RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1444500; Gene model (P.falciparum): PF3D7_1229800; Gene product: myosin J (MyoJ)
Name tag: GFP
Phenotype Oocyst;
Last modified: 8 August 2019, 18:37
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) : 31283102
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/ResearcherWall RJ, Tewari R
Name Group/DepartmentSchool of Life Sciences, Queens Medical Centre
Name InstituteUniversity of Nottingham
Name of the mutant parasite
RMgm numberRMgm-4664
Principal nameMyoJ-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 ookineteNot different from wild type
OocystMyoJ‐GFP was only observed in mature oocysts, located at the junction between the differentiating sporozoites and the oocyst bodies. Following oocyst rupture, MyoJ‐GFP remained associated with the oocyst body and was not present in sporozoites.
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

The mutant expresses a C-terminal GFP-tagged version of  MyoJ.

Protein (function)
The myosin superfamily is comprised of molecular motors present during early eukaryotic cell evolution. In unicellular parasites, they perform a wide variety of cellular functions that require movement, including differentiation, host interactions, and cell invasion. The myosin molecule contains three main domains: the N‐terminal head domain, which hydrolyses ATP and binds actin filaments; the neck domain/lever arm, which has an α‐helical structure containing up to six IQ motifs; and a tail region, which is required for cargo binding. There are six P. berghei myosins, two of these (MyoA and MyoB) have no tail region, and the remainder have a tail, which in the case of MyoF contains five WD40 repeats.
Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analyses of blood stages, ookinetes and sporozoites revealed that the Class XIV myosins MyoA (PBANKA_0135570), MyoB (PBANKA_0110300), and MyoE (PBANKA_0112200) were strongly transcribed in the invasive stages with an abundance of MyoE transcript in developing merozoites within schizonts. MyoF (PBANKA_1344100) was transcribed at all stages and was second only to MyoA in abundance. In contrast, low levels of transcription of MyoK (PBANKA_0908500) and MyoJ (PBANKA_1444500) could be seen throughout these life cycle stages.

MyoJ‐GFP was only observed in mature oocysts, located at the junction between the differentiating sporozoites and the oocyst bodies. Following oocyst rupture, MyoJ‐GFP remained associated with the oocyst body and was not present in sporozoites.

Additional information
We investigated the presence and location of the six myosin proteins throughout the life cycle, by adding a C‐terminal GFP tag to each via single homologous recombination at the corresponding myosin gene locus. MyoA‐GFP and MyoB‐GFP have been generated previously. In addition, MLC-B (PBANKA_0929400 myosin light chain B, putative) was C-terminally tagged with GFP and MyoA was C-terminally tagged with mCherry.

We used live cell imaging to detect expression of each protein throughout the life cycle and to examine their location. The Class XIV myosins were detected predominantly in the invasive stages (developing merozoites in schizonts and merozoites, ookinetes, and developing sporozoites within oocysts and sporozoites). As shown previously, MyoA‐GFP was associated with the surface pellicle of each invasive stage, whereas MyoB‐GFP was localised as an apical end dot in merozoites, mature (>20 hr) ookinetes, and late stage sporozoites. MyoE‐GFP was detected as a dot at the basal end of ookinetes and sporozoites and as a dot in merozoites. MyoE‐GFP was also expressed in liver stages, but as with merozoites, it was not possible to clearly identify the basal end of these stages. MyoF‐GFP was most abundant in the insect stages, particularly ookinetes and oocysts. In mature ookinetes, MyoF‐GFP was restricted to the apical end, whereas in oocysts, it was more evenly distributed throughout the developing sporozoites. In contrast, MyoJ‐GFP was only observed in mature oocysts, located at the junction between the differentiating sporozoites and the oocyst bodies. Following oocyst rupture, MyoJ‐GFP remained associated with the oocyst body and was not present in sporozoites. Finally, MyoK‐GFP was found exclusively associated with the nucleus of gametocytes, appearing as an arc across the cell, and in zygotes/early ookinetes, as two distinctive dots. These dots were either absent or only remnants were seen in mature ookinetes. 

Live cell imaging reveals distinct spatio-temporal profiles for each myosin during ookinete development
We showed previously that MyoA-GFP is associated with the pellicle of this protuberance during zygote to ookinete development. MyoA-GFP was only detectable from stage II of ookinete development, initially located throughout the cytoplasm until, after stage V, when it was associated with the pellicle of the growing protuberance and the mature ookinete. MyoB-GFP was not detected until the very last stage of ookinete development, appearing as a dot at the apical end of the parasite. Since PfMLC-B was also expressed at the apical end of merozoites, we next checked its location in zygotes and ookinetes. Interestingly, MLC-B-GFP was detected much earlier in ookinete development, visible as an apical dot several hours before MyoB-GFP was observed. This suggests that MLC-B-GFP is made and positioned, well before its partner, MyoB. MyoE-GFP was detectable as very faint fluorescence in the cytosol until the final stage of differentiation when it was apparent at the basal end of the ookinete. As with MyoB, the localised appearance of MyoE was only seen after the body of the retort was finally reduced to the tapered base of the mature ookinete. MyoF-GFP expression appeared to translocate from the boundary of the stage II ookinetes - where the newly formed ookinete protrudes from the retort - to the apical end of the mature ookinete. As described above, MyoK-GFP was initially detected as a single dot in zygotes, then as two dots in the developing ookinete associated with the nucleus, but the signal almost completely disappeared from mature ookinetes. To resolve the structure of the apical and basal ‘dots’ of MyoB and MyoE (respectively) in mature ookinetes, we used 3D-SIM super resolution microscopy. This showed MyoB-GFP as an apical ring with an aperture in the centre, whereas MyoE-GFP formed a continuous basal ‘cap’ .

Co-localisation of myosins with MyoA and the apical marker, ISP1
To further study the location of the GFP-tagged myosins, we expressed them in parasite lines that were also expressing either MyoA-mCherry or ISP1-mCherry. ISP1-mCherry is a marker for early ookinete development located and maintained at the protuberance that becomes the apical end of ookinetes. The MyoA-mCherry line was generated in this study and validated by integration PCR. Expression was identical to that seen previously for MyoA-GFP. MyoA-mCherry and MyoB-GFP co-localise at the apical end of ookinetes whereas MyoA-mCherry and MyoE-GFP co-localise at the basal end. These locations were confirmed by MyoB-GFP co-localised with ISP1-mCherry at the apical end, and MyoE-GFP located at the opposite end of the ookinete from ISP1-mCherry. There was no co-localisation between MyoA-mCherry and MyoJ-GFP, expressed in the sporozoite bodies of oocysts and oocyst body, respectively. However, the two myosins did overlap at the point where the sporozoites detach from the oocyst body. In contrast, MyoA-mCherry partially co-localised with MyoF-GFP in the sporozoite bodies of oocysts. Finally, and as seen with ookinetes, MyoA-mCherry also partially co-localises with MyoE-GFP in liver stage schizonts.
Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1444500
Gene Model P. falciparum ortholog PF3D7_1229800
Gene productmyosin J
Gene product: Alternative nameMyoJ
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 single 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 modificationFor GFP-tagging of each myosin/MLC-B by single homologous recombination, a region of the 3’ end of the gene, but omitting the stop codon, was PCR amplified. This fragment was inserted into p277 vector, upstream of the gfp sequence, using KpnI and ApaI restriction sites. The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine. Before transfection, the vector was linearised. The GFP tagging constructs and cell lines for MyoA and MyoB have been reported previously. MyoA-mCherry was constructed using the same primers as described previously.
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