Characterization of the putative surface protein TLP in Plasmodium berghei and Plasmodium falciparum Ryan M Harrison 1,2, Cristina K Moreria 3, Catherine Lavazec 3, Thomas J Templeton 3 1 Gateways to the Laboratory Program, Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program, New York, NY 2 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 3 Department of Immunology and Microbiology, Weill Cornell Medical College, New York, NY

Malaria in Context 41% of the World Population live in endemic areas 41% of the World Population live in endemic areas Estimated 700,000 to 2.7 million malaria deaths annually, 75% of them African children Estimated 700,000 to 2.7 million malaria deaths annually, 75% of them African children

Plasmodium Life Cycle Sporozoite Merozoite Asexual/ Gametocyte Zygote Ookinete Oocyst Plasmodium, Encyclopedia of Life Sciences, 2006

TLP (Thrombospondin Like Protein) discovered via micro array (Hayward et al., 2000) TLP (Thrombospondin Like Protein) discovered via micro array (Hayward et al., 2000) Putative surface protein? Putative surface protein? Shares conserved domains with: Shares conserved domains with: History of TLP CSPTRAPCTRPTLP membranecytoplasmmembranecytoplasmmembranecytoplasmmembranecytoplasm Salivary Gland Sporozoite ?? Hemocoel Sporozoite ?? Ookinete ?? Asexual Stages ??

Experimental Plan: 1. Determine stage TLP expressed in Wild Type - Quantitative Real-Time PCR 2. Verify TLP knockout gene disruption - Diagnostic PCR - Southern Blot Project Goals Hypothesis: The TLP protein is predominately expressed in the sporozoite life cycle stage of both P. falciparum and P. berghei

P. berghei TLP Expression

P. falciparum TLP Expression

Verification of gene disruption A B C D WTTLP P. berghei A B C D P. falciparum A B C D WTTLP A B C D Diagnostic PCR Integrated cassette gDNA

Verification of gene disruption Southern blot design gDNA NcoIHindIII Hybridization Probe Wildtype TLP knockout 7,600bp gDNA NcoIHindIII Hybridization Probe 9,700bp HindIII 2000bp Integrated Cassette

Verification of gene disruption 1,650bp1,000bp 5,000bp12,000bp TLP WT P. falciparum Southern blot

The gene disruption cassette is likely integrated into the TLP knockout parasite genome The gene disruption cassette is likely integrated into the TLP knockout parasite genome TLP is expressed predominantly in the sporozoite life cycle stage of both P. berghei and P. falciparum TLP is expressed predominantly in the sporozoite life cycle stage of both P. berghei and P. falciparum Conclusion CSPTRAPCTRPTLP membranecytoplasmmembranecytoplasmmembranecytoplasmmembranecytoplasm Salivary Gland Sporozoite Hemocoel Sporozoite Ookinete Asexual Stages

Hepatocye invasion assay Hepatocye invasion assay Reduced ability of TLG knockout sporozoites to recognize and/or invade hepatocyes ? Reduced ability of TLG knockout sporozoites to recognize and/or invade hepatocyes ? Immunoflouresence assay (IFA) Immunoflouresence assay (IFA) TLP co-localization with TRAP or CSP? TLP co-localization with TRAP or CSP? Future Directions

Acknowledgements Thomas J Templeton, PhD Cristina K Moreira, PhD Catherine Lavazec, PhD Christian, PhD Sabrina Scholz, PhD Student Templeton Lab Deitsch Lab Quadri Lab

Questions?