RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-377
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0715100; Gene model (P.falciparum): PF3D7_0815900; Gene product: dihydrolipoyl dehydrogenase, apicoplast (dihydrolipoamide dehydrogenase; PDH E3; E3)
Phenotype Liver stage;
Last modified: 5 April 2010, 11:57
  *RMgm-377
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20059687
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherY. Pei; S.H.I. Kappe
Name Group/DepartmentNot applicable
Name InstituteSeattle Biomedical Research Institute, University of Washington
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-377
Principal namee3-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageBALB/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 E3 (lipoamide dehydrogenase).

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 E3 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 E3 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 E1α (pyruvate dehydrogenase E1 alpha subunit; RMgm-376) and an independent mutant lacking expression of PDH E3 (RMgm-379)

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-371: A mutant expressing myc-tagged PDH E1α (pyruvate dehydrogenase E1 alpha subunit)
RMgm-372: A mutant expressing myc-tagged PDH E3
RMgm-376: A mutant lacking expression of PDH E1α (pyruvate dehydrogenase E1 alpha subunit)
RMgm-378: A mutant lacking expression of  PDH E1α and expressing RFP under the constitutive eef1a promoter.
RMgm-379: A mutant lacking expression of  PDH E3 and expressing RFP under the constitutive eef1a promoter.

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0715100
Gene Model P. falciparum ortholog PF3D7_0815900
Gene productdihydrolipoyl dehydrogenase, apicoplast
Gene product: Alternative namedihydrolipoamide dehydrogenase; PDH E3; E3
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid KpnI, SacII
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1AAAGGTACCCTCGCTAAAATAATTCATAA
Additional information primer 15'UTR Forward (KpnI)
Sequence Primer 2GGGAAGCTTGCTAAAAGAGAAGACGAAAATGACG
Additional information primer 25'UTR Reverse (HindIII)
Sequence Primer 3AAAACTAGTCGCATCAAGCATTAAGAGTAATCG
Additional information primer 33'UTR Forward (SpeI)
Sequence Primer 4AAACCGCGGGGTACCTGATTTATTCTCTTTTTGAG
Additional information primer 43'UTR Reverse (SacII)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6