RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5105
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1116000; Gene model (P.falciparum): PF3D7_0616500; Gene product: TRAP-like protein (TLP)
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)
Phenotype Sporozoite;
Last modified: 10 November 2021, 13:05
  *RMgm-5105
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34717635
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherBeyer K, Frischknecht F
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School
CityHeidelberg
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-5105
Principal nametlp(-)trap(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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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
SporozoiteNormal numbers of hemolymph sporozoites; strongly reduced numbers of salivary gland sporozoites; strongly reduced ('unproductive') gliding motility
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of both TLP and TRAP (see "additional remarks genetic modification' for expression of the reporter mCherry under control of the promoter of the csp gene in the different mutants)

Protein (function)
TLP belongs to the TRAP/MIC2 family of transmembrane proteins, members of which link extracellular adhesion to the intracellular actomyosin motor complex that plays a role in (gliding) motility and cell invasion.
Similar to the Plasmodium sporozoite protein, TRAP, and the ookinete protein, CTRP, TLP possesses an extracellular domain architecture that is comprised of von Willebrand factor A (vWA) and thrombospondin type 1 (TSP1) domains, plus a short cytoplasmic domain.

TRAP 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). The cytoplasmic part (tail) of the protein is postulated to interact with actin–myosin motor proteins giving the force needed for motility.
TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells as has been shown by analysis of mutant parasites lacking expression of TRAP.

Phenotype
Normal numbers of hemolymph sporozoites; strongly reduced numbers of salivary gland sporozoites; strongly reduced ('unproductive') gliding motility.

Additional information
From the paper:
'Taken together, no evidence was obtained of a synergistic or antagonistic effect of the investigated proteins, TRAP, TLP and TREP. TRAP-family proteins appear to work independently of each other to enable sporozoite infection of salivary glands (TRAP, TREP) and efficient transmission to mice (TRAP, TLP). However, the observation that sporozoites lacking all three TRAP-family adhesins could still undergo some active motion, i.e., waving and patch gliding, suggests that additional proteins are important in sporozoite adhesion and the transition from adhesion to motility."

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1116000
Gene Model P. falciparum ortholog PF3D7_0616500
Gene productTRAP-like protein
Gene product: Alternative nameTLP
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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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationGeneration of Plasmodium berghei parasite lines lacking two and three TRAP family adhesins: In order to generate parasite lines lacking more than one TRAP family gene, a strategy based on sequential positive and negative selection was employed. Vectors for deletion of trep and tlp were engineered to replace the endogenous gene with the gene encoding for the red fluorescent protein mCherry controlled by the circumsporozoite protein (csp) promoter, which is strongly expressed throughout the life of sporozoites. The vector for the deletion of trap was obtained from PlasmoGEM, which also contained a cassette enabling positive–negative selection. A clonal tlp knockout line was generated and the drug resistance cassette was recycled by negative selection. Subsequently, vectors targeting the trap and trep genes were transfected into the tlp(-) line. The trep KO vector was additionally transfected into trap(-) parasites that were generated previously yielding the line trep(-)/trap(-). A triple mutant line lacking all three TRAP family adhesins was obtained by negative selection of the trap(-)/tlp(-) line followed by deletion of trep.
Generation of plasmids and parasite lines
All generated plasmids were based on either the Pb238 or the Pb301 transfection vector containing the 5′ and 3′ UTRs of tlp or trep as well as the gene encoding the green fluorescent protein (GFP). Prior to transfection, all vectors were linearized with SacII and PmeI.

