The Study of Autophagy in <i>Plasmodium falciparum</i>
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The Ohio State University / OhioLINK
2013
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu13855866612021-08-03T06:20:51Z The Study of Autophagy in <i>Plasmodium falciparum</i> Walker, Dawn Marie Biomedical Research Parasitology Microbiology Molecular Biology Biochemistry autophagy Plasmodium falciparum malaria Atg8 antimalarials Atg7 Apicomplexa parasite The Plasmodium falciparum genome encodes a limited number of putative autophagy genes, specifically the four genes involved in Atg8 lipidation, an essential step in formation of autophagosomes. In other eukaryotic systems, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. We have confirmed that these four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite’s erythrocytic stages. We expect that these putative autophagy genes are the essential players of a functional Atg8 lipidation pathway in P. falciparum. To assess PfAtg8 localization, we have expressed mCherry tagged PfAtg8 in the parasite and observed an unusual tubular localization. This has recently been confirmed as localization to the apicoplast, a unique organelle in apicomplexan parasites. This leads to our hypothesis of PfAtg8 involvement in the maintenance of this essential organelle, an interesting and novel role for an autophagy pathway. Recent efforts by our lab have focused on dissecting the biochemistry of this pathway. We have genetically engineered parasites to allow for regulatable expression of the activating enzyme PfAtg7. Upon PfAtg7 attenuation, parasites exhibit slow growth in culture, indicating the essentiality of this enzyme for normal growth. Furthermore, we have recently acquired a stable parasite line harboring a transposable element upstream of PfATG7 that also results in slow parasite growth due to a reduction in PfATG7 expression as confirmed by quantitative PCR. We have also modified the PfATG7 locus to introduce a C-terminal hemagglutinin (HA) tag. Detection of PfAtg7-HA by western blot has revealed the presence of two species, one at the predicted 150kDa and one at 65kDa. Experiments are in process to determine which form(s) are active and to confirm E1- type ligase activity. To assess the role of PfAtg7 in apicoplast maintenance, we are evaluating inhibitors of the apicoplast’s isoprenoid biosynthesis pathway. Parasites with attenuated levels of PfATG7 are more sensitive to fosmidomycin, a drug that targets an enzyme in the non-mevalonate pathway. This suggests that PfAtg7 is involved in maintaining properly functioning apicoplasts. Measuring the cell cycle time, number of merozoites, and doubling time indicates a delayed death-like phenotype in parasites with attenuated PfAtg7, which suggests an apicoplast defect. Experiments are ongoing to determine the exact mechanism responsible for slow growth of these parasites. As to translational implications of this research, it has been shown possible to develop specific inhibitors for E1-type ligases. Such inhibitors, like the mammalian NEDD activating enzyme, are currently in clinical trials as anticancer therapeutics. We propose a similar strategy in the development of specific and selective PfAtg7 inhibitors. If successful, these inhibitors would represent a novel class of antimalarials. 2013 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1385586661 http://rave.ohiolink.edu/etdc/view?acc_num=osu1385586661 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Biomedical Research Parasitology Microbiology Molecular Biology Biochemistry autophagy Plasmodium falciparum malaria Atg8 antimalarials Atg7 Apicomplexa parasite |
| spellingShingle |
Biomedical Research Parasitology Microbiology Molecular Biology Biochemistry autophagy Plasmodium falciparum malaria Atg8 antimalarials Atg7 Apicomplexa parasite Walker, Dawn Marie The Study of Autophagy in <i>Plasmodium falciparum</i> |
| author |
Walker, Dawn Marie |
| author_facet |
Walker, Dawn Marie |
| author_sort |
Walker, Dawn Marie |
| title |
The Study of Autophagy in <i>Plasmodium falciparum</i> |
| title_short |
The Study of Autophagy in <i>Plasmodium falciparum</i> |
| title_full |
The Study of Autophagy in <i>Plasmodium falciparum</i> |
| title_fullStr |
The Study of Autophagy in <i>Plasmodium falciparum</i> |
| title_full_unstemmed |
The Study of Autophagy in <i>Plasmodium falciparum</i> |
| title_sort |
study of autophagy in <i>plasmodium falciparum</i> |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2013 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1385586661 |
| work_keys_str_mv |
AT walkerdawnmarie thestudyofautophagyiniplasmodiumfalciparumi AT walkerdawnmarie studyofautophagyiniplasmodiumfalciparumi |
| _version_ |
1719435050364174336 |