RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5184
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0309500; Gene model (P.falciparum): PF3D7_0212600; Gene product: secreted protein with altered thrombospondin repeat domain (SPATR)
MutatedGene model (rodent): PBANKA_0309500; Gene model (P.falciparum): PF3D7_0212600; Gene product: secreted protein with altered thrombospondin repeat domain (SPATR)
Details mutation: 'promoter-swap' mutant; spatr gene with the promoter of PBANKA_0603900 (hado) in neutral SIL6 locus
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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: 28 March 2022, 16:40
  *RMgm-5184
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
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 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase 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/ResearcherCosta DM, Tavares J
Name Group/DepartmentIBMC – Instituto de Biologia Molecular e Celular
Name InstituteUniversidade do Porto
CityPorto
CountryPortugal
Name of the mutant parasite
RMgm numberRMgm-5184
Principal nameSPATR KD (knock-down)
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
OocystNeither the number nor the size of the oocysts revealed any clear developmental impairments. However, a significant increase in the number of sporozoites per oocyst was observed in the SPATR KD line on day 15, indicating a defect in egress of sporozoites from the oocyst.
SporozoiteSPATR KD-infected mosquitoes showed strongly reduced sporozoite numbers. The reduced numbers of sporozoites in the salivary glands did not result in the accumulation of sporozoites in the hemolymph. Few SPATR KD sporozoites were detected within the salivary glands and these did not show obvious structural modifications by transmission electron microscopy analysis.
Liver stageTo evaluate sporozoite infectivity, sporozoites were collected from the hemolymph and injected intravenously in mice. While WT parasites successfully accomplished the liver stage and subsequent blood infections, parasite liver loads in SPATR KD-infected mice were strongly reduced and were rarely able to establish a blood infection. A milder defective phenotype was observed when we infected mice with the few SPATR KD sporozoites that we were able to collect from salivary glands, with reduced parasite liver loads and prolonged prepatent periods of approximately 1 day compared to WT.
Evidence is presented that SPATR KD sporozoites exhibit defects in hepatocyte invasion, migration and homing to the liver.
Additional remarks phenotype

Mutant/mutation
In this 'promoter-swap' mutant, first an additional copy of the spatr gene (under control of the promoter of PBANKA_0603900 (hado; haloacid dehydrogenase domain ookinete protein)) has been introduced into the silent intergenic locus in chromosome 6 (SIL6) and subsequently the endogenous spatr gene has been deleted. The mutant expresses GFP-Luc under control of the eef1a promoter.

Published in bioRxiv preprint doi: https://doi.org/10.1101/2022.03.06.483110

Protein (function)
The secreted protein with an altered thrombospondin repeat (SPATR) contains two adhesive domains: an atypical thrombospondin type-1 repeat (TSR) and a Type II epidermal growth factor (EGF)-like domain. It is conserved across Plasmodium species and has several orthologues among the Apicomplexa phylum. Although its gene is transcribed in all invasive stages (sporozoites, merozoites and ookinetes), SPATR levels are upregulated in mature sporozoites of both human and rodent infecting Plasmodium species. Moreover, immunolocalization studies detected the protein at the surface of sporozoites of several Plasmodium species, despite the lack of predicted transmembrane domains or a glycosylphosphatidylinositol anchor. Antibodies against the SPATR of P. falciparum also blocked hepatocyte invasion by sporozoites. 

Phenotype
Normal asexual blood stage development, gametocyte, ookinete and oocyst production.
Neither the number nor the size of the oocysts revealed any clear developmental impairments. However, a significant increase in the number of sporozoites per oocyst was observed in the SPATR KD line on day 15, indicating a defect in egress of sporozoites from the oocyst.
SPATR KD-infected mosquitoes showed strongly reduced sporozoite numbers. The reduced numbers of sporozoites in the salivary glands did not result in the accumulation of sporozoites in the hemolymph. Few SPATR KD sporozoites were detected within the salivary glands and these did not show obvious structural modifications by transmission electron microscopy analysis. To evaluate sporozoite infectivity, sporozoites were collected from the hemolymph and injected intravenously in mice. While WT parasites successfully accomplished the liver stage and subsequent blood infections, parasite liver loads in SPATR KD-infected mice were strongly reduced and were rarely able to establish a blood infection. A milder defective phenotype was observed when we infected mice with the few SPATR KD sporozoites that we were able to collect from salivary glands, with reduced parasite liver loads and prolonged prepatent periods of approximately 1 day compared to WT.
Evidence is presented that SPATR KD sporozoites exhibit defects in hepatocyte invasion, migration and homing to the liver.

