RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_1303400; Gene model (P.falciparum): PF3D7_1439500; Gene product: oocyst rupture protein 2, putative (ORP2; NFYC)
Details mutation: (Part of) the histone fold domain (34-107 aa) of ORP2 deleted
Phenotype Oocyst; Sporozoite;
Last modified: 21 December 2018, 17:30
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30391497
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherSiden-Kiamos I, Currà C
Name Group/DepartmentFoundation for Research and Technology-Hellas
Name InstituteInstitute of Molecular Biology and Biotechnology
Name of the mutant parasite
RMgm numberRMgm-4560
Principal nameORP2hfd(-)
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
OocystNormal oocyst production. Sporozoites are formed inside oocysts. Oocysts do not rupture and oocysts do not release sporozoites.
SporozoiteNormal oocyst production. Sporozoites are formed inside oocysts. Oocysts do not rupture and oocysts do not release sporozoites. No salivary gland sporozoites.
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a mutated orp2 gene that is fused to GFP. (Part of) the histone fold domain (34-107 aa) of the endogenous orp2 gene is deleted and the mutated orp2 gene was C-terminally fused to GFP

Protein (function)
Oocyst Rupture Proteins (ORP1 and ORP2), localize at the oocyst capsule, that are each essential for rupture of the oocysts. Both ORPs contain a histone fold domain (HFD) at the N-terminal, implicated in the mechanism of oocyst rupture, possibly through the formation of a heterodimer between the two HFDs. The HFD domain maps to aa 30-107 in ORP2

Oocysts of mutants lacking expression of ORP2 do not rupture and oocysts do not release sporozoites.

The mutant described here that lacks (part of) the histone fold domain (HFD) shows the same phenotype, i.e. oocysts do not rupture and oocysts do not release sporozoites. This result indicate that the HFD is required for rupture of the oocyst.  The GFT-tag was not expected to interfere with the function of the protein as a C-terminal mCherry fusion of the WT protein did not confer any phenotypical disadvantage to the parasite (Currà et al., 2016).

Additional information
In the paper two additional mutants are reported that express C-terminal truncated forms of ORP2. These C-terminal deletions of ORP2 reveal two important regions for function.

From the paper:
'In order to associate a function to the C-terminal half of the protein, we generated two progressive deletion mutants from the C-terminal end. The N-terminal region was kept intact while motifs 17 and 20, were removed from 414ORP2::mCherry mutant parasites. These mutants expressed truncated ORP2 fused to C-terminal mCherry for the detection of the protein. The shorter deletion in 576ORP2::mCherry contains the first 576 amino acids while 414ORP2::mCherry comprises only the first 414 amino acids. Two clones of each mutant were generated  and analyzed throughout the Plasmodium life-cycle. Truncated proteins fused to mCherry were also revealed in western blots at expected bands of 73 kDa for 414ORP2::mCherry and 91 kDa for 576ORP2::mCherry samples . As expected, both mutants developed normally in the blood stages and oocysts were formed after transmission to mosquitoes. At day 12 these had a typical appearance and the numbers of oocysts were similar to WT. The chimeric ORP2::mCherry protein was detected in the cytoplasm of the oocysts, similar to what was found for the full-length protein. At days 13-14  the oocysts of the longer C terminal truncated line 576ORP2::mCherry ruptured, releasing sporozoites and thus no oocysts of this mutant were detected at day 21. However, the oocysts from the mutant 414ORP2::mCherry persisted in the midguts at least until day 21 and no free sporozoites were detected. ORP1 was present in the oocyst capsule and DNA replication had taken place as verified by staining with DAPI. Sporozoites were detected in 414ORP2::mCherry oocysts by bright field microscopy but the oocyst capsule did not rupture.

Salivary glands were dissected from mosquitoes infected with the two parasite lines harbouring the C-terminal truncations. As expected, no sporozoites were detected in salivary glands of mosquitoes infected with the short mutant 414ORP2::mCherry. Sporozoites of the longer mutant 576ORP2::mCherry were found in salivary glands dissected at day 21, although the number of sporozoites was reduced approximately four- to six-fold compared with WT. The differences between the WT and the mutant in sporozoite motility and numbers of sporozoites were significant (P<0.0001, Student's t-test.). A gliding motility assay of 414ORP2::mCherry was carried out with midgut sporozoites mechanically released from oocysts at day 21 but no motility was detected. On the other hand, for 576ORP2::mCherry, a few sporozoites collected at day 21 from salivary glands were able to glide, but the number of gliding sporozoites compared with WT was reduced approximately four-fold 576ORP2::mCherry sporozoites were tested for their ability to establish an infection in mice; 25,000 and 8000 sporozoites injected in the tail vein of naive mice were able to establish parasitemia of 0.3% after five or 7 days, respectively, from injection, compared with the control infections with WT where parasites were detected at day three or 4. Infected mosquitoes from both C-terminally truncated mutants were also allowed to feed on naïve mice, and as expected only 576ORP2::mCherry was able to establish an infection. However, only one out of four mice became infected and the time to patency in this single mouse was also substantially longer than in the WT-infected mice suggesting that these sporozoites are strongly impaired in infectivity.'

Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1303400
Gene Model P. falciparum ortholog PF3D7_1439500
Gene productoocyst rupture protein 2, putative
Gene product: Alternative nameORP2; NFYC
Details of the genetic modification
Short description of the mutation(Part of) the histone fold domain (34-107 aa) of ORP2 deleted
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) PCR construct 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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe pBAT plasmid was used to generate the final construct pORP2hfd(-). Since the HFD (histone fold domain) of ORP2 is very close to the N-terminal of the protein, a recombinant region was selected in the 5’ untranslated region (UTR) using primers 5/6 (670 bp) and cloned using SacII and SphI restriction enzymes. The ORP2 promoter was amplified with primers 7/8 (931 bp) cloned in the ApaI and XhoI restriction sites. A third and more complex step consists of the PCR amplification of the upstream and downstream region of the HFD using, respectively, primers 9/10 (1081 bp) and 11/12 (689 bp) annealed and amplified by nested PCR with primers 13/14 (1624 bp). The fragment was cloned in XhoI and KpnI restriction sites in the final plasmid. The coding region was sequenced with appropriate primers. The plasmid was linearized with PvuI before transfection.

Generation of progressive deletion ORP2 mutants:
Two mutants expressing progressive deletions of ORP2 fused to mCherry were generated. The plasmid ORP2::mCherry (Currà et al., 2016) was modified as appropriate for generation of the plasmids used to introduce two deletions in the mutant parasites. Starting from plasmid ORP2::mCherry, the PCR fragment between the SacII and SpeI sites was substituted with PCR fragments. p414ORP2::mCherry was generated by inserting a fragment which was amplified using 1/2 primers (638 bp, amino acids 194-314) and the p576ORP2::mCherry fragment was amplified with primers 3/4 (693 bp, amino acids 641-576). In each case this resulte in the protein being in frame with mCherry. Plasmids were sequenced and linearized with PvuI before 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