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Characterization of the putative surface protein TLP in Plasmodium berghei and Plasmodium falciparum

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

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Characterization of the putative surface protein TLP in Plasmodium berghei and Plasmodium falciparum

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  1. Characterization of the putative surface protein TLP in Plasmodium berghei and Plasmodium 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, NY 2 Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 3 Department of Immunology and Microbiology, Weill Cornell Medical College, New York, NY

  2. Malaria in Context • 41% of the World Population live in endemic areas • Estimated 700,000 to 2.7 million malaria deaths annually, 75% of them African children http://www.cdc.gov/malaria/facts.htm

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

  4. History of TLP • TLP (Thrombospondin Like Protein) discovered via micro array (Hayward et al., 2000) • Putative surface protein? • Shares conserved domains with:

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

  6. P. berghei TLP Expression

  7. P. falciparum TLP Expression

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

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

  10. P. falciparum Southern blot 1,650bp 1,000bp TLP WT 12,000bp 5,000bp Verification of gene disruption

  11. Conclusion • 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

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

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

  14. Questions?

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