Summary

RMgm-4536
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0919100; Gene model (P.falciparum): PF3D7_1129100; Gene product: parasitophorous vacuolar protein 1 (PV1)
Transgene
Transgene not Plasmodium: GFP fused to the signal peptide of PBANKA_081890
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_0919100; Gene product: parasitophorous vacuolar protein 1 (PV1)
PhenotypeNo phenotype has been described
Last modified: 23 September 2018, 20:58
  *RMgm-4536
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30232409
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherMatz JM, Matuschewski K
Name Group/DepartmentDepartment of Molecular Parasitology
Name InstituteInstitute of Biology, Humboldt University
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4536
Principal namePBANKA_0919100 KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the parasitophourous vacuole protein PBANKA_0919100 (PV1) and expresses GFP under the constitutive hsp70 promoter. GFP is fused to the signal peptide of PBANKA_081890 and consequently GFP is localized to the parasitophourous vacuole  (see RMgm-1333).

Protein (function)
The protein was selected in a screen for putative PV (parasitophorous vacuole) proteins.
Proteins are targeted to the PV by means of default protein secretion, which is initiated by the recognition and cleavage of an amino-terminal signal peptide (SP). To identify novel PV candidates, we searched the Plasmodium genome database (PlasmoDB) for SP-containing proteins and sequentially removed proteins containing predicted transmembrane domains, export motifs, apicoplast targeting peptides, endoplasmic reticulum (ER) retention signals and GPI anchors, which could potentially redirect the protein to other locations. Twelfe proteins were selected. Five (PV1-5) proteins were shown to have a PV location in blood- and liver-stages.

Phenotype
No phenotype detected in blood and liver stages.

Additional information
Five (PV1-5; PBANKA_0919100; PBANKA_1441700; PBANKA_1350500; PBANKA_1349200; PBANKA_0826700) proteins were shown to have a PV location in blood- and liver-stages.

No phenotype was detected for blood and liver stages that lack expression of PV1, PV2, PV3 and PV4. Repeated attempts to delete the gene encoding PV5 were unsuccessful, indicating an essential function for blood stage growth/multiplication.

All PV features were present despite the absence of PV1, 2, 3, or 4, and general PV morphology appeared normal throughout asexual blood stage development. In the absence of PV1, 2, 3, or 4, parasites grew indistinguishable from WT during in vivo infections, indicating that these genes are entirely dispensable during asexual blood propagation. We then asked whether these proteins might promote the export of virulence factors across the PVM and, thus, quantified the export of an exemplary cargo protein. To that end, we crossed the knockout mutants in vivo with a transgenic parasite line expressing mCherry-tagged erythrocyte membrane-associated protein 1 (EMAP1) as demonstrated previously. Te protein was efficiently exported by all parasite lines as shown by live fluorescence imaging and quantitative microscopy, suggesting general protein export competence in the absence of PV1, 2, 3, or 4.
To exclude that other exported proteins may yet be retained in the parasite upon deletion of PV1, 2, 3, or 4 we used iRBC sequestration as a proxy for the parasite’s general protein export status. Since deficiencies in protein translocation across the PVM are expected to result in reduced iRBC sequestration to peripheral organs, we quantified the circulating schizonts during synchronized in vivo infections. As expected, only very few WT schizonts were detected in the circulation as most of them had sequestered to the peripheral organs, e.g. lungs, brain and adipose tissue.
Mutants devoid of PV1, 2, 3, or 4 all formed high numbers of oocysts displaying a similar morphology as the WT and produced normal numbers of sporozoites.
In the absence of PV1, 2, 3, or 4, liver stages matured normally and gave rise to detaching merosomes. We then allowed infected mosquitoes to feed on C57BL/6 mice and monitored peripheral blood daily by microscopic examination of Giemsa-stained thin blood films. All animals bitten by WT or knockout-infected mosquitoes became blood-stage positive on day 3 afer bite back.

All five proteins were found to be associated with the parasite periphery and with PV-derived tubules and vesicular structures throughout the entire asexual cycle. Strikingly, a substantial fraction of PV4 was found in the erythrocyte cytoplasm during ring and early trophozoite stages. This exported protein fraction progressively disappeared during parasite growth and was not detected in mature trophozoites or schizonts. During the schizont stage, all five proteins localised around the formed merozoites and co-localised with the hemozoin crystals of the digestive vacuoles. We next investigated whether the PV proteins are indeed associated with the vacuolar lumen rather than the parasite surface. To that end, we imaged ruptured schizonts, which have already disintegrated the iRBC membrane and the PV, thus leading to the loss of all soluble PV contents. Upon merozoite egress all five proteins localised almost exclusively to the digestive vacuoles. Upon inspection of individual emerging merozoites, PV2-5 were hardly detected, whereas PV1 was found to be associated with a distinct intraparasitic structure, possibly reflecting localisation to parasite dense granules, as previously demonstrated for other PV proteins. In no instance were the PV proteins detected on the surface of emerging merozoites further supporting the notion that they are indeed mostly luminal.
Inoculation of in vitro cultivated hepatoma cells with the transgenic sporozoites revealed that all five proteins are highly expressed in the liver stage PV. PV5 was not expressed during early pre-erythrocytic growth, but was only present in the PV of more mature parasite stages.

Due to the extracellular nature and the absence of a PV during the oocyst stage, we expected the PV proteins to be switched of throughout mosquito infection. While this was true for PV2–5, PV1 was highly expressed in the periphery of growing oocysts

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0919100
Gene Model P. falciparum ortholog PF3D7_1129100
Gene productparasitophorous vacuolar protein 1
Gene product: Alternative namePV1
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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor the generation of gene deletion constructs, 3′ fragments were amplifed from genomic DNA (ranging in size from 837 to 1,020 bp) and cloned into the pBAT-G624 or the pGFP-PV plasmid using the XhoI and KpnI restriction sites. Subsequently, 5′ fragments (ranging in size from 808 to 1,003bp) were amplifed and cloned into the intermediate vectors using SacII in combination with EcoRI or PvuII. Prior to transfection into wild-type ANKA parasites, knockout vectors were digested with XmnI and SalI
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 nameGFP fused to the signal peptide of PBANKA_081890
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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 Not available
Gene productNot available
Gene product: Alternative name
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_0919100
Gene productparasitophorous vacuolar protein 1
Gene product: Alternative namePV1
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