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Development of a Luciferase-Based Assay to Screen for Gametocyte-Specific Antimalarial Drugs

Fields, Becita Development of a Luciferase-Based Assay to Screen for Gametocyte-Specific Antimalarial Drugs. Master's Thesis, University of Pittsburgh.

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    Abstract

    For centuries, Malaria has continued to be one of the most deadly infectious diseases in the world. Almost all of the current antimalarial drugs target the asexual blood stages of the Plasmodium parasite responsible for the clinical pathology of malaria, but nearly all have no activity against the mature gametocyte or sexual stage that is responsible for the transmission of the parasite through the mosquito vector. Renewed interest in global eradication of malaria has turned some of the focus on blocking transmission. We have developed transgenic Plasmodium berghei that expresses luciferase under the control of gametocyte-specific promoters. Pb920Lux is a transgenic parasite that expresses luciferase in both male and female gametocytes, while Pb610Lux is a transgenic parasite that expresses luciferase in the male gametocyte. Immunofluorescence assay (IFA) shows luciferase is expressed in some parasites and in accordance with a previous study, suggest these may be gametocytes. These transgenic parasites were then used in a luciferase-based drug assay. Seven known antimalarial drugs were used to confirm the validity of the assay. Four of the known drugs had gametocidal activity, while three of the drugs have no gametocidal activity. We first created a dose response using the Pb920Lux and Pb610Lux along with PbGFPLuxcon as our control. From there we calculate the IC50 values of these drugs and compared them to the IC50 values calculated using PbGFPLuxcon (control). As expected, the transgenic parasites showed significant gametocidal activity in the four known drugs and no significant activity in the three non-gametocidal drugs. We confirmed this finding by calculating the percentage of parasites and gametocyte by light microscope and compared these to our findings with the luciferase-based assay. Next we analyzed four unknown drugs and found that they contain no gametocidal activity. The public health importance of developing a luciferase-based assay specific for gametocytes is to provide a simple and efficient method of detecting gametocidal drugs in order to prevent the transmission of malaria.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairTarun, AliceAST22@PITT.EDU
    Committee MemberMartinson, JeremyJMARTINS@PITT.EDU
    Committee MemberBoyle, JonBOYLEJ@PITT.EDU
    Committee MemberChakrabarti, KausikKAUSIK@ANDREW.CMU.EDU
    Title: Development of a Luciferase-Based Assay to Screen for Gametocyte-Specific Antimalarial Drugs
    Status: Published
    Abstract: For centuries, Malaria has continued to be one of the most deadly infectious diseases in the world. Almost all of the current antimalarial drugs target the asexual blood stages of the Plasmodium parasite responsible for the clinical pathology of malaria, but nearly all have no activity against the mature gametocyte or sexual stage that is responsible for the transmission of the parasite through the mosquito vector. Renewed interest in global eradication of malaria has turned some of the focus on blocking transmission. We have developed transgenic Plasmodium berghei that expresses luciferase under the control of gametocyte-specific promoters. Pb920Lux is a transgenic parasite that expresses luciferase in both male and female gametocytes, while Pb610Lux is a transgenic parasite that expresses luciferase in the male gametocyte. Immunofluorescence assay (IFA) shows luciferase is expressed in some parasites and in accordance with a previous study, suggest these may be gametocytes. These transgenic parasites were then used in a luciferase-based drug assay. Seven known antimalarial drugs were used to confirm the validity of the assay. Four of the known drugs had gametocidal activity, while three of the drugs have no gametocidal activity. We first created a dose response using the Pb920Lux and Pb610Lux along with PbGFPLuxcon as our control. From there we calculate the IC50 values of these drugs and compared them to the IC50 values calculated using PbGFPLuxcon (control). As expected, the transgenic parasites showed significant gametocidal activity in the four known drugs and no significant activity in the three non-gametocidal drugs. We confirmed this finding by calculating the percentage of parasites and gametocyte by light microscope and compared these to our findings with the luciferase-based assay. Next we analyzed four unknown drugs and found that they contain no gametocidal activity. The public health importance of developing a luciferase-based assay specific for gametocytes is to provide a simple and efficient method of detecting gametocidal drugs in order to prevent the transmission of malaria.
    Defense Date: 22 November 2011
    Approval Date: 30 January 2012
    Submission Date: 29 November 2011
    Release Date: 30 January 2012
    Access Restriction: No restriction; Release the ETD for access worldwide immediately.
    Patent pending: No
    Number of Pages: 67
    Institution: University of Pittsburgh
    Thesis Type: Master's Thesis
    Refereed: Yes
    Degree: MS - Master of Science
    Uncontrolled Keywords: Plasmodium, gametocytes, luciferase, antimalarial, pyrimethamine, primaquine, atovaquone, tetracycline, amodiaquine, artesunate, quinine, fatty acids, Plasmodium berghei
    Schools and Programs: Graduate School of Public Health > Infectious Diseases and Microbiology
    Date Deposited: 30 Jan 2012 13:41
    Last Modified: 31 Jan 2012 01:15

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