RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1140700; Gene model (P.falciparum): PF3D7_1364900; Gene product: ferrochelatase (FC)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 26 June 2017, 18:22
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) : 23935500
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherNagaraj, VA; Padmanaban, G.
Name Group/DepartmentDepartment of Biochemistry
Name InstituteIndian Institute of Science
Name of the mutant parasite
RMgm numberRMgm-925
Principal namePbFCKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystStrongly reduced oocyst production
SporozoiteStrongly reduced oocyst and no sporozoite production.
Mice remained negative when infected by mosquito bite or after i.v. injection of sporozoites (see also additional remarks phenotype)
Liver stageStrongly reduced oocyst and no sporozoite production.
Additional remarks phenotype

The mutant lacks expression of ferrochelatase (FC)

Protein (function)
The malaria parasite is capable of de novo heme biosynthesis despite its ability to acquire heme from red blood cell (RBC) hemoglobin. The first enzyme of the de novo heme-biosynthetic pathway, δ-aminolevulinate synthase (ALAS) and the last two enzymes, Protoporphyrinogen IX oxidase (PPO) and Ferrochelatase (FC) localize to the mitochondrion. The enzymes that catalyze the intermediate steps: ALA dehydratase (ALAD), Porphobilinogen deaminase (PBGD) and Uroporphyrinogen III decarboxylase (UROD) localize to the apicoplast whereas, the next enzyme Coproporphyrinogen III oxidase (CPO) is cytosolic. Earlier studies showed that host ALAD and FC are imported into the parasites in the intraerythrocytic stages, suggesting that the host machinery may augment parasite heme synthesis.

Mutant parasites showed normal blood stage development, indicating a non-essential role of FC during blood stage development (and for gametocyte and ookinete production). Mutant parasites showed strongly reduced oocyst and no sporozoite production .

Additional information
Evidence is presented that blood stage parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Evidence is presented that de novo pathway is essential for parasite development in the mosquito and liver stages. PbKO parasites formed strongly reduced oocysts and did not form sporozoites in the salivary glands.
Oocyst production in PbALASKO parasites (RMgm-924) recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement.

Other mutants
RMgm-924: A mutant lacking expression of delta-aminolevulinic acid synthetase (ALAS; PBANKA_145920)

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1140700
Gene Model P. falciparum ortholog PF3D7_1364900
Gene productferrochelatase
Gene product: Alternative nameFC
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 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
Additional information primer 1PbFCKO 5’UTR(F)
Additional information primer 2PbFCKO 5’UTR(R)
Additional information primer 3PbFCKO 3’UTR(R)
Additional information primer 4PbFCKO 3’UTR(R)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6