Summary

RMgm-4173
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1106900; Gene model (P.falciparum): PF3D7_0507300; Gene product: PIMMS2 protein, putative (Midgut Invasion Ookinete Protein Screen candidate 2)
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; Liver stage;
Last modified: 8 June 2017, 17:30
  *RMgm-4173
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) : 28559405
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/DepartmentLaboratory of Insect Immunogenomics, Department of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4173
Principal nameΔpbpimms2
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. Normal ookinete motility. Significantly reduced oocyst numbers/production.
SporozoiteNormal ookinete production. Significantly reduced oocyst numbers/production.Significantly reduced salivary gland numbers/production. Sporozoites were infective to mice as shown by bite-back experiments
Liver stageSignificantly reduced salivary gland numbers/production. Sporozoites were infective to mice as shown by bite-back experiments
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PIMMS2 and expresses GFP under the constitutive eef1a promoter.

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
Normal ookinete production. Normal ookinete motility. Significantly reduced oocyst numbers/production.Significantly reduced salivary gland numbers/production. Sporozoites were infective to mice as shown by bite-back experiments.
Evidence is presented that ookinetes fail to traverse the midgut epithelium (see below)

Additional information
PIMMS2 transcripts are limited to mosquito midgut stages. Transcripts were as early as 1 h post gametocyte activation and peaked as the ookinete development progressed. No PIMMS2 transcripts were detected in the oocyst.
Immunofluorescence assays revealed a prominent surface distribution of PIMMS2 in in vitro produced ookinetes that were permeabilized using Triton. This signal was largely abolished in non-permeabilized ookinetes suggesting that the subtilisin-like domain of PIMMS2, against which the antibody was raised, is located on the internal side of the ookinete surface. The surface distribution of P28, which was used as a control, showed no difference between permeabilized and non-permeabilized ookinetes, and it was not affected by the disruption of PIMMS2.

From the paper 'Next, we examined whether the Δpbpimms2 ookinetes fail to traverse the mosquito midgut epithelium or are eliminated after they traverse the epithelium, prior to their development to oocysts. Infections with Δpbpimms2 and c507 control parasites were carried out in A. gambiae L3-5 mosquitoes that are known to melanize P. berghei ookinetes immediately after they traverse the epithelium, upon exiting into the basal sub-epithelial space. Therefore, this assay provides a powerful means of quantifying the ookinete invasive ability. To control for variation in gametocyte numbers, membrane feeds of equal numbers of in vitro cultured ookinetes were performed. The results revealed that there were significantly lower numbers of melanized Δpbpimms2 ookinetes compared to c507 controls, indicating that the Δpbpimms2 phenotype is due to the failure of mutant ookinetes to traverse the midgut epithelium'.

Other mutants
 
RMgm-4174 - a mutant expressing 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).


  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 nameMidgut Invasion Ookinete Protein Screen candidate 2
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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