Summary

RMgm-4458
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1106900; Gene model (P.falciparum): PF3D7_0507300; Gene product: PIMMS2 protein, putative (PIMMS2; SOPT)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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: 29 June 2018, 17:31
  *RMgm-4458
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) : 29873127
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-29
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase 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/ResearcherArmistead JS, Boddey JA
Name Group/DepartmentDepartment of Medical Biology
Name InstituteThe University of Melbourne
CityParkville 3052, Victoria
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4458
Principal name1482cl1
Alternative namePbΔSOPT/PIMMS2
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
Oocyst(Strongly) Reduced oocyst formation
Sporozoite(Strongly) Reduced sporozoite formation
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PIMMS2/SOPT and expresses GFP-luciferase under control of the constitutive eef1a promoter

Protein (function)
SOPT and one other gene have a conserved location between SUB1 (subtilisin 1; PF3D7_0507500) and SUB3 (subtilisin 3; PF3D7_0507200) in different Plasmodium species. SOPT is predicted to encode an 893 amino acid protein of 107 kDa that contains a signal peptide (SignalP prediction 23FLL↓ KQ27), a PEXEL-like sequence (38RILEE42) and a putative transmembrane helix (position 490-505). A homology model of SOPT revealed a 387 amino acid subtilisin-like fold from the S8 peptidase family, similar to that of SUB1 from P. falciparum. The model was sufficient to reveal the conserved positions of putative catalytic residues. However, the catalytic triad in SUB1 (D372, H428 and S606) was absent from SOPT, with only the nucleophile serine present (G172, E247 and S492). In contrast, P. berghei SOPT/PIMMS2 possessed a canonical triad with potential to form a charge-relay network typical of subtilisins (D155, H222, S414). A multiple sequence alignment of Plasmodium orthologs revealed that catalytic residues were not conserved, with SOPT from P. malariae, P. reichenowi, P. yoelii and P. chabaudi chabaudi lacking either the catalytic aspartate and/or histidine residue, like P. falciparum, while orthologs in P. vivax, P. ovale, P. knowlesi and P. gallinaceum possessed a canonical triad, as in P. berghei. The putative transmembrane helix internal to SOPT is also predicted in some orthologs; however, it is positioned within 5 amino acids from the nucleophile serine residue suggesting that the transmembrane helix prediction may be incorrect. No protein domain in the C-terminus of SOPT was identified by computational methods or modelling. Given the divergent catalytic triad across the genus, it is likely that SOPT is a pseudoprotease rather than an active protease in Plasmodium spp.

Phenotype
Normal ookinete formation; (Strongly) reduced oocyst and sprozoite formation, sporozoites have normal infectivity to mice

Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1106900
Gene Model P. falciparum ortholog PF3D7_0507300
Gene productPIMMS2 protein, putative
Gene product: Alternative namePIMMS2; SOPT
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct 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 modificationTo generate a P. berghei mutant line lacking expression PBANKA_110690, we targeted the gene locus for deletion using a linear construct, generated using a 2-step anchor tagging PCR method (Lin et al., 2011). The 5’- and 3’ targeting regions of the gene were PCR amplified from genomic DNA using primer pairs 4744/4745 and 4746/4747 (for primer details see Table S4). Primers 4745 and 4747 have 5’-terminal extensions homologous to the hdhfr selectable marker cassette. Primers 4744 and 4747 both have a 5’-terminal overhang with an anchor-tag which serves as a primer site in the 2nd PCR reaction. The target fragments from the first PCR reaction were annealed to either side of the selectable marker cassette by PCR with anchor-tag primers 4661 and 4662, resulting in the 2nd PCR product. To remove the ‘anchor’, the final PCR fragment was digested with Asp718 and ScaI (primers 4744 and 4747 contained Asp718 and ScaI restriction enzyme sites, respectively), resulting in construct pL1500. The hdhfr selectable marker cassette used in this reaction was digested from pL0040 using restriction enzymes XhoI and NotI (pL0040 is available from The Leiden Malaria Research Group).
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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