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?