RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1358700; Gene model (P.falciparum): PF3D7_1345800; Gene product: conserved Plasmodium protein, unknown function
PhenotypeNo phenotype has been described
Last modified: 17 April 2023, 16:10
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 36898988
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/ResearcherRashpa R, Brochet M
Name Group/DepartmentFaculty of Medicine, Department of Microbiology and Molecular Medicine
Name InstituteUniversity of Geneva
Name of the mutant parasite
RMgm numberRMgm-5340
Principal namePBANKA_1358700-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expresses of PBANKA_1358700

Protein (function)
PBANKA_1358700 was enriched in SCF(FBXO1) immuno-precipitates. SCF complexes contain three invariant proteins, RBX1, Cullin and SKP1, in addition to a variable F-box protein. Genome mining in Plasmodium identified SKP1, Cullin1, and RBX1 together with two F-box domain-containing proteins. See below for more information.

Normal asexual blood stage development/growth, gametocyte production and male gamete formation (exflagellation)

Additional information
From the paper:
'Plasmodium genome mining previously identified orthologues of four proteins possibly belonging to Skp1/Cullin/F-box (SCF) complexes: Cullin1 (CUL1 - PBANKA_1426500), RBX1 (PBANKA_0806200), SKP1 (PBANKA_1142900), Nedd8 (PBANKA_1411400), and a second cullin domain-containing protein (CUL2 - PBANKA_1128600).

We first set out to identify whether these proteins form a complex in Plasmodium using affinity purification of triple HA-tagged proteins. Transgenic P. berghei lines expressing triple HA-tagged RBX1 (RMgm-5335), SKP1 (RMgm-5333) and CUL1 (RMgm-5334). To identify interacting proteins for RBX1-HA and SKP1-HA, we used affinity purification of triple HA-tagged proteins from synchronised mitotic gametocytes 4–6min post-activation followed by label-free semiquantitative mass spectrometry. Supporting the notion of an SKP1/CUL1 complex, CUL1, Nedd8 and polyubiquitin (PolyUb – PBANKA_ 0610300) were enriched proteins co-purifying with RBX1-HA and SKP1-HA. Among the proteins most enriched in both immunoprecipitates were also two proteins containing either F-box (FBXO1 – PBANKA_1118900) or leucine-rich repeats (FBXL2 or LRR11,  PBANKA_0925100) that could represent possible adaptors of the SKP1/CUL1 complex. We also identified the IMC sub-compartment protein 1 (ISP1 - PBANKA_1209400), suggesting that this protein, which is important to define the polarity of ookinetes, may be regulated by SCF-dependent ubiquitination.
A protein of unknown function, PBANKA_1358700 was also enriched in all immunoprecipitates.'
'To confirm FBXO1 and FBXL2 are components of SKP1/CUL1 complexes, we generated transgenic P. berghei lines expressing endogenously triple HA-tagged alleles of FBXO1 (RMgm-5336) and FBXL2 (RMgm-5337). 
Immunoblotting confirmed the expression of fusion proteins in gametocytes with the expected mobility. IFA showed a similar cytoplasmic distribution of FBXO1-HA and FBXL2-HA in both macro- and microgametes with slightly brighter staining at structures that likely correspond to mitotic spindles and forming axonemes in the microgametes. Immunoprecipitations of FBXO1-HA or FBXL2-HA enriched peptides from SKP1, CUL1, RBX1, PolyUb and Nedd8, while the most enriched protei nwas ISP1. CDPK1, a regulator of merozoite formation, gamete egress and ookinete development, was also  slightly enriched in FBXO1-HA immuno-precipitates.'

'These results indicate that gametocytes express a conserved SKP1/RBX1/CUL1 complex with at least two possible adaptor proteins, FBXO1 and FBXL2. The enrichment of CDPK1 in FBXO1-HA and SKP1-HA immuno-precipitates  suggests a possible interplay between phosphorylation and ubiquitination to regulate sexual development.'

