RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1457700; Gene model (P.falciparum): PF3D7_1244500; Gene product: PIMMS57 protein (PIMMS57)
Name tag: GFP
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 20 June 2022, 14:05
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 33791240
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherUkegbu CV, Vlachou D
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
Name of the mutant parasite
RMgm numberRMgm-5001
Principal namec57::gfp
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 ookineteSee additional information below
OocystSeverely reduced sporozoite numbers, suggesting that insertion of GFP compromises the function of the protein.
SporozoiteSeverely reduced sporozoite numbers, suggesting that insertion of GFP compromises the function of the protein.
Liver stageNot tested
Additional remarks phenotype

The mutant c57::gfp expresses a C-terminal GFP-tagged version of PIMMS57

Protein (function)
PfPIMMS57 encodes a 810 amino acid-long (94 kDa) protein with a predicted signal peptide (aa 1–26) that overlaps with a putative transmembrane domain albeit with low probability according to Phobius (0.343). A second transmembrane domain close to the carboxy terminus of PfPIMMS57 (aa 677–697) is predicted with very high probability (0.874). The P. berghei orthologue, PbPIMMS57, encodes a protein of 774 amino acid-long (90 kDa) protein with two putative transmembrane domains (aa 6–23 and aa 633–653) predicted with high probability (0.984 and 0.967, respectively). These data suggest that both proteins are putatively membrane-bound. Higher sequence conservation between PIMMS57 orthologues is observed in the second half of the proteins compared to the first half, pointing to a conserved functional role served by this region. A previous in silico analysis identified in PfPIMMS57, two P. falciparum serine/threonine protein phosphatase type I catalytic (PfPP1c) binding motifs, the RVxF motif, RRKVNF (aa 347–352) and the Fxx[RK] x[RK] motif, FNKILKR (aa 488–494). These motifs are not conserved in the other PIMMS57 orthologues   

Severely reduced sporozoite numbers, suggesting that insertion of GFP compromises the function of the  protein. Also HA-tagging resulted in reduced oocyst and sporozoite numbers.
See RMgm-4998 for a mutant lacking expression of PIMMS57, showing reduced oocyst and sporozoite formation

Additional information
Three genes were selected for analysis that exhibit highly abundant transcripts 24 h post blood feeding in the midguts of Anopheles coluzzii mosquitoes.
PIMMS01: PF3D7_0112100; PBANKA_0201700 
PIMMS57: PF3D7_1244500; PBANKA_1457700
PIMMS22: PF3D7_0814600; PBANKA_1422900 
((PIMMS: Plasmodium Infection of the Mosquito Midgut Screen)

PbPIMMS01 and PbPIMMS57 transcripts were highly abundant in purified mature ookinetes and not detectable in mixed blood stages and purified gametocytes. While PbPIMMS57 appears to be specific for ookinetes, low levels of PbPIMMS01 transcripts are also detected in mature oocysts and midgut sporozoites at 10 days and 15 days post blood-feeding. Like PfPIMMS22, expression of PbPIMMS22 starts in gametocytes and continues at high levels at 24 hour and peaks in midgut sporozoites at day 15.

Using the non-functional c57::gfp parasite line and the α-GFP antibody in western blot analysis, high levels of the PbPIMMS57::GFP fusion protein were detected in mature ookinetes, at the expected molecular weight of 117 kDa. This band was not detected in MBS or gametocytes or in the ANKA 2.34 background parasite line. Similarly, western blot analysis using the c57::3xha transgenic line and the α-HA antibody confirmed  expression of the PbPIMMS57::3xHA protein in the ookinete at the expected molecular weight and its absence from gametocytes. The protein was mostly detected in the Triton insoluble fraction, indicating membrane association. No band was detected in the c507 reference line. To validate the ookinete expression of PbPIMMS57, the αPbc57 peptide antibody was utilized. In westerns, the α-Pbc57 antibody detected in ookinetes a band at about 90 kDa, close to the predicted molecular weight of PbPIMMS57. This band was not seen in blood stages, gametocytes. Fractionation assays revealed that, in ookinetes, PbPIMMS57 is mostly found in the Triton X-100 insoluble fraction with low amounts also seen in the Triton X-100 soluble fraction, suggesting that Pbc57 is membrane associated. These results agree with the prediction of transmembrane domains in this protein. The α-Pbc57 peptide antibody did not work in IFAs. IFAs on the developmentally compromised c57::gfp and c57::3xha transgenic lines revealed cytoplasmic localization of the PbPIMMS57::GFP and PbPIMMS57::3xHA fusion proteins with bias for the ookinete convex side, especially for the former, pointing to a possible involvement of the protein with ookinete motility or invasion machinery. However, given that both types of fusions lead to developmentally compromised parasites, these data must be interpreted with caution.

From the abstract:
PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1457700
Gene Model P. falciparum ortholog PF3D7_1244500
Gene productPIMMS57 protein
Gene product: Alternative namePIMMS57
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor GFP tagging of Pbc01 in the 2.34 line, a 603 bp ApaI/HindIII 5’ and a 770 bp EcoRI/BamHI 3’ homology arm region were amplified from P. berghei 2.34 genomic DNA using the primer pairs P1/P2 and P3/P4, respectively. For GFP tagging of Pbc57 in the 2.34 line, a 919 bp ApaI/SacII 5’ homology arm corresponding to the most 3’ region of the CDS without the stop codon and a 359 bp XhoI/XmaI 3’ homology arm region corresponding to the 3’ UTR of the gene were amplified using the primer pairs P5/P6 and P7/P8, respectively. For GFP tagging of Pbc22 in the 2.34 line, a 753 bp ApaI/HindIII 5’ homology arm corresponding to the most 3’ region of the CDS without the stop codon and a 530 bp EcoRI/BamHI 3’ homology arm region corresponding to the 3’ UTR of the gene were amplified using the primer pairs P9/P10 and P11/P12, respectively. The Pbc01 and Pbc22 fragments were cloned into the pBS-TgDHFR vector which carries a modified Toxoplasma gondii dihydrofolate gene (TgDHFR/TS) cassette that confers resistance to pyrimethamine (Dessens et al., 1999). The Pbc57 fragments were cloned into plasmid pL0035 which carries the hDHFR selection cassette. Finally, to put GFP tag in frame with the 3’ region of the CDS, a HindIII or SacII GFP- P. berghei DHFR 3’UTR fragment was amplified from the pL00018 vector (MRA787, MR4) using primers P13/P14 or P15/P16, respectively.
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