RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_0833000; Gene model (P.falciparum): PF3D7_0932200; Gene product: profilin (PFN, PRF)
Details mutation: P. berghei profilin replaced by different chimeric forms of profilin
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 6 April 2020, 16:11
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) : 32034083
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
Name of the mutant parasite
RMgm numberRMgm-4746
Principal nameprofilin chimeras
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageSee below
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteSee below
Liver stageSee below
Additional remarks phenotype

Different mutants are described that express mutated (chimeric) forms of profilin. We designed the profilin acidic loop chimeras such that a P. falciparum profilin contained the loop of either P. berghei (Pf PfnPb loop) or T. gondii (Pf PfnTg loop) and also included a P. berghei profilin with a loop of P. falciparum (Pb PfnPf loop).

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.

Compared to human actin, Plasmodium profilin contains several additional motifs, including a β-hairpin arm motif and an acidic loop. Comparison between different Apicomplexan species shows that there is little difference in the arm motif while T. gondii profilin has only a very short acidic loop compared with the profilins in Plasmodium spp. The regions bordering the acidic loop are highly conserved between P. falciparum and P. berghei profilin (100% over the 15 amino acid residues following the loop and only one A to E difference in the 15 amino acid residues preceding the loop). In contrast, 6 of the 8 amino acid residues of the acidic loop are not conserved. The T. gondii profilin loop sequence is more divergent, consisting of just four aspartic acid residues.

To investigate a potential role of the acidic loop in stabilizing the overall
structure of profilin and in actin binding, we performed molecular dynamics simulations on a series of chimeric profilins similar to those described for the arm-motif mutations . We designed the profilin acidic loop chimeras such that a P. falciparum profilin contained the loop of either P. berghei (Pf PfnPb loop) or T. gondii (Pf PfnTg loop) and also included a P. berghei profilin with a loop of P. falciparum (Pb PfnPf loop).
To test the chimeras in vivo, we generated a series of P. berghei strain ANKA parasite lines expressing the chimeras in place of the endogenous profilin. As the absence of  introns did not influence life cycle progression or motility of parasites we introduced intron-free genes.
As we previously also showed that a C-terminal fluorescent protein tag could slow down parasites, we further opted for exchanging the genes without such a tag. After isolating clones from the different lines, we next investigated the progression of these along the life cycle.

We first compared the blood stage growth rates of the chimeric parasite lines to those of wild type P. berghei and the previously reported P. berghei line expressing P. falciparum profilin. This showed that the parasites expressing the P. berghei profilin with the P. falciparum loop grew as fast as wild type P. berghei. Those lines expressing P. falciparum profilin and P. falciparum profilin with the P. berghei profilin loop grew somewhat faster but at comparable rates (Table 1). Intriguingly, expression of the P. falciparum chimera featuring the T. gondii loop slowed blood stage growth indicating that this chimeric profilin might not perform as efficiently in vivo. 

Infection of Anopheles stephensi mosquitoes however revealed similar infection rates and numbers of sporozoites.

Infections of mice by mosquito bite showed a mild reduction in infectivity of the parasite lines expressing P. falciparum profilins containing the loops of P. berghei or T. gondii.

P. berghei sporozoites expressing P. falciparum profilin migrated faster than wild type P. berghei sporozoites although fewer parasites were gliding.

Examination of the parasite lines expressing loop chimeras showed that sporozoites of all chimeras moved in the typical circular fashion of wild type parasites. About the same percentage of sporozoites were gliding, but curiously, those expressing P. falciparum profilin containing the loop of T. gondii showed a higher percentage of persistently moving sporozoites than those just expressing the P. falciparum profilin. The quantification of their average and instantaneous speeds showed however, that all sporozoites expressing chimeras were over 50% slower than their respective control parasite lines. 

Additional information
From the Abstract
' Here we show that different mutations in profilin, not affecting actin binding in vitro, still generate lower force during Plasmodium sporozoite migration. Lower force generation inversely correlates with increased retrograde flow suggesting that, like in mammalian cells, the slow-down of flow to generate force is the key underlying principle governing Plasmodium gliding motility.' 

Other mutants

  Mutated: Mutant parasite with a mutated 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
Short description of the mutationP. berghei profilin replaced by different chimeric forms of profilin
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
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 modificationVectors used for in vivo work are based on the b3D+ vector. We modified the vector for homologous recombination in the profilin (PBANKA_0833000) locus on chromosome 8 as follows. The P. berghei profilin 5’ upstream region (871 bp) was amplified from P. berghei ANKA WT genomic DNA using primer combination 5 (see Table S1) and subsequently inserted into b3D+ via SacII and NotI digestion and ligation. The profilin 3’ downstream region (805 bp) was amplified with primer combination 6 and inserted using ClaI and KpnI.
P. berghei wild type profilin was amplified with primer combination 3 and cloned with NotI and XbaI. P. falciparum wild type was amplified with primer combination 4 and cloned into b3D+ using NotI and XbaI. Profilin chimeras were generated by overlap extension PCR where the respective loop regions were encoded by the interior primers generating two fragments (A and B). The loop regions present in both fragments were then used to anneal and fuse the fragments together. The Pb Pfn loop was encoded in primers 7a and 7b, the Pf Pfn loop in primers 8a and 8b and the Tg Pfn loop by primers 9a and 9b.
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