'To study the function of SCFF(BXO1) complex, we first attempted to knock-out skp1 (RMgm-5330), cul1 (RMgm-5331), and rbx1 (RMgm-5332). These attempts were unsuccessful indicating an essential function during asexual blood stage growth/multiplication'.

'The endogenous  skp1, cul1 and rbx1 genes were taged with an auxin-inducible degron (AID) coupled to an HA epitope tag that allows degradation of the fusion protein in the presence of auxin in a strain expressing the Tir1 protein (RMgm-1305; Philip and Waters, 2015). However, no significant degradation of the targeted proteins nor defects in exflagellation could be observed upon auxin addition in non-clonal populations This prevented  to further use this system to interrogate the functions of these proteins across multiple stages.'

'To infer potential function in gametocytes, we then opted for stage-specific knockdowns by placing the endogenous cul1 or rbx1 genes under the control of the promoter of the blood-stage schizont-specific promoter ama1 (PBANKA_0915000), which is active in schizonts but silent in gametocytes. Cloned parasite lines expressing cul1 (Pama1CUL1; RMgm-5338) and rbx (Pama1RBX; RMgm-5339) under control of the ama-1 promoter were  obtained, but none of these showed quantifiable defects in exflagellation (possibly due to sufficient expression levels?). However, the Pama1CUL1 clone mainly formed retort ookinetes, a phenotype that was partially rescued by fertilisation with competent Nek4-KO microgametes, suggestive of a role for cul1 in either sexual lineage.'

We then interrogated the requirement for the putative adaptor proteins FBXO1, FBXL2 and the protein of unknown function PBANKA_1358700 that was enriched in SCFFBXO1 immuno-precipitates. We were able to obtain a KO clonal line for PBANKA_1358700 (RMgm-5340), but no defects in asexual blood stages nor in exflagellation were detected.
No FBXL2-KO transient populations could be observed (RMgm-5341) and no major protein degradation was observed in an FBXL2-AID/HA line upon treatment with auxin. However, no exflagellation defect could be detected upon auxin treatment preventing us from further functional analysis of this protein.
We obtained a clonal FBXO1-AID/HA line as well as a clonal line, FBXO1-GD (for Gene Disruption; RMgm-5342), in which 207 fbxo1 bases were deleted, disrupting the coding sequence at the 353rd amino acid. Phenotypic analysis of FBXO1-GD showed that FBXO1 is required for normal growth of asexual blood stages, microgametogenesis and ookinete development. Slight growth/multiplication delay of FBXO1-GD asexual blood stages and strongly impaired microgamete formation as determined by the percentage of microgametocytes leading to exflagellating microgametes. Flagellar movement of the forming microgametes commenced while the parasite was still inside the host cell and delayed egress of male gametes from the host erythrocyte. 
FBXO1-GD parasites did not form banana-shaped ookinetes and only retort stages were observed.
Evidence is presented that that: 
- FBXO1 expression during gametocytogenesis is required for macro- and microgamete egress and centrosome partitioning during microgametogenesis.
- FBXO1-GD gametocytes show significantly reduced ubiquitination levels upon activation
- FBXO1 playse a role in maintaining the integrity of the pellicle and the subpellicular microtubule network during ookinete development.
- FBXO1 and CDPK1 share a molecular environment and show related cellular requirements during sexual development
- CDPK1 negatively regulates FBXO1 function by downregulating its expression level in gametocytes

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1358700
Gene Model P. falciparum ortholog PF3D7_1345800
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative name
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption 3xHA, KO (disruption) or AID/HA targeting vectors were generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of knockout and tagging constructs were 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 and goi KO-F x goi KO-R for 3xHA, AID/HA tagging and KO targeting vectors, respectively. Insertion of the GWcassette following the gateway reaction was confirmed using primer pairs GW1 x goiQCR1 and GW2 x goiQCR2.The modified library inserts were then released from the plasmid backbone using NotI. The AID/HA targeting vectors were transfected into the 615 parasite line, while the KO/GD and triple HA targeting vectors were transfected into the 2.34 line unless otherwise specified.
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
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