Summary

RMgm-4183
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0714900; Gene model (P.falciparum): PF3D7_0816100; Gene product: phosphopantothenoylcysteine decarboxylase, putative (PPCDC)
Transgene
Transgene not Plasmodium: eGFP
Promoter: Gene model: PYYM_0712000; Gene model (P.falciparum): Not available; Gene product: heat shock protein, putative (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PY17X_0616300; Gene product: phosphopantothenate--cysteine ligase, putative (PPCDC)
Phenotype Oocyst; Sporozoite;
Last modified: 8 July 2017, 09:28
  *RMgm-4183
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28676844
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 line
The mutant parasite was generated by
Name PI/ResearcherHart RJ; Aly ASI
Name Group/DepartmentDepartment of Tropical Medicine
Name InstituteTulane University
CityNew Orleans
CountryUS
Name of the mutant parasite
RMgm numberRMgm-4183
Principal namePyppcdc(−)
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
OocystNormal ookinete development/production. Significant reduction in the number of oocysts (already at day 4 after feeding). Reduced size of oocysts. No sporozoite production inside oocyst
SporozoiteSignificant reduction in the number of oocysts (already at day 4 after feeding). Reduced size of oocysts. No sporozoite production inside oocyst. No salivary gland sporozoites.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PPCDC and expresses GFP under the control of the strong and constitutive hsp70 promoter.

Protein (function)
CoA is an essential cofactor for all prokaryotes and eukaryotes to support a large number of metabolic processes including fatty acid biosynthesis and oxidation, as well as carbohydrate and amino acid metabolism. In many living cells, the transport and utilization of pantothenic acid (vitamin B5, pantothenate in ionic form) is essential for CoA biosynthesis. The cellular machinery for the biosynthesis of CoA from exogenous pantothenate involves a putative pantothenate transporter (PAT) and five enzymes, PanK (Pantothenate Kinase), PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase), PPAT (Phosphopantetheine Adenylyltransferase), and DPCK (Dephospho-CoA Kinase).
Previous studies showed that PAT, PanK1, and PanK2 genes are dispensable for blood stage development in mice but are crucial for oocyst development and sporozoite formation in the mosquito.
In this study the following was shown: 'The intermediate enzymes PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase; RMgm-4183) were shown to be dispensable for asexual and sexual blood stage development, but they were essential for oocyst development and the production of sporozoites. However, the last two enzymes of this pathway, PPAT (Phosphopantetheine Adenylyltransferase; RMgm-4184) and DPCK (Dephospho-CoA Kinase; RMgm-4185), were essential for blood stage development. These results indicate alternative first substrate requirement for the malaria parasite, other than the canonical pantothenate, for the synthesis of CoA in the blood but not inside the mosquito midgut. Collectively, these studies indicate that CoA de novo biosynthesis is essential for both blood and mosquito stages'.

Phenotype
Normal blood stage development (including gametocytes). Normal ookinete development/production.Significant reduction in the number of oocysts (already at day 4 after feeding). Reduced size of oocysts. No sporozoite production inside oocyst. No salivary gland sporozoites.

Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0714900
Gene Model P. falciparum ortholog PF3D7_0816100
Gene productphosphopantothenoylcysteine decarboxylase, putative
Gene product: Alternative namePPCDC
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo create individual gene deletion of PyPPCS and PyPPCDC, a targeting vector, pAA20, containing the enhanced GFP (eGFP) reporter cassette and the
human DHFR resistance selection marker cassette was used to clone 5′ and 3′ regions of each gene.
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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameeGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PYYM_0712000
Gene Model P. falciparum ortholog Not available
Gene productheat shock protein, putative
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY17X_0616300
Gene productphosphopantothenate--cysteine ligase, putative
Gene product: Alternative namePPCDC
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4