Summary

RMgm-4174
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1106900; Gene model (P.falciparum): PF3D7_0507300; Gene product: PIMMS2 protein, putative (Midgut Invasion Ookinete Protein Screen candidate 2)
Details mutation: Asp155, His222 and Ser414, which make up the catalytic triad of subtilases, replaced with alanines
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;
Last modified: 23 June 2017, 17:41
  *RMgm-4174
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28559405
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4173
Other information parent lineThe parent line is a mutant line that lacks the PIMMS2 gene
The mutant parasite was generated by
Name PI/ResearcherUkegbu CV, Vlachou D
Name Group/DepartmentLaboratory of Insect Immunogenomics, Department of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4174
Principal nameΔpbpimms2::pimms2(mut)
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
OocystNormal ookinete production. The Δpbpimms2::pimms2mut parasites exhibited oocysts numbers that were significantly less than those of control parasite lines parasites but higher than those of the Δpbpimms2 parasites (RMgm-4173). These data suggest that the amino acid residues that form the putative catalytic triad of subtilases are important but not essential for the function of PIMMS2.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of PIMMS2 (the conserved Asp155, His222 and Ser414 residues of PIMMS2, which make up the catalytic triad of subtilases, were replaced with alanine residues by site-directed mutagenesis). In addition, it expresses GFP under the constitutive eef1a promoter.
The mutated pimms2 gene has been introduced in a mutant lacking the pimms2 gene.

Protein (function)
PBANKA_1106900, is mapped between genes encoding two known subtilisin-like proteases, SUB1 and SUB3, and encodes an 836 amino acid long protein. Bioinformatics analysis predicted with high probability two hydrophobic regions of alpha helix structure encompassing the amino acid residues 6-25 and 416-427, respectively. Additionally,  a cleavage site at amino acid residue 30 was predicted suggesting an N-terminal signal peptide and a transmembrane domain located at the center of the protein. Protein sequence analysis predicted a peptidase S8 domain (IPR000209) that ends shortly after the predicted transmembrane domain and is followed by a long C-terminal extension (CTE)  with no known domain homology. A highly significant (100% confidence) structural similarity was predicted of the predicted peptidase S8 domain region to the P. vivax SUB1, and P. falciparum SUB1. PIMMS2 sequence alignments with PvSUB1 and the bacterial subtilisin BPN’ identified several conserved amino acid residues within the predicted subtilisin-like domain including D155, H122 and S414, which correspond to the catalytic triad of subtilisin-like serine proteases. A conserved asparagine residue N328 that serves in stabilizing the putative oxyanion hole was also identified. However, the conserved motifs HGT and GTS, which encompass the catalytic His and Ser residues of subtilases, respectively, are not present in PIMMS2, suggesting that the putative catalytic triad is likely to be non-functional and thus PIMMS2 is a potential pseudoenzyme.
The subtilisin-like domain architecture of PbPIMMS2 is conserved only in PvPIMMS2; the  rest of the PIMMS2 orthologs appear to lack one or more of the conserved amino acid residues or motifs.

Phenotype
Analyses of a mutant lacking the wild type pimms2 gene (RMgm-4173) showed significantly reduced oocyst numbers/production. Evidence is presented that ookinetes fail to traverse the midgut epithelium in the absence of PIMMS2 expression (see below)

Analysis of the mutant expressing the mutated PIMMS2 showed the following:
Normal ookinete production. The Δpbpimms2::pimms2mut parasites exhibited oocysts numbers that were significantly less than those of control parasite lines parasites but higher than those of the Δpbpimms2 parasites (RMgm-4173). These data suggest that the amino acid residues that form the putative catalytic triad of subtilases are important but not essential for the function of PIMMS2.

Additional information


Other mutants
RMgm-4173 - a mutant lacking expression of PIMMS2.


  Mutated: Mutant parasite with a mutated 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 nameMidgut Invasion Ookinete Protein Screen candidate 2
Details of the genetic modification
Short description of the mutationAsp155, His222 and Ser414, which make up the catalytic triad of subtilases, replaced with alanines
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutated pimms2 gene has been introduced in a mutant lacking the pimms2 gene (RMgm-4173). The mutated gene is introduced into the pimms2 gene locus.
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 modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
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