Characterization of the putative surface protein TLP in Plasmodium berghei and Plasmodium falciparum...
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Characterization of the putative Characterization of the putative surface protein TLP in surface protein TLP in Plasmodium Plasmodium
bergheiberghei and and Plasmodium falciparumPlasmodium falciparum
Ryan M Harrison1,2, Cristina K Moreria3, Catherine Lavazec3, Thomas J Templeton3
1 Gateways to the Laboratory Program, Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY2 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD3 Department of Immunology and Microbiology, Weill Cornell Medical College, New York, NY
Malaria in ContextMalaria in Context
41% of the World Population live in 41% of the World Population live in endemic areasendemic areas
Estimated 700,000 to 2.7 million malaria Estimated 700,000 to 2.7 million malaria deaths annually, 75% of them African deaths annually, 75% of them African childrenchildren
http://www.cdc.gov/malaria/facts.htm
Plasmodium Life CyclePlasmodium Life Cycle
Sporozoite
Merozoite
Asexual/
Gametocyte
Zygote
Ookinete
OocystPlasmodium, Encyclopedia of Life Sciences, 2006
TLP (Thrombospondin Like Protein) discovered via TLP (Thrombospondin Like Protein) discovered via micro array (Hayward et al., 2000)micro array (Hayward et al., 2000)
Putative surface proteinPutative surface protein?? Shares conserved domains with:Shares conserved domains with:
History of TLPHistory of TLP
CSPCSP TRAPTRAP CTRPCTRP TLPTLP membranemembrane cytoplasmcytoplasm membranemembrane cytoplasmcytoplasm membranemembrane cytoplasmcytoplasm membranemembrane cytoplasmcytoplasm
Salivary Salivary Gland Gland SporozoiteSporozoite
++ -- ++ ++ -- -- ?? ??
Hemocoel Hemocoel SporozoiteSporozoite ++ -- ++ -- -- -- ?? ??OokineteOokinete -- -- -- -- ++ ++ ?? ??Asexual Asexual StagesStages -- -- -- -- -- -- ?? ??
Experimental Plan:Experimental Plan:
1. 1. Determine stage TLP expressed in Wild Determine stage TLP expressed in Wild TypeType
- Quantitative Real-Time PCR- Quantitative Real-Time PCR
2. 2. Verify TLP knockout gene disruptionVerify TLP knockout gene disruption- Diagnostic PCR- Diagnostic PCR
- Southern Blot- Southern Blot
Project GoalsProject Goals
Hypothesis:Hypothesis: The TLP protein is The TLP protein is predominately expressed in the sporozoite predominately expressed in the sporozoite life cycle stage of both life cycle stage of both P. falciparumP. falciparum and and P. P. bergheiberghei
P. bergheiP. berghei TLP Expression TLP Expression
P. falciparumP. falciparum TLP Expression TLP Expression
Verification of gene disruptionVerification of gene disruption
A B C D
WT TLP
P. berghei
A B C D
P. falciparum
A B C D
A B C D
WT
TLP
A
B
C
D
Diagnostic PCRIntegrated cassettegDNA gDNA
Verification of gene disruptionVerification of gene disruption
Southern blot design
gDNA gDNA
NcoI HindIII
Hybridization Probe
Wildtype
TLP knockout
7,600bp
gDNA gDNA
NcoI HindIII
Hybridization Probe
9,700bp
HindIII HindIII2000bp
Integrated Cassette
Verification of gene disruptionVerification of gene disruption
1,650bp 1,000bp
5,000bp12,000bp
TLP
WT
P. falciparum Southern blot
The gene disruption cassette is likely The gene disruption cassette is likely integrated into the TLP knockout parasite integrated into the TLP knockout parasite genomegenome
TLP is expressed predominantly in the TLP is expressed predominantly in the sporozoite life cycle stage of both sporozoite life cycle stage of both P. bergheiP. berghei and and P. falciparumP. falciparum
ConclusionConclusion
CSPCSP TRAPTRAP CTRPCTRP TLPTLP membranemembrane cytoplasmcytoplasm membranemembrane cytoplasmcytoplasm membranemembrane cytoplasmcytoplasm membranemembrane cytoplasmcytoplasm
Salivary Salivary Gland Gland SporozoiteSporozoite
++ -- ++ ++ -- -- ++
Hemocoel Hemocoel SporozoiteSporozoite ++ -- ++ -- -- -- -- --OokineteOokinete -- -- -- -- ++ ++ -- --Asexual Asexual StagesStages -- -- -- -- -- -- -- --
Hepatocye invasion assayHepatocye invasion assay Reduced ability of TLG knockout sporozoites to Reduced ability of TLG knockout sporozoites to
recognize and/or invade hepatocyes recognize and/or invade hepatocyes ?? Immunoflouresence assay (IFA)Immunoflouresence assay (IFA)
TLP co-localization with TRAP or CSPTLP co-localization with TRAP or CSP??
Future DirectionsFuture Directions
AcknowledgementsAcknowledgements
Thomas J Templeton, PhDCristina K Moreira, PhDCatherine Lavazec, PhDChristian, PhDSabrina Scholz, PhD StudentTempleton LabDeitsch LabQuadri Lab
Questions?Questions?