RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5066
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS; CSP)
Details mutation: glutamine in region I of CSP replaced with alanine (Q92A)
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)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 25 August 2022, 16:43
  *RMgm-5066
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-4681
Other information parent lineThe mutant lacks expression of CSP. In the mutant, the endogenous P. berghei csp gene has been deleted by replacing the csp gene with the drug-selectable marker cassette hdhfr-yfcu. It also expresses mCherry and luciferase under constitutive promoters
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-5066
Principal name3299cl1; 3300cl2
Alternative namePbcsp(mut1); Pbcsp(mut2)
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 numbers of oocysts are produced. On day 10 after infection melanized oocysts are present in 26-48% of CSP(mut) infected mosquitoes (1-35 melanized oocysts/mosquito), while such oocysts were absent in WT-infected mosquitoes.
SporozoiteReduced 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 CSP(mut) sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization.
Liver stageReduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Wild type development of liver stages in vitro.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of CSP with glutamine in region I of CSP replaced with alanine (Q92A). In addition, it expresses mCherry and luciferase under control of constitutive promoters. The mutated csp gene is introduced in the by GIMO transfection in the 'CSP-GIMO' line RMgm-4681.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Normal numbers of oocysts are produced. On day 10 after infection melanized oocysts are present in 26-48% of CSP(mut) infected mosquitoes (1-35 melanized oocysts/mosquito), 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 CSP(mut) sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Wild type development of liver stages in vitro.

Additional information
From the paper:
'Circumsporozoite surface protein (CSP) is the most abundant protein on the sporozoite surface. We therefore hypothesized that CSP could be a prime target for immune recognition of QC-null parasites. We analyzed published proteomes for pGlu modification of CSP and identified multiple CSP peptides with pGlu at the N-terminus with the majority (>90%) having pGlu modification of the glutamine (Q) located in region 1 of CSP. This short, five amino acid region (KLKQP), contains a proteolytic cleavage site and the glutamine is conserved across different Plasmodium species. CSP cleavage is known to occur at the sporozoite surface and this processing step is essential for host cell invasion. To establish whether CSP is a main target for immune recognition of QC-null sporozoites, two independent P. berghei lines (csp(mut)1,2) were generated that express a mutated CSP with glutamine in region 1 replaced with alanine (Q92A), thereby preventing pGlu formation. Both csp(mut) mutants produced similar numbers of oocysts as WT parasites. No melanization of csp(mut) oocysts was observed until day 10,whereas at day 14-16 p.i., melanized oocysts were detected in 26-48% of csp(mut)-infected mosquitoes (1-35 melanized oocysts/mosquito), mainly rupturing oocysts. Clusters of melanized sporozoites were found in the hemocoel and csp(mut) sg-sporozoite numbers were reduced compared to WT, although not statistically significant. We confirmed CSP processing in both WT and csp(mut) sg-sporozoites by Western analysis and csp(mut) sg-sporozoites showed WT-like infectivity to cultured hepatocytes. These observations demonstrate that the absence of the single glutamine residue in CSP region 1 results in immune recognition of viable/infective sporozoites in the hemocoel, resulting in melanization'.

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_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCS; CSP
Details of the genetic modification
Short description of the mutationglutamine in region I of CSP replaced with alanine (Q92A)
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 markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationTo generate two independent P. berghei mutants (Pbcsmut) that express a mutated CS with the glutamine in region I replaced with an alanine (Q92A), DNA construct pL2360 was generated. The cs 5’utr and part of the orf (until the mutation in region I) was amplified as fragment-1 using the primers 9771 and 9772. The remainder of the cs orf and 3’utr were amplified as fragment-2 using the primers 9773 and 9774. A unique restriction site EcoO109I was introduced in the mutated region using primers 9772 and 9773. Fragment-1 was digested with PstI/EcoO109I and fragment-2 with EcoO109I/SacI and subsequently introduced into the Pst/SacI sites of pUC19 vector by three fragment ligations. This resulted in construct pL2360 that was sequenced to confirm the presence of the point mutation and absence of undesired mutations. The plasmid pL2360 was linearized with XhoI and 431 transfected into PbΔcsp-GIMO parasites (https://www.pberghei.eu/ index.php?rmgm=4681) in two independent experiments, followed by applying negative selection with 5-fluorocytosine (5-FC) and cloning of selected parasites. This resulted in selection of the csp(mut) parasites (line 3299cl1, csp(mut1) and 3300cl1, csp(mut2)) where the hdhfr::yfcu SM in the csp locus of the PbΔcsp-435 GIMO line is replaced by the Q92A mutated Pbcsp orf.
Additional remarks selection proceduresee above
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