Communication Between

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Communication Between Toxoplasma gondii and Its
Host Cell
Ira Blader Microbiology and Immunology OUHSC
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Host Processes Targeted By Infectious Pathogens

• Protein Synthesis
• Antigen Presentation
• Apoptosis
• Metabolism
• Chemotaxis
• Transcription

HOW are pathways targeted?
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Toxoplasma gondii

• Parasite in cat litter danger to pregnant women.
• Obligate intracellular eukaryote.
• Infects most warm-blooded animals.
• Related to Plasmodium (Malaria), Cryptosporodium
  (Cryptosporidiosis), and Eimeria (Coccidiosis).

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What Makes Toxoplasma A Good System To Study The
Interaction Between Parasites and Host Cells?

• Genetics - Haploid, linkage maps
• Animal and Tissue Culture - Can infect most cell
  types, natural pathogen of mice.
• Biochemistry - High numbers of purified
  parasites.
• Genomics - Genome sequenced.

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Toxoplasma Is Medically Important

• Severe birth defects in the developing fetus.
• Severe disease in immune compromised individuals
  e.g. AIDS, transplant, lymphoma, etc...
• High prevalence. Up to 85 in some countries.
  In USA, 10- 30.

Chorioretinitis
Hydrocephalus
Encephalitis
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Toxoplasma In The Intermediate Host
Digest cyst-containing meat
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Question What Is The Molecular Basis For
Communication Between Toxoplasma And Its Host
Cell?

• How do the parasite and host cell recognize each
  other?
• How does the parasite disseminate in the host?
• How does the parasite grow inside of a host cell?
• How does the host maintain an immune response?

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Toxoplasma Growth In A Host Cell

• Induces immune responses.
• Scavenges nutrients from host cell glucose,
  cholesterol, purine nucleosides, tryptophan.
• Prevents host cells from dying.

DNA Microarrays To Address The Molecular Basis
For These Phenomena.
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Which Host Genes Are Modulated During Infection?
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1
2
4
6
24
7 (1191 / 18000) of genes spotted changed.
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Proinflammatory EarlyResponse Transcripts

• GRO1
• NF-kB, p50
• Matrix metalloproteinase 3
• REL-B
• TNF-alpha-induced protein 2
• TNF-alpha-induced protein 3
• GRO2
• Interleukin 6
• Interleukin 1, beta
• MCP1
• Leukemia inhibitory factor
• MCP3
• IkB, alpha
• Superoxide dismutase 2
• c-MYC

• Cyclooxygenase
• Plasminogen activator, urokinase
• ICAM1
• 5' nucleotidase (CD73)
• B-cell CLL/lymphoma 3
• jun-B proto-oncogene
• CD53 antigen
• Thrombomodulin
• Plasminogen activator inhibitor, type II
• AP-1 transcription factor
• Metallothionein 1L
• Dopachrome tautomerase
• Interferon regulatory factor 1
• Pre-B-cell colony-enhancing factor

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What Else Is Modulated?

• Trafficking
• Growth and Differentiation
• Cytoskeleton
• Apoptosis
• Metabolism

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HOW TO USE THE MICROARRAY DATA
Role of Pathway in Growth/Virulence
Microarray Data
Potential Pathways
How is Pathway Activated
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GLYCOLYSIS
Glucose
Hexokinase (3.3 ?)
Glucose 6-Phosphate
PGI (NC)
Fructose 6-Phosphate
PFK-P (3.5 ?)
Fructose 1,6-Bisphosphate
Aldolase (NC)
TPI (3.1 ?)
GAP
DHAP
GAPDH (3.0 ?)
1,3-Bisphosphoglycerate
PGK (4.0 ?)
3-Phosphoglycerate
PGAM (3.6 ?)
2-Phosphoglycerate
Enolase (2.6 ?)
Phosphoglycerate
? - Upregulated NC - No change PS - Poor spot
PK (NC)
Pyruvate
PDH (NC)
LDH-A (2.4 ?)
Acetyl-CoA
Lactate CO2
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Up-Regulation Of Glycolytic Genes Is Specific.

• No significant induction of genes involved in
• TCA cycle.
• Amino acid metabolism.
• Implies anaerobic glycolysis is upregulated?

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Glycolysis
Glucose
Hexokinase
Glucose 6-phosphate
PFK, GAPDH, ENO1...
Pyruvate
Lactate Dehydrogenase
Lactate
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Toxoplasma Activates HIF1
Control Lysate
- Toxo
Toxo
150
100
75
50
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Toxoplasma Activates HIF1
5000
Mock Infected
Toxoplasma
4000
CoCl2
3000
LUC Units
2000
1000
0
HIF1aWT
HIF1aKO
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Host Gene Expression is Regulated by HIF1 During
Infection
HIF1aWT
HIF1aKO
- Toxo
Toxo
GAPDH
ACTIN
Fold Change
2.5
1.2
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DOES HIF1 ACTIVATION REQUIRE PARASITE CONTACT
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Only Live Intact Parasites Activate HIF1
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15
Fold Induction
10
5
0
Live Parasites
Mock
ESA
Heat Killed
CM
Whole Parasite Lysate
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SUPEROXIDE ACTIVATES HIF1
35
30
25
20
Fold Induction
15
10
5
0
Control
Toxo
Toxo SOD
Toxo Catalase
Toxo Mannitol
Toxo Hemoglobin
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Which Host Genes Are Modulated During Infection?
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Defective Toxoplasma Growth in HIF1 Knockout Cells
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Acknowledgements
STANFORD John Boothroyd Amato Giaccia Denise
Chan Stanford Microarray Database UCSD Randy
Johnson
OUHSC Terry Coursey Elizabeth Charles Wade Spear