RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5096
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (sporozoite surface protein 2; SSP2; SSP-2; TRAP)
DisruptedGene model (rodent): PBANKA_1310700; Gene model (P.falciparum): PF3D7_1446900; Gene product: glutaminyl-peptide cyclotransferase, putative (glutamyl cyclase, QC)
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;
Last modified: 25 August 2022, 16:36
  *RMgm-5096
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption, 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-5057
Other information parent lineThis mutant (line 3172cl1(2nd)) lacks expression of QC and expresses luciferase under the control of the eef1α (PBANKA_1133300) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_0711900) promoter. Both reporter cassettes are introduced into the silent 230p locus, using a single DNA construct. The mutant does not contain a drug-selectable marker (SM) that has been removed by negative selection from mutant RMgm-5056 (line 2930cl1).
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-5096
Principal name3365cl1
Alternative namePbΔqcΔtrap
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. Sporozoite formation inside oocysts. Normal hemolymph (midgut) sporozoite production. Melanization of oocysts and sporozoites (comparable to single gene-deletion mutant lacking expression of QC).
SporozoiteNormal numbers of oocysts are produced. Sporozoite formation inside oocysts. Normal hemolymph (midgut) sporozoite production. Melanization of oocysts and sporozoites (comparable to single gene-deletion mutant lacking expression of QC).
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of QC and TRAP and expresses mCherry and luciferase under control of constitutive promoters.
The TRAP gene has been deleted in mutant RMgm-5057 (line 3172cl1(2nd)) that lacks expression of QC and expresses luciferase under the control of the eef1α (PBANKA_1133300) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_0711900) promoter. Mutant RMgm-5057 does not contain a drug-selectable marker (SM) that has been removed by negative selection from mutant RMgm-5056 (line 2930cl1). 


Protein (function)
QC: 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. 

TRAPTRAP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells.

Phenotype
Normal numbers of oocysts are produced. Sporozoite formation inside oocysts. Normal hemolymph (midgut) sporozoite production. Melanization of oocysts and sporozoites (comparable to single gene-deletion mutant lacking expression of QC).

In mosquitoes infected with QC 'single knockout' mutants (RMgm-5056RMgm-5057RMgm-5058) melanized oocysts are present on day ten after infection in 55-65% of QC-null infected mosquitoes (1-85 melanized oocysts/mosquito), while such oocysts were absent in WT-infected mosquitoes. In addition, reduced numbers of salivary gland sporozoites are present.  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. 
 

From the paper about  ‘double knock-out’ QC-null mutants:
'The absence of melanized QC-null oocysts before day 10 p.i. and absence of distinct deposition of melanin on the oocyst capsule may suggest that QC-null sporozoites, either still inside rupturing oocysts or after release into the hemocoel, are specifically recognized by the immune system. If this is the case, abolishing sporozoite formation inside QC-null oocysts or blocking egress of OC-null sporozoites would prevent oocyst melanization. To test this hypothesis we deleted genes encoding proteins involved in sporozoite formation (CSP, ROM3) or in sporozoite egress (ECP1, CRMP4) in P. berghei QC-null parasites. These ‘double knock-out’ QC-null mutants (RMgm-5062RMgm-5063RMgm-5064RMgm-5065) produced WT numbers of oocysts; however, melanized oocysts were completely absent. In contrast, melanized oocysts/sporozoites were observed in 79% of mosquitoes infected with a ‘double knock-out’ QC-null mutant (RMgm-5066) lacking the trap gene that releases sporozoites into  the hemocoel that cannot invade the salivary glands (1-60 melanized oocysts/mosquito). These observations confirm that melanization only occurs when QC-null oocysts rupture and sporozoites are released into the hemocoel'.

Additional information
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

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1349800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2; TRAP
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-338811
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe hdhfr::yfcu SM was first recycled from PbΔqc1 (RMgm-5056; line 2930cl1) parasites by negative selection with 5-Fluorocytosine (5-FC). This treatment selects for parasites that have undergone homologous recombination between the two 3’-UTR sequences of Pbdhfr, present in the integrated construct PbGEM-342996 (pL2243) that flank the hdhfr::yfcu SM cassette and thereby removing the SM. Selection and cloning of the parasites resulted in the SM-free ‘single knockout’ mutant PbΔqc1(-sm) (line 3172cl1(2nd)).
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

  Disrupted: Mutant parasite with a disrupted 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
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-342996
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) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe hdhfr::yfcu SM was first recycled from PbΔqc1 (RMgm-5056; line 2930cl1) parasites by negative selection with 5-Fluorocytosine (5-FC). This treatment selects for parasites that have undergone homologous recombination between the two 3’-UTR sequences of Pbdhfr, present in the integrated construct PbGEM-342996 (pL2243) that flank the hdhfr::yfcu SM cassette and thereby removing the SM. Selection and cloning of the parasites resulted in the SM-free ‘single knockout’ mutant PbΔqc1(-sm) (line 3172cl1(2nd)).
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