A genetic analysis to elucidate the function of the Plasmodium falciparum parasitophorous vacuole protein, PfPV1

Malaria is one of the most lethal infectious diseases worldwide. Understanding the biology of the causative agent Plasmodium will lead to better control of the disease. The biogenesis and maintenance of the parasitophorous vacuole (PV) within the infected erythrocyte is an essential factor for...

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Bibliographic Details
Main Author: Chu, T. T. Trang
Contributors: Lingelbach, Klaus (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2009
Biologie
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Summary:Malaria is one of the most lethal infectious diseases worldwide. Understanding the biology of the causative agent Plasmodium will lead to better control of the disease. The biogenesis and maintenance of the parasitophorous vacuole (PV) within the infected erythrocyte is an essential factor for parasite survival. The PV has been postulated to be involved in various pivotal functions, however little is known about the PV contents and their respective functions. Our group had previously provided the first PV’s proteome research and have continuously exposed more members of this important compartment. The protein PfPV1 was a newly discovered PV localisation protein, encoded by the PF11_0302 gene. In order to address the function of this protein a gene knock-out strategy was applied. A search for the interacting proteins of PfPV1 was also carried out using the GST pull-down assay. The first attempt to knock out the encoding gene was the double-crossover strategy in the presence of a negative selection. The knocked-out parasite was unable to obtain. However, the integration into the PfPV1 locus did occur, evidenced by the presence of both endogenous and knock-out band in the specific southern blot hybridisation. The PfPV1 gene was therefore assumed to be essential for in vitro growth, thus the targeting into the endogenous locus was accompanied by a duplication event for maintaining expression of the gene. The assumption was further validated by the second knock-out strategy, using the complementing experiment. The result had been expected to be able to disrupt the endogenous gene locus through the knock-out vector while concomitantly expressing a copy of PfPV1 under the control of a foreign promoter from an episomal plasmid, which should not recombine with the endogenous gene locus. However, the gene was still resistant to be disrupted. Various clones were isolated from the double transfected parasites. One of the clones has lost the episomal copy of the PfPV1 gene and showed the same southern blot result as the single transfected parasite, indicating that the parasite needs to maintain the expression of the endogenous gene. Other clones, if keeping the episomal copy of the PfPV1, did not show the specific integration. The result strongly suggests that the PfPV1 expression needs to be controlled by its endogenous promoter to be fully active. The data has also proved for the first time that in some cases of the negative selection strategy, upon the integration, the Plasmodium parasite might rearrange the thymidine kinase encoding sequence in order to inactivate its activity, therefore prevent the lethal effect from converting ganciclovir to toxic metabolite. In the GST pull-down assay, no interacting protein was obtained. However, the experiment was carried out with the cell extract from trophozoite-stage parasites, thus might not detect interactions at other stages. In conclusion, the data suggest that PfPV1 is a conserved, unique protein with unknown but essential function during the intraerythrocytic cycle.
DOI:https://doi.org/10.17192/z2009.0710