Pb262 transfection vector: This vector uses the hDHFR-yFCU selection marker cassette and the reporter gene mCherry under the control of the csp promoter. Using the Pb238 as parental vector, the selection marker cassette was replaced with the dhfr 3′UTR–ef1α 5′UTR–hDHFR–yFCU–dhfr 3′UTR from PlasmoGEM transfection vector, which was amplified with P600 and P601 and cloned with EcoRV HindIII into Pb238. Next, the 5′UTR of csp was amplified from P. berghei gDNA with primers P207 and P208 and cloned into the vector with EcoRI and NdeI. The open reading frame of the red fluorescent protein encoding gene mCherry was amplified with primers P238 and P232 and inserted into the vector with NdeI/BamHI. The resulting vector is termed Pb262.
trap(-) and trap(-)NS: For the generation of trap(-) parasites the plasmid PbGEM-107890 was requested from PlasmoGEM. It replaces most of the TRAP coding sequence with the positive–negative selection marker hDHFR-yFCU. For negative selection the drinking water of mice was supplemented with 1 mg/ml 5-FC. The parasite line was already generated and characterized in a previous study and used here as basis to generate the double mutant.
trap(-)/trep(-)/tlp(-)mCherry: The final TLP-KO transfection vector (PbCSmCherryYFCU_TLP-KO) was generated by amplifying the 5′ UTR of tlp from Plasmodium berghei gDNA with primers P159 and P160 and cloning into Pb238 via SacII/ NotI. The tlp 3′ UTR was amplified with primers P161 and P162 and then cloned via HindIII/KpnI. Next, using HindIII and NotI restriction sites of the ‘TLP plasmid’ and Pb262, the selection cassette and the mCherry reporter gene of the Pb262 plasmid was inserted to create the final TLP-KO transfection plasmid.
tlp(-)mCherryNS: For negative selection the drinking water of mice infected with tlp(-)mCherry parasites was supplemented with 1 mg/ml 5-FC. Subsequently, clonal parasites which lost the resistance cassette by homologous recombination were obtained by limiting dilution.
tlp(-)mCherry|trap(-): The PlasmoGem vector PbGEM-107890 was transfected into selection marker free tlp(-)mCherry parasites. Clonal parasites were obtained by limiting dilution.
tlp(-)mCherry|trep(-): In order to remove trep in the tlp(-)mCherryNS parasites, the 5′ UTR of trep was amplified with primers P100 and P101, and cloned into Pb238 via SacII / NotI. The 3′ UTR was amplified with primers P102 and P103 and then cloned via HindIII/KpnI. The resulting vector and Pb262 were digested with HindIII and NotI to insert the CSPmCHerryYFCU cassette, yielding PbCSmCherryYFCU_TREP-KO. This vector was used to create trap(-)|trep(-)mCherry (see below). For two of the planned knockout lines that already expressed mCherry in the tlp locus [TLP(-)/TREP(-) and TLP(-)/TRAP(-)/TREP(-)], a TREP(-) transfection vector [PbYFCU_TREP(-)] was created that lacked the mCherry reporter gene. In order to remove the mCherry reporter gene and the CSP promoter, PbCSmChYFCU_TREP-KO was digested with NotI and EcoRV, blunted to get rid of any overhangs and finally re-ligated.
tlp(-)mCherry|trep(-)NS: The vector PbYFCU_TREP-KO was transfected into selection marker free tlp(-)mCherry parasites. Clonal parasites were obtained by limiting dilution. For negative selection the drinking water of mice infected with tlp(-)mCherry|trep(-) parasites was supplemented with 1 mg/ml 5-FC. Subsequently, clonal parasites which lost the resistance cassette by homologous recombination were obtained by limiting dilution.
trap(-)|trep(-)mCherry: The PbCSmChYFCU_TREP-KO transfection vector was transfected into trap(-)NS parasites. Clonal parasites were obtained by limiting dilution.
tlp(-)mCherry|trep(-)|trap(-): The PlasmoGem vector PbGEM-107890 was transfected into selection marker free tlp(-)mCherry|trep(-) parasites. Clonal parasites were obtained by limiting dilution.
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
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  Disrupted: Mutant parasite with a disrupted gene
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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
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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-107890
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 modificationGeneration of Plasmodium berghei parasite lines lacking two and three TRAP family adhesins: In order to generate parasite lines lacking more than one TRAP family gene, a strategy based on sequential positive and negative selection was employed. Vectors for deletion of trep and tlp were engineered to replace the endogenous gene with the gene encoding for the red fluorescent protein mCherry controlled by the circumsporozoite protein (csp) promoter, which is strongly expressed throughout the life of sporozoites. The vector for the deletion of trap was obtained from PlasmoGEM, which also contained a cassette enabling positive–negative selection. A clonal tlp knockout line was generated and the drug resistance cassette was recycled by negative selection. Subsequently, vectors targeting the trap and trep genes were transfected into the tlp(-) line. The trep KO vector was additionally transfected into trap(-) parasites that were generated previously yielding the line trep(-)/trap(-). A triple mutant line lacking all three TRAP family adhesins was obtained by negative selection of the trap(-)/tlp(-) line followed by deletion of trep.
Generation of plasmids and parasite lines
All generated plasmids were based on either the Pb238 or the Pb301 transfection vector containing the 5′ and 3′ UTRs of tlp or trep as well as the gene encoding the green fluorescent protein (GFP). Prior to transfection, all vectors were linearized with SacII and PmeI.

