RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_1105300; Gene model (P.falciparum): PF3D7_0505700; Gene product: conserved Plasmodium membrane protein, unknown function (akratin)
Details mutation: C-terminal EYKK motif changed into four alanines and GFP tag
Phenotype Gametocyte/Gamete; Oocyst;
Last modified: 28 October 2020, 18:24
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4885
Other information parent lineIn this mutant the akarin gene has been deleted. This mutant does not contain a drug selectable marker (SM). The SM has been removed by negative selection
The mutant parasite was generated by
Name PI/ResearcherKehrer J, Frischknecht F
Name Group/DepartmentCenter for Infectious Diseases, Integrative Parasitology
Name InstituteHeidelberg University Medical School
Name of the mutant parasite
RMgm numberRMgm-4886
Principal nameakratinEYKY/AAAA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot tested
Gametocyte/GameteSlightly reduced exflagellation
Fertilization and ookineteNot different from wild type
OocystAbsence of oocyst formation
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a mutated form of akratin that is C-terminally GFP-tagged. In the mutated akratin the C-terminal EYKK motif has been changed into four alanines and a GFP tag is added (C-terminal). The mutated and tagged akratin gene has been introduced into the akratin locus of a mutant (see RMgm-4885) in which the akratin gene has been deleted.
Published in bioRxiv preprint doi: https://doi.org/10.1101/2020.09.29.318857.

Protein (function)
PbANKA_1105300 was found to be conserved among Plasmodium spp. It shares 82% identity with its orthologue in P. yoelii, but only 35% and 32% with its orthologues in P. falciparum and P. vivax, respectively. In contrast to P. berghei and P. falciparum, the orthologues in P. yoelii and P. vivax are predicted to contain only two TMDs. Furthermore, the P. falciparum protein contains about two times more amino acids than the P. berghei protein. No orthologue was found outside Plasmodium spp

Mutation of a C-terminal motif of akratin uncoupled akratin function in gametocytes and ookinetes (see mutant RMgm-4885). In the mutant expressing mutated akratin, ookinetes were formed but no oocysts. Evidence is presented that the mutant ookinetes are defective in midgut-wall traversal.
The mutant lacking expression of akratin (see mutant RMgm-4885) fails to produce male gametes (and ookinetes)

Additional information
In mammalian cells the C-terminal sorting sequence YXXf (with Y, tyrosine; X, any amino acid and f, hydrophobic amino acid) is important for protein trafficking. A similar motif was also shown to be important for trafficking of the micronemal sporozoite protein TRAP (Bhanot et al., 2003), although the sequence is annotated as being located in the trans-membrane domain. Somewhat similar tyrosine-containing C- terminal motifs (SYHYY and EIEYE) from the Toxoplasma gondii adhesin MIC2 were also shown to target the protein to micronemes. We noticed in the C-terminal, likely cytoplasmic domain of P. berghei akratin a short sequence (YKKL) representing the described YXXf pattern. However, since the P. falciparum orthologue contains a leucine instead of a tyrosine and a conserved glutamic acid to the left we chose to mutate the sequence EYKK instead of YKKL to investigate a possible role for akratin trafficking. To this end, we generated a mutant parasite line expressing akratin-GFP with the EYKK motif changed into four alanines.

The Akratin-EYKK/AAAA-GFP mutant showed similar staining patterns in asexual and sexual parasite stages compared with akratin-GFP. (see mutant RMgm-4888) However, in contrast to the mostly peripheral and vesicular localization of akratin-GFP in ookinetes the akratin-EYKK/AAAA GFP signal was diffusely localized within the cytoplasm

Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1105300
Gene Model P. falciparum ortholog PF3D7_0505700
Gene productconserved Plasmodium membrane protein, unknown function
Gene product: Alternative nameakratin
Details of the genetic modification
Short description of the mutationC-terminal EYKK motif changed into four alanines and GFP tag
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 5`UTR together with the entire ORF of PbANKA_1105300 was amplified from wt gDNA using primers JK66 and JK152 and inserted into the pL28 plasmid using KpnI and NdeI leading to the replacement of the selection marker. A TgDHFR selection cassette was amplified using primers JK153 and JK154 and inserted between the GFP and 3`UTR using NotI and EcorV resulting in plasmid 361 pL59.
The 5`UTR together with the entire ORF was amplified from wt gDNA using primers JK66 and a reverse Primer introducing the mutations and inserted into the pL59 plasmid using KpnI and NdeI resulting in pL84.
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