RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_0112200; Gene model (P.falciparum): PF3D7_0613900; Gene product: myosin E, putative (myoE)
Name tag: triple-HA
Phenotype Asexual bloodstage;
Last modified: 23 October 2018, 15:54
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) : 30315162
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/ResearcherFang H, Billker O, Brochet M
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine, Faculty of Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-4555
Principal namePbmyoE-3xHA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageEndogenously tagged MyoE localises to the cell periphery of merozoites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal HA-tagged version of MyoE.

Protein (function)
Six myosins genes are now annotated in the Plasmodium falciparum Genome Project. Malaria myosins have been named alphabetically. RT-PCR on blood stage parasite mRNA amplifies a specific product for all six myosins and each shows developmentally regulated transcription. Pfmyo-A and Pfmyo-B genes are transcribed throughout development; Pfmyo-C is predominant in trophozoites; Pfmyo-D occurs in trophozoites and schizonts; Pfmyo-E though barely present in earlier stages is abundant in schizonts; Pfmyo-F increases steadily throughout development and maturation.

Endogenously tagged MyoE localises to the cell periphery of merozoites

The mutant in this study was generated to screen for genetic interactions among protein kinases. In this study a role of CDPK4 during erythrocytic (asexual blood stage) proliferation has been found, which was dependent on a negative interaction with PKG.

From the paper:
'While calcium levels can account for the functional interaction between pkg and cdpk4 mutations, they do not explain how CDPK4 can affect RBC invasion by the merozoite. To address this question, we tagged CDPK4 in PbANKA 2.33, a line unable to produce gametocytes, and confirmed its expression in erythrocytic schizonts. Immunofluorescence localisation shows a signal excluded from the nucleus with a slight enrichment at the merozoite periphery. In CDPK4-3xHA immunoprecipitates following crosslinking, 19 proteins are enriched over wild-type controls, including GAP40, MyoA and GAC, three proteins essential for the IMC biogenesis or gliding motility. In addition to MyoA, CDPK4 also immunoprecipitates an uncharacterised Plasmodium-specific myosin, MyoE (PBANKA_0112200). Altogether, this further suggests that the redundant role of CDPK4 observed in invasion could be linked to the regulation of the molecular machinery that forms the IMC or provides the force for invasion.
Endogenously tagged GAP40 localises to the parasite periphery. Among the 20 proteins coimmunoprecipitated with GAP40-3xHA is CDPK4, in addition to known IMC and glideosome components.
GAP40-3xHA also coprecipitates another member of the CDPK family involved in merozoite invasion, CDPK1, and again, MyoE. Epitope tagged CDPK1 and MyoE are enriched at the cell periphery, and both proteins co-immunoprecipitate multiple glideosome or IMC components. Collectively, these results suggest MyoE may act as an alternative myosin of the motor complex and that both CDPK1 and CDPK4 are at the interface between the glideosome and the IMC. Immunoprecipitation of GAP40 or MyoE recovers multiple peptides from a protein of unknown function which, like GAP40 itself, was one of a small number of hits that emerged from our recent biochemical screen for substrates of CDPK4 (SOC proteins) in parasite lysates. This protein, SOC6 (PBANKA_0707700), has since been shown to interact with the IMC protein Phil1 in P. berghei schizonts and with MyoA in P. falciparum schizonts, and may thus provide a molecular link between CDPK4 and invasion. SOC6 is characterised by a C-terminal stretch of 106 amino acids that are relatively conserved across the syntenic orthologues of different malaria parasites, but lacks obvious homologues in other apicomplexan genomes. SOC6 is further characterised by 4 to 15 tandem amino acid repeats that show sequence and position variability across species. In P. berghei, the serine residue that CDPK4 phosphorylates in vitro lies in one such repeat of 54 amino acids, which has prevented us from mutagenising specifically the phosphosite. Endogenously tagged SOC6-3xHA localises to the cell periphery of merozoites and immunoprecipitates peptides from multiple IMC, glideosomeassociated proteins and glideosome proteins. Altogether, this indicates that SOC6 is also at the interface between the IMC and the glideosome. A SOC6-KO line shows a significant growth defect compared with wild type. While segmented SOC6-KO schizonts display the same number of merozoites as wild type, they show a reduced capacity to transform into ring stage parasites, while no accumulation of circulating SOC6-KO schizonts is observed. This indicates SOC6 is important either at the final stage of schizont maturation or to invade new RBCs. TEM of purified SOC6-KO schizonts reveals a discontinuous IMC as observed for PKGT619Q-3xHA/CDPK4-KO transgenic, suggesting that SOC6 is important for the formation or the stability of the IMC in merozoites. To investigate the function of SOC6 further, we turned to the ookinete stage, which in P. berghei offers a tractable model to study the molecular motor that powers gliding motility. Ookinetes emerge from the zygote that forms after fertilisation of macrogametes by microgametes in the mosquito blood meal. Male gamete formation does not require SOC6, but the SOC6-KO nevertheless fails to form typical banana-shaped ookinetes. Again, TEM of SOC6-KO cells reveals either a discontinuous IMC or the complete absence of an IMC below the plasma membrane, suggesting that SOC6 plays a conserved role to control the IMC formation or stability at multiple stages of the malaria lifecycle.

From the Abstract:
Most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito.

Additional information

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0112200
Gene Model P. falciparum ortholog PF3D7_0613900
Gene productmyosin E, putative
Gene product: Alternative namemyoE
Details of the genetic modification
Name of the tagtriple-HA
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 usedYes
Name of PlasmoGEM construct/vector533211
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPreparation of targeting vectors. 3xHA tagging, knockout and allelic replacement constructs in P. berghei were generated using phage recombineering in Escherichia coli tryptic soy agar (TSA) bacterial strain with PlasmoGEM vectors (http://plasmogem.sanger.ac.uk/). For final targeting vectors not available in the PlasmoGEM repository, generation tagging constructs was performed using sequential recombineering and gateway steps. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R for 3xHA tagging. The modified library inserts were released from the plasmid backbone using NotI.
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