RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5061
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1310700; Gene model (P.falciparum): PF3D7_1446900; Gene product: glutaminyl-peptide cyclotransferase, putative (glutamyl cyclase, QC)
Details mutation: the P. berghei qc gene replaced with the qc gene of P. falciparum
Transgene
Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: Luciferase
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 (P230p)
PhenotypeNo phenotype has been described
Last modified: 25 August 2022, 16:40
  *RMgm-5061
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35994647
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-5058
Other information parent lineThis mutant lacks expression of QC and expresses mCherry and luciferase under control of constitutive promoters. In this QC-GIMO line the qc open reading frame is replaced with a hdhfr-yfcu selectable marker cassette.
The mutant parasite was generated by
Name PI/ResearcherKolli SK, Janse CJ
Name Group/DepartmentMalaria Research Group, Department of Parasitology,
Name InstituteLeiden University medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-5061
Principal name3213cl1
Alternative namePfqc(c))
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
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
In the mutant the P. berghei qc gene has been replaced with the qc gene of P. falciparum. In addition, the mutant expresses mCherry and luciferase under control of constitutive promoters. The P. falciparum qc  gene has been introduced by GIMO transfection in mutant PbΔqc-G (3198cl1; RMgm-5058).

Protein (function)
N-terminal modification of glutamine or glutamic acid residues to pyroglutamic acid (pGlu; 5-oxo-L-46 proline) is a posttranslational modification (PTM), catalyzed by glutaminyl cyclases (QCs) found in eukaryotes and prokaryotes. Two evolutionary unrelated classes exist; mammalian QCs and QCs of bacteria, plants and parasites, which share no sequence homology, supporting a different evolutionary origin. Mammalian cells can express two forms, the secreted glutaminyl‐peptide cyclotransferase (QPCT) or its iso-enzyme (QPCTL), localized in the Golgi complex. pGlu is implicated in maturation and stabilization of mammalian proteins such has neuropeptides and cytokines. QC activity has been associated in humans with pathological processes such as amyloidotic diseases and QPCTL is critical for pGLu formation on CD47, facilitating myeloid immune evasion.

A single gene encoding a glutaminyl cyclase (QC), named glutaminyl-peptide cyclotransferase, has been identified by electronic annotation in all sequenced Plasmodium genomes. Plasmodium QCs share 70-76% sequence similarity and 50-54% identity and contain a transmembrane domain. QC of the human malaria parasite P. falciparum (PfQC) shows 21-27% identity to QCs of various 64 bacteria and the plant Carica papaya (CpQC). All 9 amino acids of the catalytic site of bacterial and plant QCs are conserved in Plasmodium QC. 

Phenotype
The phenotype analyses indicate that  P. falciparum QC can complement the function of P. berghei QC. No melanized oocysts or sporozoites were observed.

In mutants lacking expression of QC (RMgm-5056RMgm-5057RMgm-5058) the following phenotype was observed:
'Normal numbers of oocysts are produced. On day 10 after infection melanized oocysts are present in >50% of QC-null infected mosquitoes, while such oocysts were absent in WT-infected mosquitoes.
Reduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Light-microscopy analysis of oocysts between day 14 and 21 showed the presence of aberrant, enlarged, melanized sporozoites, either still inside or during release from oocysts.
On day 21 p.i. non-motile, enlarged sporozoites covered by melanin were found in the hemocoel, often in clusters or attached to salivary glands. In contrast, most QC-null sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization'.


Additional information
Published expression data indicate that QC is expressed in gametocytes, ookinetes, oocysts, and sporozoites. QC expression in mosquito/transmission stages was confirmed by analyzing a transgenic rodent malaria parasite (P. berghei; Pbqc::cmyc), expressing a cmyc-tagged QC (RMgm-5059).

From the Abstract:
'We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites'.

Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1310700
Gene Model P. falciparum ortholog PF3D7_1446900
Gene productglutaminyl-peptide cyclotransferase, putative
Gene product: Alternative nameglutamyl cyclase, QC
Details of the genetic modification
Short description of the mutationthe P. berghei qc gene replaced with the qc gene of P. falciparum
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 parasiteNo selectable marker
Promoter of the selectable markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationFor complementation of PbΔqc QC-null parasites with the qc gene of P. falciparum (Pfqc), the PbΔqc-GIMO parasite line (line 3198cl1) was used. For complementation with Pfqc, DNA construct pL2326 was generated. The Pbqc 5’ utr and 3’ utr were amplified from P. berghei gDNA using primers 9483/9484 368 and 9485/9486 respectively and introduced sequentially into pBSKS+ plasmid (Stratagene) at 369 XhoI/BamHI and EcoRI/SacII restriction sites respectively, to obtain intermediate plasmid SKK94. The Pfqc orf was amplified from WT P. falciparum NF54 gDNA using primers 9487/9488 and ligated into SKK94 using BamHI and EcoRI restriction sites, to obtain pL2326. The DNA-construct pL2326 was sequenced to confirm absence of mutations and the plasmid was linearized with XhoI and SacII before transfection. Transfection of PbΔqc-GIMO parasites with the construct, followed by applying negative selection with 5-fluorocytosine (5-FC) and subsequent cloning of the parasites resulted in selection of complemented parasites where the hdhfr::yfcu SM in the qc locus of PbΔqc-GIMO is replaced by the orf of either Pfqc (line 3213cl1; Pfqc(c)).
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 namemCherry
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
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 nameP230p
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