RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4683
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0202700; Gene model (P.falciparum): PF3D7_0111000; Gene product: kinesin-8B, putative
Name tag: GFP
Phenotype Gametocyte/Gamete;
Last modified: 27 October 2019, 14:16
  *RMgm-4683
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) : 31634979
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/ResearcherDepoix D, Kohl L
Name Group/DepartmentCNRS Molécules de Communication et Adaptation des Micro-organismes
Name InstituteMuséum National d'Histoire Naturelle, Sorbonne Universités
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-4683
Principal namePbkin8B-gfp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of male and female gametocytes. Normal male and female gametocyte production.
No GFP expression in blood stages and female gametocytes. In unactivated male gametocytes, cytosolic expression. Upon activation, the localization of the Pbkin8B-gfp signal changes and is seen as punctiform lines with spatial and temporal dynamics similar to the axonemal marker α-tubulin II. Expression in male gametes.
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of kinesin-8B

Protein (function)
Plasmodium axonemes display a classical “9+2” structure: 9 doublets of microtubules arranged in a circular pattern surrounding a central pair of singlet microtubules. Their mode of assembly is unusual: Plasmodium axonemes are assembled in the cytoplasm of the microgametocyte independent of intraflagellar transport, a bidirectional transport machinery essential for the construction of flagella in most other eukaryotes. The components of the axoneme, such as tubulin, are ubiquitous in the cytoplasm and are assembled upon activation.  The nuclear envelope does not break down during the 3 mitotic replications. The axonemes are however linked to the 8 replicated haploid genomes in the nucleus through spindle poles situated in nuclear pores, thus forming 8 flagellated motile microgametes, which, following violent axonemal ‘swimming’ are expelled from the microgametocyte in an exceptionally fast process called “exflagellation”. These dynamic processes will certainly require motor proteins, such as kinesins and dyneins, to transport cargoes, assemble the structure and generate the force for motility. Most dyneins involved in motility, are part of the complex organisation of outer and inner dynein arms present on each doublet microtubule. In Plasmodium, inner dynein arms are seen less often than outer dynein arms and appear thinner in electron microscopy studies.
Only 10 kinesin encoding genes are found in the P. berghei genome, fewer than in other eukaryotes. Among the kinesins identified by proteomic  studies of sexual stages, only three are present in male gametocytes and gametes. According to the comprehensive study of kinesins across eukaryotes by Wickstead et al. (2010), they belong to the families kinesin-8 (subfamily 8B), -13 and -15. Those kinesins are among the microtubule motors phosphoregulated during  P. berghei gametocyte activation. While most kinesins display predominantly one activity, either transport along microtubules or depolymerization, kinesin-8 family members are reportedly multitalented. They can walk on microtubules, but also regulate microtubule length, during mitosis and/or ciliary and flagellar assembly.
PbKIN8B encompasses a classical motor domain positioned centrally [aa 779- 1118] with 8 predicted ATP binding sites [aa 787, 872, 875, 877, 878, 879, 880, 1018] and 3 predicted microtubule interaction sites [aa 1071, 1074, 1077]. With the exception of the kinesin motor domain and of coiled-coil motives in the C-terminal region, no additional protein signatures or nuclear localisation signals (NLS) were detected.

Phenotype
Analysis of a mutant lacking expression of kinesin-8B (RMgm-4682) showed the following: Normal production of male and female gametocytes. No male gamete formation as shown by absence of exflagellation centers. Strongly reduced fertilisation, ookinete and oocyst formation.

Analysis of Pbkin8B-gfp parasites showed: Normal production of male and female gametocytes. Normal male and female gametocyte production. No GFP expression in blood stages and female gametocytes. In unactivated male gametocytes, cytosolic expression. Upon activation, the localization of the Pbkin8B-gfp signal changes and is seen as punctiform lines with spatial and temporal dynamics similar to the axonemal marker α-tubulin II. Expression in male gametes.

Additional information

Other mutants
a mutant lacking expression of kinesin-8B (RMgm-4682)
 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0202700
Gene Model P. falciparum ortholog PF3D7_0111000
Gene productkinesin-8B, putative
Gene product: Alternative name
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationhe Pbkin8B-gfp plasmid was generated by amplifying the final portion of the PbKIN8B coding sequence [nt1978-nt4378] with primers 111 and 112b. A unique restriction site for SacII in the middle of this region was used for single digestion and single crossover. The construct was inserted into the vector pl0016 (MRA-785 (BEI Resources)). The stop codon was removed and the gfp coding sequence was fused in-frame to the coding sequence. The plasmid also contained the T. gondii dhfr/ts resistance marker conveying resistance to pyrimethamine.
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