| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene disruption,
Introduction of a transgene
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 20059687
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| MR4 number |
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| Parent parasite used to introduce the genetic modification |
| Rodent Malaria Parasite | P. yoelii |
| Parent strain/line | P. y. yoelii 17XNL |
| Name parent line/clone |
Not applicable
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| Other information parent line | 17XNL is a non-lethal strain of P. yoelii |
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| The mutant parasite was generated by |
| Name PI/Researcher | Y. Pei; S.H.I. Kappe |
| Name Group/Department | Not applicable |
| Name Institute | Seattle Biomedical Research Institute, University of Washington |
| City | Seattle |
| Country | USA |
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| Name of the mutant parasite |
| RMgm number | RMgm-378 |
| Principal name | e1α-rfp |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Not different from wild type |
| Fertilization and ookinete | Not different from wild type |
| Oocyst | Not different from wild type |
| Sporozoite | Not different from wild type |
| Liver stage | BALB/c mice intravenously injected with 1x10(4)-5x10(4) salivary gland sporozoites did not develop blood stage infections.
Analysis of in vitro cultured liver stages showed significant defects in terms of growth and nuclear division. At 24 h pi, no difference was observed between mutant and wild type liver stages with regard to protein expression and parasite size. During late liver stage development (43 and 52 h pi), no expression was detected of the merozoite-specific protein MSPI in mutant parasites. Mutant parasites showed significant defects in terms of growth and nuclear division and the average size of the mutant parasites at 43 h pi was approximately 30% of wild type parasites. |
| Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of PDH E1α (pyruvate dehydrogenase E1 alpha subunit ) and expresses RFP under the constitutive eef1a promoter..
Protein (function)
Acetyl-CoA is synthesized from pyruvate by the enzyme complex pyruvate dehydrogenase (PDH). PDH is a member of the α-ketoacid dehydrogenase multienzyme complexes and consists of three subunits: pyruvate dehydrogenase (E1), dihydrolipoyl acetyltransferase (E2) and lipoamide dehydrogenase (dihydrolipoyl dehydrogenase; E3). Acetyl-CoA is an essential precursor of fatty acid synthesis in plastids and fuels the tricarboxylic acid (TCA) cycle in mitochondria, which generates energy in the form of ATP.
In most eukaryotes PDH localizes to the mitochondria where it converts pyruvate into acetyl-CoA and NADH that then enter the TCA cycle for complete oxidization. In plants, a second unique PDH complex is harboured by the plastid and its function is to provide acetyl-CoA for fatty acid synthesis. In Plasmodium there is no evidence for the existence of a mitochondrial PDH complex and the sole Plasmodium PDH complex is targeted to the apicoplast.
In previous studies it has been shown that apicoplast-targeted proteins include enzymes of the FAS II pathway. The presence of this pathway indicates that as well as taking up lipids from its host, Plasmodium also utilizes its own de novo fatty acid synthesis. Transcriptome and proteome analyses of Plasmodium liver stages have demonstrated the expression of apicoplast-targeted enzymes involved in FAS II in liver stages, indicating an important role of de novo FAS II in the developing liver stage. It has been shown that deletion of genes encoding FAS II elongation enzymes (i.e. FabI, FabB/F, FabZ.) caused no defects in parasite blood stage replication and mosquito stage development but livers stage development was severely reduced in the FAS II-deficient parasites.
Phenotype
The phenotype analyses indicate a non-essential role of PDH E1α for blood stages and mosquito stages but an important role during late liver stage development. Analysis of in vitro cultured liver stages showed significant defects in terms of growth and nuclear division. Disruption of PDH E1α showed a similar phenotype to mutants lacking FAS II elongation enzymes (i.e. FabI, FabB/f; FabZ). This suggests that in Plasmodium the sole function of PDH is to provide the acetyl-CoA necessary for de novo fatty acid synthesis. See also the phenotype of a mutant lacking PDH E3 (lipoamide dehydrogenase; RMgm-377) and an independent mutant lacking expression of PDH E1α (RMgm-376)
Additional information
Mutant parasites develop normal apicoplasts in early liver stages. However, although the apicoplasts were still developing beyond 25 h pi, they developed at a much slower rate when compared with the apicoplast in liver stages of wild type parasites.
Analyses of mutants expressing cmyc-tagged version of PDH E1α (RMgm-371) and PDH E3 (RMgm-372) showed no significant expression in blood stages. PHD E1α-myc and PHD E3-myc expression was observed in mosquito salivary gland sporozoites. The myc-tagged proteins were localized to a small circular structure next to the nucleus. This localization pattern is similar to the localization of epitope-tagged FAS II elongation enzymes that are expressed in the apicoplast . To confirm apicoplast localization of E1α and E3, an antibody was generated against P. yoelii FabI to be used as an apicoplast marker. IFA analyses demonstated the colocalization of E1α-myc and E3-myc with FabI confirming that PDH, as previously shown for P. falciparum, is exclusively targeted to the apicoplast in P. yoelii. In liver stages E1α-myc staining was observed at 24 hour as tadpole-shaped structures surrounding the dividing nuclei . The complexity of the apicoplast increased after 24 h and exhibited intensive staining at 30 and 43 hi, which coincides with the extensive expansion of the apicoplast during schizogony. At 52 hi, E1α-myc expression appeared as small dots in a similar number and size to the individual nuclei present in the developing merozoites, indicating that each mature merozoite contains an individual apicoplast. IFA analyses of liver stages of parasites expressing PDH E3-myc showed similar expression patterns.
Other mutants
RMgm-376: An independent mutant lacking expression of PDH E1α
RMgm-371: A mutant expressing myc-tagged PDH E1α
RMgm-372: A mutant expressing myc-tagged PDH E3 (lipoamide dehydrogenase)
RMgm-377: A mutant lacking expression of PDH E3 (lipoamide dehydrogenase)
RMgm-379: A mutant lacking expression of PDH E3 and expressing RFP under the constitutive eef1a promoter.
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*RMgm-378
