RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1342500; Gene model (P.falciparum): PF3D7_1327300; Gene product: conserved Plasmodium protein, unknown function (SPM3)
Phenotype Sporozoite;
Last modified: 11 January 2023, 11:55
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) : 36625655
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/ResearcherWichers-Misterek JS, Gilberger TW
Name Group/DepartmentCentre for Structural Systems Biology
Name InstituteCentre for Structural Systems Biology
Name of the mutant parasite
RMgm numberRMgm-5264
Principal namePbSPM3-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 different from wild type
SporozoiteNormal sporozoite formation inside oocysts. Strongly reduced sporozoite numbers in salivary glands. Strongly reduced (aberrant) motility.
Liver stageNot different from wild type
Additional remarks phenotype

The mutant lacks expression of SPM3

Protein (function)
Mature gametocytes of Plasmodium (P.) falciparum display a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated to the inner membrane complex (IMC). Microtubule associated proteins (MAPs) define MT populations and modulate interaction to pellicular components. Several MAPs have been identified in Toxoplasma gondii and homologues can be found in the genome of Plasmodium species, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genus Plasmodium. 
The number of SPMTs varies across life cycle stages. For instance, merozoites of P. falciparum possess 2–4 SPMTs, whereas ookinetes have 60, sporozoites 14 and gametocytes 21 SPMTs

Normal sporozoite formation inside oocysts. Strongly reduced sporozoite numbers in salivary glands. Strongly reduced (aberrant) motility.
Evidence is presented that:
- SPM3 deletion disturbs subpellicular microtubule (SPMT) architecture in sporozoites

Additional information
Conditional knockdown and targeted gene disruption of Pfspm3 in P. falciparum cause severe morphological defects during gametocytogenesis leading to round, non-falciform gametocytes with an aberrant subpellicular microtubules (SPMT) pattern. In contrast, Pbspm3 knockout in P. berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in sporozoite invasion of salivary glands leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the inner membrane complex (IMC) in Pbspm3 knockout parasites suggesting a function of SPM3 in anchoring microtubules (MTs) to the inner membrane complex (IMC).

Analysis of a mutant expressing a C-terminal GFP-tagged version of SPM3 (RMgm-5265) showed expression in oocysts and sporozoites. In sporozoites, the fluorescent signal extended from the apical end towards the rear around the nucleus. Subsequent co-staining with an anti-tubulin antibody revealed co-localization of PbSPM3-GFP with tubulin (Figure 4A) in midgut and salivary gland sporozoites. C-terminal tagging of PbSPM3 with GFP did not affect parasite life cycle progression, as SPM3-GFP parasites showed normal midgut infection and midgut oocyst numbers as well as numbers of both midgut and salivary gland sporozoites comparable to wild-type.

For P. falciparum SPM3 evidence is presented that:
- PfSPM3 is a Plasmodium-specific microtubule-associated protein in P. falciparum merozoites
- PfSPM3 is dispensable for intraerythrocytic development
- PfSPM3 is important for falciform gametocyte morphology

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1342500
Gene Model P. falciparum ortholog PF3D7_1327300
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameSPM3
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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor gene deletion of PbSPM3 (PBANKA_1342500), both a 3’ and a 5’ homology region (750 and 717 bps) were amplified from PbANKA wild-type-genomic DNA and cloned into the Pb262 vector using HindIII/XhoI and EcoRI/EcoRV restriction 476 sites. The Pb262 vector contains the hDHFR gene that allows for positive selection using the drug pyrimethamine. Prior to transfection, the vector was linearized using SacII and PmeI restriction enzymes followed by ethanol-precipitation.
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