Effects on growth, hemoglobin metabolism and paralogous gene expression resulting from disruption of genes encoding the digestive vacuole plasmepsins of Plasmodium falciparum
| Autor: | John B. Dame, Pedro A. Moura, Tonya D. Bonilla, J. Alfredo Bonilla, Charles A. Yowell, David A. Fidock |
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| Rok vydání: | 2007 |
| Předmět: |
Hemeproteins
Erythrocytes Blotting Western Genes Protozoan Plasmodium falciparum Mutant Plasmepsin Paralogous Gene Biology Transfection Hemoglobins Gene expression Animals Aspartic Acid Endopeptidases Gene Gene knockout Southern blot Life Cycle Stages Reverse Transcriptase Polymerase Chain Reaction biology.organism_classification Molecular biology Blotting Southern Infectious Diseases Gene Expression Regulation Parasitology Gene Deletion |
| Zdroj: | International Journal for Parasitology. 37:317-327 |
| ISSN: | 0020-7519 |
| DOI: | 10.1016/j.ijpara.2006.11.008 |
| Popis: | Four of the plasmepsins of Plasmodium falciparum are localised in the digestive vacuole (DV) of the asexual blood stage parasite (PfPM1, PfPM2, PfPM4 and PfHAP), and each of these aspartic proteinases has been successfully targeted by gene disruption. This study describes further characterisation of the single-plasmepsin knockout mutants, and the creation and characterisation of double-plasmepsin knockout mutants lacking complete copies of pfpm 2 and pfpm 1 or pfhap and pfpm 2. Double-plasmepsin knockout mutants were created by transfecting pre-existing knockout mutants with a second plasmid knockout construct. PCR and Southern blot analysis demonstrate the integration of a large concatamer of each plasmid construct into the targeted gene. All mutants have been characterised to assess the involvement of the DV plasmepsins in sustaining growth during the asexual blood stage. Analyses reaffirmed that knockout mutants Δ pfpm 1 and Δ pfpm 4 had lower replication rates in the asexual erythrocytic stage than the parental line (Dd2), but double-plasmepsin knockout mutants lacking intact copies of either pfpm 2 and pfpm 1, or pfpm2 and pfhap , had normal growth rates compared with Dd2. The amount of crystalline hemozoin produced per parasite during the asexual cycle was measured in each single-plasmepsin knockout to estimate the effect of each DV plasmepsin on hemoglobin digestion. Only Δ pfpm 4 had a statistically significant reduction in hemozoin accumulation, indicating that hemoglobin digestion was impaired in this mutant. In the single-plasmepsin knockouts, no statistically significant differences were found in the steady state levels of mRNA from the remaining intact DV plasmepsin genes. Disruption of a DV plasmepsin gene does not affect the accumulation of mRNA encoding the remaining paralogous plasmepsins, and Western blot analysis confirmed that the accumulation of the paralogous plasmepsins in each knockout mutant was similar among all clones examined. |
| Databáze: | OpenAIRE |
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