RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_1232400; Gene model (P.falciparum): PF3D7_0517600; Gene product: F-actin-capping protein subunit beta, putative (CPβ, capping protein beta; UIS17, upregulated in infectious sporozoites gene 17)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 17 January 2015, 12:19
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) : 19682250
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherM. Ganter; K. Matuschewski
Name Group/DepartmentDepartment of Parasitology
Name InstituteHeidelberg University School of Medicine
Name of the mutant parasite
RMgm numberRMgm-328
Principal namecpβ(-)
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 ookineteNormal rate of fertilization and ookinete production. Ookinetes showed reduced motility. Mutant ookinetes showed an average speed of 3.7 µm/min and wild type ookinetes an average speed of 6.5 µm/min. Mutant ookinetes transiently showed fast locomotion comparable to wild type.
OocystStrongly reduced oocyst formation in Anopheles stephensi mosquitoes, both after feeding mosquitoes on infected mice and by membrane feeding of in vitro cultured mature ookinetes to mosquitoes. The oocysts formed, produced normal numbers of sporozoites.
SporozoiteSporozoites have lost the ability to invade salivary glands. Midgut-associated sporozoites were non-infectious to mice after intravenous injection. Sporozoites showed strongly reduced motility and never displayed productive (forward) locomotion.
Liver stageMidgut-associated sporozoites were non-infectious to mice after intravenous injection.
Additional remarks phenotype

The mutant lacks expression of CPβ (capping protein beta;  UIS17, upregulated in infectious sporozoites gene 17)

Protein (function)
In the genome of Plasmodium two genes, cpα (PFE1420w) and cpβ, are present that encode two F-actin capping protein subunits. CPβ has been identified in a screen for transcripts that are upregulated in infectious sporozoites (Matuschewski et al., 2002, J. Biol. Chem.).

The phenotype analyses indicate a reduced transformation of mature ookinetes into the oocyst stage, possibly as a result of reduced ookinete motility and penetration of the mosquito midgut epithelium. In addition, sporozoites showed strongly reduced motility and were unable to invade the salivary gland. These results indicate a role of CPβ in the motility (forward locomotion) of ookinetes and sporozoites.

Additional information
RT-PCR analyses showed transcription of cpβ in merozoites, ookinetes and sporozoites.

To test whether the phenotype of mutant parasites during mosquito development can be reversed by inheritance of a wild type cpβ copy during sporozoite formation, cpβ(−) and wild type parasites were crossed and the mixed parasites genotyped in comparison with clonal parasites before and after mosquito transmission. In mixed inoculations the cpβ(−) genotype was recovered after life cycle completion. These findings led us to conclude that the essential function of Plasmodium CPβ is restricted to the insect vector stages, and this deficiency can be rescued by transient complementation of one wild type copy during oocyst development, where a heterozygous cell undergoes multiple rounds of replication prior to sporozoite budding.

Additional information on the protein
The actin-based microfilament system drives motile processes, such as cell motility, cytokinesis and vesicle transport in eukaryotic cells. These processes require dynamic interconversion of pools of monomeric and filamentous actin (G- and F-actin respectively), regulated by a large number of accessory proteins. Capping protein (CP) is a heterodimeric protein that is a central component of actin polymerization-driven cell motility, as it restricts growth of a subset of filaments thereby allowing fast, directed polymerization from a pool of unpolymerized actin. Malaria and related parasites, such as Toxoplasma gondii, employ their own actin/myosin motor machinery to propel themselves into the host cell. In addition, actin-based motility drives parasite locomotion and transmigration en route to the final target cell. The actin motor machinery of Plasmodium features the short tailless motor MyoA, tethered to the inner membrane complex by accessory proteins, and very short polymers of actin that are linked to thrombospondin-related anonymous protein (TRAP)/MIC2-family invasins via aldolase and possibly other proteins. This arrangement mediates gliding on the substratum, apparently by moving F-actin–receptor complexes from the apical tip backwards along the parasite's longitudinal axis. The regulation of this motor machinery remains elusive. Plasmodium and related Apicomplexa encode only a fraction of the conventional microfilament regulators, with many protein families missing entirely. Given the intrinsic instability of parasite actin polymers F-actin end-capping proteins are expected to be required for both orchestrated F-actin assembly and sustained filament stability. In Plasmodium genomes, two capping protein subunits can be identified. Protein sequences of the putative beta capping protein subunits from different Plasmodium species (PFE0880c and PBANKA_123240 for P. falciparum and P. berghei CPβ respectively) share around 25% sequence identity with those of yeast, chicken and human. These genes are among the most divergent within the family. However, the majority of the key residues stabilizing the heterodimer and those implicated in actin binding are present. The Plasmodium genomes also encode the corresponding putative capping protein alpha-subunit (CPα; f-actin capping protein alpha subunit, putative; PFE1420w; PBANKA_124310; ).
Evidence in the paper is presented that PbCP displays bona fide F-actin capping activity in vitro.

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1232400
Gene Model P. falciparum ortholog PF3D7_0517600
Gene productF-actin-capping protein subunit beta, putative
Gene product: Alternative nameCPβ, capping protein beta; UIS17, upregulated in infectious sporozoites gene 17
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 SacII, KpnI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
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
Additional information primer 1PbCPβ_forI (SacII); 5' flanking region
Additional information primer 2PbCPβ_revII (NotI); 5' flanking region
Additional information primer 3PbCPβ_revIII (HindIII); 3' flanking region
Additional information primer 4PbCPβ_revIV (KpnI); 3' flanking region
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6