Summary

RMgm-4824
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1119200; Gene model (P.falciparum): PF3D7_0620000; Gene product: PIMMS43 protein (secreted ookinete protein 25 (PSOP25), ookinete surface-associated protein 8 (PSOP8))
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
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 Oocyst; Sporozoite;
Last modified: 20 June 2020, 19:45
  *RMgm-4824
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) : 32165544
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherUkegbu CV, Vlachou D
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4824
Principal nameΔc43
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystIn Anopheles coluzzi no oocyst formation
SporozoiteIn Anopheles coluzzi no oocyst formation; no sporozoite formation (in A stephensi low numbers of oocysts were observed, but no sporozoites).
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PIMMS43

Protein (function)
PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43).
PIMMS43 encode deduced proteins of 505 and 350 amino acids, respectively. N-terminal signal peptides (amino acids 1 to 25 for PfPIMMS43 and 1 to 22 for PbPIMMS43) and C-terminal transmembrane domains (amino acids 482 to 504 for PfPIMMS43 and 327 to 350 for PbPIMMS43) are predicted for both proteins. The transmembrane domains are predicted by PredGPI to also contain signals for attachment of a GPI lipid anchor with 99% probability.

Our transcriptomic profiling of field P. falciparum isolates from Burkina Faso in the midgut of sympatric Anopheles coluzzii (previously Anopheles gambiae M form) and Anopheles arabiensis mosquitoes and a laboratory P. berghei strain in the midgut of A. coluzzii identified hundreds of genes exhibiting conserved and differential expression during gametocyte-to-oocyst development. Several of them encoding putatively secreted or membraneassociated proteins were made part of a screen to identify genes that function during parasite infection of the mosquito midgut. These genes were given a candidate gene number preceded by the acronym PIMMS, for “Plasmodium Infection of the Mosquito Midgut Screen.”
Here, we report the characterization of P. falciparum and P. berghei PIMMS43 that encodes a membrane-bound protein found on the surface of ookinetes and sporozoites. The gene was first reported in P. berghei to be a target of the transcription factor AP2-O, has a role in mosquito midgut invasion and oocyst development, and was named POS8. A later study by another group reported the gene as being important for ookinete maturation, designating it as PSOP25. Evidence is presented that PIMMS43 has no detectable function in ookinete maturation or mosquito midgut invasion but plays a key role in ookinete evasion of the mosquito complement-like response.

Phenotype
Normal blood stage development; normal production of gametocytes, gametes and ookinetes. In Anopheles coluzzi no oocyst formation; no sporozoite formation (in A stephensi low numbers of oocysts were observed, but no sporozoites).

Additional information
Evidence is presented that:
- PfPIMMS43 prominently localizes on the surface of female gametes or early stage zygotes found in the A. coluzzii blood bolus , as well as on the surface of ookinetes traversing the mosquito midgut epithelia and sporozoites found in the mosquito salivary gland lumen.
- Ookinetes Lacking PIMMS43 Are Killed by the Mosquito Complement-Like Response upon Midgut Traversal.
- PIMMS43 Is Additionally Required for Oocyst Maturation and Sporozoite Development
- PIMMS43 KO Leads to Compromised Ookinete Fitness and Attack by the Complement-Like Response
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Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1119200
Gene Model P. falciparum ortholog PF3D7_0620000
Gene productPIMMS43 protein
Gene product: Alternative namesecreted ookinete protein 25 (PSOP25), ookinete surface-associated protein 8 (PSOP8)
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 genePartial
Additional remarks partial/complete disruption 50% of the coding region deleted
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPartial ko of P. berghei c43 CDS was carried out by double crossover homologous recombination in the c507 and 1804cl1 lines. For partial disruption in the c507 line, a 765 bp ApaI/HindIII 5’ homology arm and a 528 bp EcorI/BamHI 3’ homology arm was amplified from P. berghei 2.34 genomic DNA using the primer pairs P1/P2 and P3/P4 respectively. These fragments were cloned into the pBS-TgDHFR vector which carries a modified Toxoplasma gondii dihydrofolate gene (TgDHFR/TS) cassette that confers resistance to pyrimethamine. The targeting cassette was released by ApaI/BamHI digestion and it allows ko of 50% of P. berghei c43 CDS at the 5’ region. For partial disruption in the 1804cl1 line, the target vector containing the human DHFR selection cassette was used (kindly provided by plasmoGEM, vector design ID, PbGEM042760; http://plasmogem.sanger.ac.uk/). hDHFR confers resistance to the drugs pyrimethamine and WR92210. The targeting cassette was released by NotI digestion and allows ko of 74% of PbPIMMS43 CDS leaving a small part of the 3’ region of the CDS
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
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 modification
Additional remarks selection procedure
Other details transgene
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
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