the modular nature of transcription
  factor proteins, genetically separating the
  physical ability to bind specific cis-acting DNA
  sequences from the physical ability to recruit
  the full transcriptional machinery
  (trans-activate) to a transcription competent gene

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YTHS Movie
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Schematic of the Gal4 based YTHS
Off
Ade2 open reading frame
UAS Gal2
Off
His3 open reading frame
UAS Gal1
Off
Mel 1/LacZ open reading frame
UAS MEL1
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The YTHS Screen

• A library screen scale transformation of strain
  AH109your bait yielding gt 1 X 106 primary
  transformants
• Plated at low density on SC-H-L-W
• Primaries selected and patched to SC-Ade-H-L-W
• Number of Survivors determines next course of
  action

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Use of TgCRK2 as bait

• Cloned the ORF of TgCRK2 in pBD- Gal4 Cam
• Tested for auto-activation in AH109
• Transformed the AH109 strain containing
  pBD-Gal4TgCRK2 with Toxoplasma cDNA plasmid
  library

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Primary YTHS Screen Plate

• 6 x 107 yeast cells
• 3 x 104 LW transformants
• 22 colonies

SC-H-L-W
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Secondary Patch Plate
Non-survivor
SC-Ade-H-L-W
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Transformation Results

• 3.2 x 106 primary transformants on 100 SC-H-L-W
  plates
• 235 colonies were patched to SC-Ade-H-L-W
  plates
• 12 clones remained able to grow under stringent
  conditions
• 8 survived further passage and were tested in a
  liquid ß-gal assay

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Liquid Assay Results
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Reproduction of Interaction(s)
TgCdk2/ Clone 3
p53/ pSV40
p53/ plaminC
TgCdk2/ Clone 1
Legend
SC-L-W
SC-H-L-W
SC-Ade-H-L-W
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Clone 1 Interactor Analysis

• Sequence the AD fusion insert
• 1.350 kb BamHI/XhoI insert
• 232 amino acids before a stop
• Screen a lambda phage library with this insert
• 2.0 kb cDNA
• 579 bp coding region
• 192 amino acid protein
• pI of 7.26
• MW of 21.97 kDa

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BlastP of SwissProt Database

• G2/MITOTIC-SPECIFIC CYCLIN A
• D. melanogaster
• G1/S-SPECIFIC CYCLIN D3
• H. sapiens
• CYCLIN A2 (CYCLIN A)
• M. musculus

• G2/MITOTIC-SPECIFIC CYCLIN A
• C. viridissima (hydra)
• CYCLIN A2 (CYCLIN A)
• M. auratus (golden hamster)
• G1/S-SPECIFIC CYCLIN D3
• Rattus norvegicus

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Cyclin A/D Box Alignment
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50 1-TgCyc2 box
.....LRSLK LEMFALIEQE EQLDVSTVAC AWVYFERLVQ
MGAVDKSVRK 2-DmCycA box DISHNMRSIL IDWLVEVSEE
YKLDTETLYL SVFYLDRFLS QMAVVRSKLQ 3-HsCycD3 box
EIKPHMRKML AYWMLEVCEE QRCEEEVFPL AMNYLDRYLS
CVPTRKAQLQ 4-MmCycA box .ITNSMRAIL VDWLVEVGEE
YKLQNETLHL AVNYIDRFLS SMSVLRGKLQ 5-CvCycA(B) box
DINSSMRAIL IDWLVEVSEE YKLIPQTLYL SVSYIDRFLS
HMSVLRGKLQ 6-RnCycD3 box EIKPHMRKML AYWMLEVCEE
QRCEEDVFPL AMNYLDRYLS CVPTRKAQLQ
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100 1-TgCyc2 box LFAGACLLLA FKFNQNGEPK
LVQRLCSLLK HLDRQRGLPA HALCQAEFTV 2-DmCycA box
LVGTAAMYIA AKYEEIYPPE VGEFVFLTDD SYTKAQ....
..VLRMEQVI 3-HsCycD3 box LLGAVCMLLA SKLRETTPLT
IEKLCIYTDH AVSPRQ.... ..LRDWEVLV 4-MmCycA box
LVGTAAMLLA SKFEEIYPPE VAEFVYITDD TYSKKQ....
..VLRMEHLV 5-CvCycA(B) box LVGAACMLVA AKFEEIYPPE
VAEFVYITDD TYTAKQ.... ..VLRMEHLI 6-RnCycD3 box
LLGTVCLLLA SKLRETTPLT IEKLCIYTDQ AMAPWQ....
..LREWEVLV 101
150 1-TgCyc2 box
FGLLGFSLQL SIEHVLPHIL HYLESKDTAF EEVYGSPETA
FFSA...... 2-DmCycA box LKILSFDLCT PTAYVFINTY
AVLCDM..PE KLKYMTLYIS ELSLMEGETY 3-HsCycD3 box
LGKLKWDLAA VIAHDFLAFI LHRLSLPRDR QALVKKHAQT
FLALCATDYT 4-MmCycA box LKVLAFDLAA PTVNQFLTQY
FLHLQP.ANC KVESLAMFLG ELSLIDADPY 5-CvCycA(B) box
LKTLAFDLSV PTCRDFLSRY LFAANAKPES QLKYLAEYLS
ELTLINCDIS 6-RnCycD3 box LGKLKWDLAA VIAHDFLALI
LHRLSLPSDR QALVKKHAQT FLALCATDYT
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Interactor Clone 3 Analysis

• Sequencing AD Fusion Insert
• 1.962 kb BamHI/XhoI insert
• 560 amino acids before a stop
• 28 of the first 34 amino acids in the fusion
  protein were serine
• Probing a lambda library with insert
• 2.600 kb cDNA
• 1.749 kb coding region
• 582 amino acid protein
• pI of 7.9
• MW of 62.296 kDa

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BlastP of SwissProt Database

• Cyclin CYC1
• Plasmodium falciparum
• Cyclin CCL1
• S. cerevisiae
• Cyclin MCS2
• S. pombe
• Cyclin H
• X. larvis

• Cyclin H
• H. sapiens
• Cyclin H
• Rattus norvegicus
• G1/S-SPECIFIC CYCLIN C
• Oryza sativa (rice)

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Cyclin H Box Alignment
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100 Tg CYC1 FSSSWPGCSS HFRRWIFRPE
ALQALRGHTH EAACVSLLRL KRCRKRASDT Pf Cyc1
..MNYPEDTT HMKKWFFK.. .......... ...SRKEIDN
ICLKKYNDFK Hs CYCH ....MYHNSS QKRHWTFSSE
.........E QLARLRADAN RKFRCKAVAN 101

150 Tg CYC1 SEVADASASF PSLRSLLDLT EYFAFQLVLI
CKRKR..VKL PVIETACVYL Pf Cyc1 EEFKDYNIKI
PKYNDVEKTK VYFCYQLVHF CEIKM..LRP HIVECATILY Hs
CYCH GKVLPNDPVF LEPHEEMTLC KYYEKRLLEF CSVFKPAMPR
SVVGTACMYF 151
200 Tg CYC1 HRFFCMRSPL
AFDIRLVIFA CLLLALKAED VARHYSLGDL LG....DIAE Pf
Cyc1 NRFYLKEIIL EYDPRILIFT CIVLAIKIEG YGRLYKINEF
F.....NDID Hs CYCH KRFYLNNSVM EYHPRIIMLT
CAFLACKVDE FNVSSPQFVG NLRESPLGQE 201

250 Tg CYC1 LDIGEVLRLE LPVCEALDFH MLVLHTRGPL
ATLLRQIFFL SLGAERANGS Pf Cyc1 INLDKVLEHE
NIVCSSLNFE LNFLYTK... ....ECIFYI KSQFEKYIN. Hs
CYCH KALEQILEYE LLLIQQLNFH LIVHNPYRPF EG...FLIDL
KTRYPILEN.
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Two-Hybrid Screen Results

• 4 screens performed employing 3 different cDNA
  libraries and 2 two-hybrid vector systems against
  TgCRK2
• More than 8 million Leu Trp transformants
• 2 putative novel TgCRK2 interactors discovered
• Cyclin 1
• Cyclin 2

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Complementation of S .cerevisae cell cycle mutants

• DL1 has the three G1/S cyclin homologs deleted
  (Cln1, Cln2, and Cln3), and Cln2 replaced under
  the control of the inducible GAL1 promoter
• K3418 has 4 of the 6 G2/M cyclins (Clb 3-6)
  deleted and Clb5 replaced under the GAL1 promoter

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The Experiment

• Construct an MET inducible TgCyc1 ORF in a vector
  with a Ura marker
• Transform DL1 and K3418 with construct and select
  for plasmid replication only
• Turn on plasmid-borne cyclin while simultaneously
  turning off the chromosomal cyclin

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Trans-complementation
DL1
DL1/16 TgCYC1
DL1/31 TgCYC1
Galactose MM
Galactose MM -ura
Glucose MM -ura
Glucose MM FOA
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Toxoplasma cyclins in yeast
MM -met -ura Glucose
MM 10x met -ura Glucose
MM 10x met -ura Galactose
G1/S
DL1
K3418
G2/M
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Other Apicomplexan Cyclins

• Penn Plasmodium Genome Resource
• Minnesota Cryptosporidium parvum Genome Project
• Text search for Cyclin
• Found 2 Plasmodium and 3 Crypto cyclin-like genes

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Alignment of Apicomplexan Cyclins
Cyclin A/D
Tg Cyc2
Pf Cyc2
G1/S
Pf Cyc3
PREG/ Pho85
Pf Cyc4
Tg Cyc1
Pf Cyc1
CAK
Cyclin H
Cp Cyc1
Cp Cyc2
G2/M
Cyclin B
Cp Cyc3
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Summary

• T. gondii has at least 2 genes homologous to
  cyclins discovered by interaction with a T.
  gondii cdc2 related kinase
• TgCyc1 functionally complements a G1/S yeast
  defect but not a G2/M
• TgCyc2 does not functionally complement a yeast
  G2/M defect or a G1/S context
• There are at least 5 other undiscovered
  apicomplexan cyclins based on a functional
  genomics strategy

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Goals for teaching and research atSt. Cloud
State University

• Incorporate Bioinformatics, Functional Genomics,
  and Proteomics into the Genetics Curriculum
• Use of the yeast two-hybrid system and yeast
  complementation as a teaching tool
• By using established yeast strains in traditional
  genetic lab exercises
• By mentoring students performing genetic screens
  in summer internships

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Collaborators

• Dr. Michael White
• Dr. Jay Radke
• Mick Guerini
• Maria Jerome

USDA/CREES/NRICGP
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TEST