Additional information
SPATR is expressed in blood stages, ookinetes and in sporozoites and has an essential function in blood stages. To achieve downregulation of expression in sporozoites while maintaining blood stage expression, a promoter swap approach was used to generate a mutant where the Plasmodium berghei spatr gene was placed under transcriptional control of the hado gene promoter.
Available RNA-seq datasets showed higher abundance of spatr transcripts in blood schizonts, ookinetes and salivary gland sporozoites. Based on these criteria, we searched genome-wide RNA-seq data and selected the genes that encode the haloacid  dehydrogenase domain ookinete protein (HADO).

Transcripts were found to be significantly less abundant in SPATR KD oocysts and sporozoites from days 11–12 post-infection than in the control lines. Protein levels were drastically reduced in SPATR KD hemolymph sporozoites.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0309500
Gene Model P. falciparum ortholog PF3D7_0212600
Gene productsecreted protein with altered thrombospondin repeat domain
Gene product: Alternative nameSPATR
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-270402
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 modificationTo generate the SPATR KD line, we resorted to a two-step strategy: 1) introduction of a second copy of the spatr gene under transcriptional control of the intergenic regions upstream of the genes encoding the haloacid dehydrogenase domain ookinete protein (hado; PBANKA_0603900) into a silent intergenic locus in chromosome 6; 2) deletion of the endogenous copy of spatr, performed using the PbGEM-270402 vector (PlasmoGEM, Sanger).
The first step required the insertion of the 5’ (490 bp) and 3’ (474 bp) homology regions amplified by PCR with primer pairs P15 + P16 and P17 + P2 into the KpnI/ClaI and SpeI/SacII cloning sites, respectively, of the pL0001 vector. The genomic region comprised of the open reading frame (1,083 bp) and 396 bp of the downstream sequence were cloned into the construct using the primer pair P18 + P19 and the BamHI and SpeI restriction enzymes. The 5’ intergenic regions of hado (2,414 bp) were amplified with primer pairs P20 + P21, and inserted into the plasmid using the EcoRV/BamHI restriction sites. Transfections were performed as before, and selection of transfectants was achieved using pyrimethamine as previously described (step 1) or 10–16 mg/kg/day of WR99210 (Jacobus Pharmaceutical Company) injected subcutaneously for 3 consecutive days (step 2). Parasites were then cloned 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

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0309500
Gene Model P. falciparum ortholog PF3D7_0212600
Gene productsecreted protein with altered thrombospondin repeat domain
Gene product: Alternative nameSPATR
Details of the genetic modification
Short description of the mutation'promoter-swap' mutant; spatr gene with the promoter of PBANKA_0603900 (hado) in neutral SIL6 locus
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate the SPATR KD line, we resorted to a two-step strategy: 1) introduction of a second copy of the spatr gene under transcriptional control of the intergenic regions upstream of the genes encoding the haloacid dehydrogenase domain ookinete protein (hado; PBANKA_0603900) into a silent intergenic locus in chromosome 6; 2) deletion of the endogenous copy of spatr, performed using the PbGEM-270402 vector (PlasmoGEM, Sanger).
The first step required the insertion of the 5’ (490 bp) and 3’ (474 bp) homology regions amplified by PCR with primer pairs P15 + P16 and P17 + P2 into the KpnI/ClaI and SpeI/SacII cloning sites, respectively, of the pL0001 vector. The genomic region comprised of the open reading frame (1,083 bp) and 396 bp of the downstream sequence were cloned into the construct using the primer pair P18 + P19 and the BamHI and SpeI restriction enzymes. The 5’ intergenic regions of hado (2,414 bp) were amplified with primer pairs P20 + P21, and inserted into the plasmid using the EcoRV/BamHI restriction sites. Transfections were performed as before, and selection of transfectants was achieved using pyrimethamine as previously described (step 1) or 10–16 mg/kg/day of WR99210 (Jacobus Pharmaceutical Company) injected subcutaneously for 3 consecutive days (step 2). Parasites were then cloned 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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