Pb262 transfection vector: This vector uses the hDHFR-yFCU selection marker cassette and the reporter gene mCherry under the control of the csp promoter. Using the Pb238 as parental vector, the selection marker cassette was replaced with the dhfr 3′UTR–ef1α 5′UTR–hDHFR–yFCU–dhfr 3′UTR from PlasmoGEM transfection vector, which was amplified with P600 and P601 and cloned with EcoRV HindIII into Pb238. Next, the 5′UTR of csp was amplified from P. berghei gDNA with primers P207 and P208 and cloned into the vector with EcoRI and NdeI. The open reading frame of the red fluorescent protein encoding gene mCherry was amplified with primers P238 and P232 and inserted into the vector with NdeI/BamHI. The resulting vector is termed Pb262.
trap(-) and trap(-)NS: For the generation of trap(-) parasites the plasmid PbGEM-107890 was requested from PlasmoGEM. It replaces most of the TRAP coding sequence with the positive–negative selection marker hDHFR-yFCU. For negative selection the drinking water of mice was supplemented with 1 mg/ml 5-FC. The parasite line was already generated and characterized in a previous study and used here as basis to generate the double mutant.
trap(-)/trep(-)/tlp(-)mCherry: The final TLP-KO transfection vector (PbCSmCherryYFCU_TLP-KO) was generated by amplifying the 5′ UTR of tlp from Plasmodium berghei gDNA with primers P159 and P160 and cloning into Pb238 via SacII/ NotI. The tlp 3′ UTR was amplified with primers P161 and P162 and then cloned via HindIII/KpnI. Next, using HindIII and NotI restriction sites of the ‘TLP plasmid’ and Pb262, the selection cassette and the mCherry reporter gene of the Pb262 plasmid was inserted to create the final TLP-KO transfection plasmid.
tlp(-)mCherryNS: For negative selection the drinking water of mice infected with tlp(-)mCherry parasites was supplemented with 1 mg/ml 5-FC. Subsequently, clonal parasites which lost the resistance cassette by homologous recombination were obtained by limiting dilution.
tlp(-)mCherry|trap(-): The PlasmoGem vector PbGEM-107890 was transfected into selection marker free tlp(-)mCherry parasites. Clonal parasites were obtained by limiting dilution.
tlp(-)mCherry|trep(-): In order to remove trep in the tlp(-)mCherryNS parasites, the 5′ UTR of trep was amplified with primers P100 and P101, and cloned into Pb238 via SacII / NotI. The 3′ UTR was amplified with primers P102 and P103 and then cloned via HindIII/KpnI. The resulting vector and Pb262 were digested with HindIII and NotI to insert the CSPmCHerryYFCU cassette, yielding PbCSmCherryYFCU_TREP-KO. This vector was used to create trap(-)|trep(-)mCherry (see below). For two of the planned knockout lines that already expressed mCherry in the tlp locus [TLP(-)/TREP(-) and TLP(-)/TRAP(-)/TREP(-)], a TREP(-) transfection vector [PbYFCU_TREP(-)] was created that lacked the mCherry reporter gene. In order to remove the mCherry reporter gene and the CSP promoter, PbCSmChYFCU_TREP-KO was digested with NotI and EcoRV, blunted to get rid of any overhangs and finally re-ligated.
tlp(-)mCherry|trep(-)NS: The vector PbYFCU_TREP-KO was transfected into selection marker free tlp(-)mCherry parasites. Clonal parasites were obtained by limiting dilution. For negative selection the drinking water of mice infected with tlp(-)mCherry|trep(-) parasites was supplemented with 1 mg/ml 5-FC. Subsequently, clonal parasites which lost the resistance cassette by homologous recombination were obtained by limiting dilution.
trap(-)|trep(-)mCherry: The PbCSmChYFCU_TREP-KO transfection vector was transfected into trap(-)NS parasites. Clonal parasites were obtained by limiting dilution.
tlp(-)mCherry|trep(-)|trap(-): The PlasmoGem vector PbGEM-107890 was transfected into selection marker free tlp(-)mCherry|trep(-) parasites. Clonal parasites were obtained by limiting dilution.
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren