RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
DisruptedGene model (rodent): PY17X_0809500; Gene model (P.falciparum): PF3D7_0319000; Gene product: P-type ATPase, putative (ATPase7)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 29 July 2021, 13:05
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) : 34301597
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/ResearcherYang Z, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name InstituteXiamen University
CityXiamen, Fujian
Name of the mutant parasite
RMgm numberRMgm-5046
Principal nameΔATPase7
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal numbers of ookinetes are produced with wild-type gliding motility and morphology. ookinetes invade mosquito midgut epithelium. Evidence is presented that ookinetes are eliminated (within hours) during epithelium traversal.
OocystNo oocyst formation
SporozoiteNo oocyst and sporozoite formation
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of ATPase7
The mutant has been generated using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095

Protein (function)
A 1764–amino acid protein with 10 predicted transmembrane helixes with extensive identity to key P4-ATPase subdomains.
Asymmetric distribution of lipid molecules across the two leaflets of biological membranes is an important feature of eukaryotic cells. To establish asymmetry, eukaryotic organisms ranging from yeast to human encode a specific type of membrane transporters known as flippases that translocate phospholipids to the cytosolic side of membranes in a reaction driven by adenosine triphosphate (ATP). These flippases belong to the P4-ATPase subfamily of P-type adenosine triphosphatases (ATPases)), and CDC50 proteins act as essential P4-ATPase cofactors. Primate malaria parasites encode four putative canonical P4-ATPases, while rodent malaria parasites encode three.

Normal numbers of ookinetes are produced with wild-type gliding motility and morphology. ookinetes invade mosquito midgut epithelium. Evidence is presented that ookinetes are eliminated (within hours) during epithelium traversal.
No oocyst and sporozoite formation

Additional information
Evidence is presented that: 
- Elimination of Δatp7 ookinetes is independent of mosquito complement system 
- Ookinete microinjection into the mosquito hemocoel rescues the Δatp7 defects
- ATP7 is localized in the ookinete plasma membrane, and flippase activity is essential for its function (see below) 
- Phosphatidylcholine uptake defect of Δatp7 ookinetes 
- ATP7 colocalizes and interacts with the CDC50C cofactor in ookinetes
- CDC50C depletion in ookinetes phenocopies ATP7 deficiency during mosquito transmission

ATP7 was expressed in gametocytes, mosquito midgut oocysts, and salivary gland sporozoites but was not detected in asexual blood-stage parasites. ATP7 was detected in the cytoplasm of gametocytes and oocysts but displayed a peripheral localization in sporozoites. Costaining of the atp7::6HA gametocytes with -tubulin (male gametocyte specific) and HA antibodies showed that ATP7 was expressed only in female gametocytes. During zygote to ookinete differentiation in vitro, ATP7 was distributed in both the cytoplasm and the cell periphery from zygote to retort but was mostly localized to the cell periphery of mature ookinetes.

To test whether a conserved flippase motif within ATP7 is required for its function, we generated parasites carrying atp7 mutations that are predicted to compromise its flippase activity but not subcellular localization. It is known that the conserved motif Asp- Gly-Glu-Ser/Thr (DGES/T) in the actuator domain are critical for the catalytic activity of P4-ATPases and that E to Q mutation in these residues abolish the flippase activity. Accordingly, we replaced E210 with Q in the atp7::6HA strain in an attempt to generate an enzymatically inactive mutant designated as atp7m1. A control strain (atp7m2) was also generated with a silent mutation still encoding “DGES”. The E210Q substitution had no effect on the protein level or localization of ATP7 in atp7m1 ookinetes compared to the parental strain. The atp7m1 parasites produced comparable levels of ookinetes as the atp7::6HA strain but were eradicated during midgut traversal  and therefore developed no oocysts in the mosquitoes. These results suggest that ATP7 is a functional flippase and that its activity is required for midgut traversal of ookinetes.

To determine the female inheritance of ATP7 function,  genetic crosses of the Δatp7 with either Δmap2 (male gamete-deficient) or Δnek4 (female gamete- deficient) parasites were performed. Normal numbers of midgut oocysts were observed in mosquitoes on day 7 pi in the Δatp7 × Δmap2 but not the Δatp7 × Δnek4 cross, in agreement with the specific expression of ATP7 in female gametocytes.

From the Abstract:
'Disruption of ATP7 blocks the parasite infection of mosquitoes. ATP7 is localized on the ookinete plasma membrane. While ATP7-depleted ookinetes are capable of invading the midgut, they are eliminated within the epithelial cells by a process independent from the mosquito complement-like immunity. ATP7 colocalizes and interacts with the flippase cofactor CDC50C. Depletion of CDC50C phenocopies ATP7 deficiency. ATP7- depleted ookinetes fail to uptake phosphatidylcholine across the plasma membrane. Ookinete microinjection into the mosquito hemocoel reverses the ATP7 deficiency phenotype.'

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0809500
Gene Model P. falciparum ortholog PF3D7_0319000
Gene productP-type ATPase, putative
Gene product: Alternative nameATPase7
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationCRISPR-Cas9 plasmid pYCm was used for all the parasite genetic modification. To construct vectors for gene editing, we amplified 5′ and 3′ genomic sequence (400 to 500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. Oligonucleotides for single guide RNAs (sgRNAs) were mixed in pairs, denatured at 95°C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. DNA fragments encoding 6HA, 4Myc, 3V5, and GFP or mCherry were inserted between the left and right arms in frame with the gene of interest. For each gene, two sgRNAs were designed to target sites close to the C- or N-terminal part of the coding region. Infected red blood cells (iRBCs) were electroporated with 5 to 10 μg of plasmid DNA using the Lonza Nucleofector . Transfected parasites were immediately intravenously injected into a naïve mouse and were exposed to pyrimethamine (6 mg/ml) 24 hours after transfection.
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