RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1310200; Gene model (P.falciparum): PF3D7_1446500; Gene product: conserved Plasmodium protein, unknown function (nucleoporin (Nup; FG-NUP)(NUP313))
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Oocyst; Sporozoite;
Last modified: 15 August 2018, 14:08
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) : 30050042
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/ResearcherKehrer J, Mair GR, Frischknecht F
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School
Name of the mutant parasite
RMgm numberRMgm-4468
Principal nameNUP313::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageGFP expression. Evidence for perinuclear localisation (see 'Additional remarks phenotype').
Gametocyte/GameteGFP expression. Evidence for perinuclear localisation (see 'Additional remarks phenotype').
Fertilization and ookineteNot tested
OocystGFP expression. Evidence for perinuclear localisation (see 'Additional remarks phenotype').
SporozoiteGFP expression. Evidence for perinuclear localisation (see 'Additional remarks phenotype').
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal GFP-tagged version of FG NUP313

Protein (function) and Phenotype
The nuclear pore complex (NPC) is a large macromolecular assembly of around 30 different proteins, so-called nucleoporins (Nups).
Nups are classified into two major groups: Firstly, the intrinsically disordered Nups with frequent FG (phenylalanine-glycine) repeats that line the central channel and interact with translocating cargo complexes. Secondly scaffold Nups that make up the cylindrical architecture grouped around the central channel. Transmembrane (TM) Nups – NDC1, GP210 and POM121 – anchor the NPC within the nuclear membrane.

In P. falciparum during the intraerythrocytic developmental cycle 3–7 nuclear pores are present per nucleus in early ring stage parasites. As the parasite grows the number increases transiently up to 60 in the trophozoite before they are being distributed among the 16–32 daughter progeny.

In Plasmodium only two putative Nup homologs had been identified: PBANKA_1445400 (SEC13, previously characterized in P. falciparum) and PBANKA_ 0416300. Failing to identify further Nups by primary sequence homology to yeast or human Nups,  the P. berghei genome was scanned for proteins containing FG di-amino-acid repeats. This screen revealed PBANKA_0416300 (Nup100), PBANKA_0107600, PBANKA_0417900, PBANKA_1140100 and PBANKA_13102000 as potential FG-Nups

In keeping with the nomenclature in other organisms, they were assigned the following names reflecting their predicted molecular weights:
Nup637 (PBANKA_0107600);
Nup313 (PBANKA_1310200);
Nup221 instead of Nup100 (PBANKA_0416300);
Nup205 (PBANKA_1140100);
Nup138 (PBANKA_0417900).

In this study all, except for Nup313 were C-terminally GFP-tagged for localisation studies. In addition, PBANKA_1445400 (SEC13) was C-terminally mCherry-tagged

Live microscopy of the transgenic lines identified GFP-fluorescence in asexual stage parasites as well as gametocytes. The staining ranged from polar and clustered, to perinuclear and contiguous, and reflects the distribution of the NPC in the different stages identified during the intraerythrocytic developmental cycle of the related human malaria parasite P. falciparum. In ring stage P. falciparum parasites, the few nuclear pores (3–7)
are typically clustered in a single location, a characteristic reflected in the ring stage fluorescent images. In the trophozoite the number of nuclear pores increases up to 12–58 per cell in P. falciparum. The fluorescence for each of the FG Nups increases with the size of the nucleus in our P. berghei lines. In general the associated NPC signal is more evenly distributed across the nucleus. With the development of up to 32 daughter merozoites from a single trophozoite and the concomitant decrease in pores per parasite to 2–6 as determined in P. falciparum, the GFP signal appears again focused on one side of the nucleus. Lastly, gametocytes (sexual precursor cells) display an atypical Nup localization distant from the DNA stain. This may perhaps reflect the specific nature of these forms, which are morphologically highly distinct between P. falciparum (elongated) and P. berghei (round). When taken up during a mosquito blood meal, a single cell-cycle arrested male gametocyte will, in less than 15 minutes, produce eight individual motile gametes able to fertilize a female. In mosquito stages fluorescence was evident in oocysts, large syncytial assemblies that produce sporozoites. Sporozoites isolated from the midgut oocysts displayed a fluorescent signal closely associated with the nuclear stain. In salivary gland derived sporozoites the distribution of the signal did not alter.

While the exclusive perinuclear staining patterns of all four FG Nups was consistent with similar studies from other organisms such as T. brucei, SEC13 (PBANKA_1445400) displayed frequent speckled localization sites away from the nucleus in several life cycle stages consistent with its inclusion in COPII vesicles. 

Tagging with mCherry of SEC31  yielded a parasite population with a signal distant from the nuclear DAPI-staining consistent with its function as a COPII vesicle protein at the tER. The distribution of staining for the COPII coat assembly protein SEC16 tagged with mCherry at the C-terminus was again similar to the one for SEC31.
SEC13 was prominently localized away from the DAPI-stained nucleus. The signal was particularly strong and widespread in the trophozoite and the gametocyte. As for the FG Nups, we also followed SEC13 and SEC31 fluorescence throughout parasite development in the mosquito. These experiments revealed an intriguing change in fluorescence signal distribution during the maturation of midgut into salivary gland sporozoites. While a single fluorescent spot is present in a juxta-nuclear position in the majority of sporozoites isolated from midguts, a second, equally strong signal appeared on the opposite side of the nucleus in sporozoites isolated from salivary glands. The staining of these two proteins was different in respect to the FG Nup staining as all FG Nups showed more than just one localization spot in midgut derived sporozoites. This indicates that SEC13 and SEC31 are not associated with (all) NPCs at the sporozoite stage.

Additional information

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1310200
Gene Model P. falciparum ortholog PF3D7_1446500
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative namenucleoporin (Nup; FG-NUP)(NUP313)
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 parasitetgdhfr
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
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