RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4172
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0833000; Gene model (P.falciparum): PF3D7_0932200; Gene product: profilin (PFN, PRF)
Name tag: mCherry
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Sporozoite;
Last modified: 2 June 2017, 18:43
  *RMgm-4172
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) : 28552953
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/ResearcherMoreau CA, Frischknecht F
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4172
Principal nameprofilin-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageImaging parasites at the ring, trophozoite, and sporozoite stages revealed uniform profilin localization in the cytosol and the nucleus, while in gametocytes and ookinetes the profilin-mCherry fluorescence was more pronounced in the nucleus.
Gametocyte/GameteImaging parasites at the ring, trophozoite, and sporozoite stages revealed uniform profilin localization in the cytosol and the nucleus, while in gametocytes and ookinetes the profilin-mCherry fluorescence was more pronounced in the nucleus.
Fertilization and ookineteImaging parasites at the ring, trophozoite, and sporozoite stages revealed uniform profilin localization in the cytosol and the nucleus, while in gametocytes and ookinetes the profilin-mCherry fluorescence was more pronounced in the nucleus.
OocystNot tested
SporozoiteImaging parasites at the ring, trophozoite, and sporozoite stages revealed uniform profilin localization in the cytosol and the nucleus, while in gametocytes and ookinetes the profilin-mCherry fluorescence was more pronounced in the nucleus.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of profilin.

Protein (function)
Profilins are ubiquitous and essential actin monomer binding proteins, known to interact with proline-rich regions in a variety of proteins and to regulate actin filament formation. For fast actin polymerization at selected sites, profilin is an essential control element by recruiting actin monomers in a polymerizable form to actin polymerization machineries. Profilins are usually defined by their shared, highly conserved structure and by their ability to bind actin, proline-rich sequences.

Phenotype
Analysis of two mutants expressing C-terminal mCherry-tagged versions of P. berghei profilin (one mutant with the tagged profilin with introns and one without introns) showed the following:

The fluorescent parasites progressed through the life cycle in a manner largely comparable to wild-type parasites, suggesting that the mCherry fusion did not detectably impair parasite viability. Imaging parasites at the ring, trophozoite, and sporozoite stages revealed uniform profilin localization in the cytosol and the nucleus, while in gametocytes and ookinetes the profilin-mCherry fluorescence was more pronounced in the nucleus. While the speed of motile ookinetes was not affected, the speed of sporozoites expressing mCherry-tagged profilin both with and without introns was significantly reduced. The speed of sporozoites could be restored to wild type levels in parasites that expressed a longer linker between profilin and mCherry. However, in these parasites, the mCherry tag was largely cleaved off, leading to a majority of non-tagged profilin. In all tagged lines, Pfn expression was under the control of the endogenous promoter and the 3'UTR from PbDHFR, suggesting that the 3'UTR did not influence the level of profilin expression.

Additional information
The mutant that expresses P. falciparum profilin in stead of P. berghei profilin (RMgm-4169) shows normal development throughout the complete life cycle indicating that P. falciparum profilin complements P. berghei profilin.

In the paper two additional mutants are analysed in which the endogenous P. berghei profilin gene is replaced by a mutated form of P. falciparum profilin. Mutant RMgm-4170 and mutant RMgm-4171 expresses mutated forms where the residues 64EDE66 were exchanged to QNQ or AAA. These mutations were chosen in order to weaken (QNQ) or abolish (AAA) the interactions of the acidic arm motif of profilin with actin (compared to classical profilins, apicomplexan profilins contain an additional arm-like β-hairpin motif).

Expression of the QNQ and AAA mutants in the parasite had no detrimental effect on life cycle progression. Whereas only minor differences in the speed of wild type and transgenic ookinetes were observed, a striking effect was seen in sporozoites. We found no difference between the wild type and QNQ sporozoites in their capacity to glide on a flat substrate, but the AAA mutants largely failed to move progressively. Pf Pfn-expressing sporozoites moved faster but less persistently than wild type P. berghei sporozoites, and this was also seen for the QNQ mutant. Also the AAA mutant ookinetes moved at lower speeds than the QNQ mutant ookinetes.
Together with the biochemical data and the molecular dynamics simulations, these data on sporozoite motility confirm that the parasite-specific arm in Pf Pfn is crucial for actin binding and, thereby, sporozoite motility.

Other mutants
See PF3D7_0932200


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0833000
Gene Model P. falciparum ortholog PF3D7_0932200
Gene productprofilin
Gene product: Alternative namePFN, PRF
Details of the genetic modification
Name of the tagmCherry
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 modificationThe profilin-mCherry gene is under control of the profilin 5' promoter region and the 3'UTR of P. berghei dhfr/ts
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