RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5367
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1431500; Gene model (P.falciparum): PF3D7_1215800; Gene product: radial spoke head protein 9, putative (RSP9)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 3 July 2023, 17:21
  *RMgm-5367
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37288670
MR4 number
top of page
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line that expresses GFP under the control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
top of page
The mutant parasite was generated by
Name PI/ResearcherRamakrishnan C, Sinden RE
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
top of page
Name of the mutant parasite
RMgm numberRMgm-5367
Principal nameΔPbrsp9-gfp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
top of page
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal numbers of mature male and female gametocyte numbers are produced. Male gametocyte formation (exflagellation) was absent (female gametes are fertile).
Fertilization and ookineteDespite the observed widespread impairment in male gamete formation, Δrsp9 parasites are able to form a small number of ookinetes
OocystThe number of oocysts obtained in the Δrsp9-gfp-infected mosquitoes were significantly lower than in the background/control line, correlating with the lower number of ookinetes produced. Mosquitoes infected with wt-gfp or Δrsp9-gfp parasites were also assessed for sporozoite loads and these were shown to be very variable in both the wild type and mutant line; additionally, infected mosquitoes could successfully transmit Δrsp9-gfp to naïve mice
SporozoiteThe number of oocysts obtained in the Δrsp9-gfp-infected mosquitoes were significantly lower than in the background/control line, correlating with the lower number of ookinetes produced. Mosquitoes infected with wt-gfp or Δrsp9-gfp parasites were also assessed for sporozoite loads and these were shown to be very variable in both the wild type and mutant line; additionally, infected mosquitoes could successfully transmit Δrsp9-gfp to naïve mice
Liver stageThe number of oocysts obtained in the Δrsp9-gfp-infected mosquitoes were significantly lower than in the background/control line, correlating with the lower number of ookinetes produced. Mosquitoes infected with wt-gfp or Δrsp9-gfp parasites were also assessed for sporozoite loads and these were shown to be very variable in both the wild type and mutant line; additionally, infected mosquitoes could successfully transmit Δrsp9-gfp to naïve mice
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of RSP9 and expresses GFP under control of the constitutive eef1a promoter

Protein (function)
The core of cilia and flagella is the axoneme, a cylindrical structure composed of 9 outer microtubule doublets. Motile cilia/flagella normally have two central single microtubules - the central pair, resulting in an overall “9+2” axoneme organisation. Radial spokes (RSs) assemble on the peripheral microtubule doublets and project towards to the central pair complex which is composed of the central pair and associated proteins. Plasmodium species have a flagellum in one critical stage of the life cycle: during sexual development, when flagellate male gametes fertilise female gametes. Only 3 RS proteins (RSPs) could be identified in Plasmodium: RSP3 (PBANKA_1039000), RSP9 (PBANKA_1431500) and RSP4/6 (PBANKA_0942300).

Phenotype
Normal numbers of mature male and female gametocyte numbers are produced. Male gametocyte formation (exflagellation) was absent (female gametes are fertile). Despite the observed widespread impairment in male gamete formation, Δrsp9 parasites are able to form a small number of ookinetes.
The number of oocysts obtained in the Δrsp9-gfp-infected mosquitoes were significantly lower than in the background/control line, correlating with the lower number of ookinetes produced. Mosquitoes infected with wt-gfp or Δrsp9-gfp parasites were also assessed for sporozoite loads and these were shown to be very variable in both the wild type and mutant line; additionally, infected mosquitoes could successfully transmit Δrsp9-gfp to naïve mice

Additional information
GFP-tagging of PbRSP9 was unsuccessful after several attempts, however anti-PfRSP9 were available. We thus determined RSP9 localisation in the human parasite P. falciparum using two specific already available anti-PfRSP9 antisera. In exflagellating gametocytes, the anti-PfRSP9 antibody signal colocalized with the anti-α-tubulin II marker onto the axoneme. No signal was seen in the blood stages.

To investigate if the loss of RSP9 only affected male and not female gametocytes, genetic crossing using a male-defective line that produces normal female gametes, was performed. The parasite line Δcdpk4 yielded similar numbers of ookinetes as Δrsp9 alone, whilst fertilisation with the female-defective line Δnek4 resulted in similar numbers of ookinetes as a cross of Δcdpk4 with Δnek4. This indicates that female Δrsp9 gametocytes/gametes are fully functional while male Δrsp9 gametocytes, although formed, are severely impaired in their function.

We investigated both axoneme formation and DNA replication in activated male gametocytes by simultaneous staining with an anti-α-tubulin II antibody that specifically stains Plasmodium male gametocytes and gametes as well as DAPI to visualize the eight newly replicated copies of the genome. At 15 min post-activation, free wild type (WT) microgametes were clearly stained with both anti-α-tubulin II antibody and DAPI revealing a wavy structure typical of motile gametes. Nuclei are mostly associated to flagella. While Δrsp9 show extensive staining with anti-α-tubulin II antibody and have clearly distinguishable nuclei their flagella seem to protrude only partially. Mitosis therefore appears to be completed normally, while subsequent axoneme motility appears to be abolished.

To assess whether these ookinetes were capable of completing the life cycle, mosquitoes were fed on mice infected with wt-gfp or Δrsp9-gfp parasites respectively, and oocyst numbers in the midguts were assessed. The number of oocysts obtained in the Δrsp9-gfp-infected mosquitoes were significantly lower than in the background/control line, correlating with the lower number of ookinetes produced. Mosquitoes infected with wt-gfp or Δrsp9-gfp parasites were also assessed for sporozoite loads and these were shown to be very variable in both the wild type and mutant line; additionally, infected mosquitoes could successfully transmit Δrsp9-gfp to naïve mice

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
top of page
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1431500
Gene Model P. falciparum ortholog PF3D7_1215800
Gene productradial spoke head protein 9, putative
Gene product: Alternative nameRSP9
top of page
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 parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate a loss of function mutant in P. berghei, the 5’ and 3’ UTR of rsp9 were cloned into pOB90 (courtesy of O. Billker) each gene fragment flanking the pyrimethamine resistance marker tgdhfr-ts. The 5’UTR was amplified using 5'UTRF and 5'UTRR and the 3’ UTR using 3'UTRF and 3'UTRR amplifying 955 or 985 bp from genomic DNA respectively. The PCR products of the 5’UTR and 3’UTR were digested with KpnI and ApaI or BamHI and XbaI and cloned into KpnI/XbaI digested pOB90. To obtain deletion mutants, plasmid PbRsp9ko was digested with KpnI and XbaI
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
top of page
  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
top of page
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
top of page
Other details transgene
top of page
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
top of page
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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
top of page
Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren