Summary

RMgm-950
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0941300; Gene model (P.falciparum): PF3D7_1105600; Gene product: translocon component PTEX88
Name tag: mCherry-3xMyc
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: PBANKA_0941300; Gene product: translocon component PTEX88
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 11 October 2013, 18:34
  *RMgm-950
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24076174
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherTWA. Kooij: JM Matz
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-950
Principal nameptex88::tag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagePTEX88::mCherry expression in asexual blood stages
Gametocyte/GametePTEX88::mCherry expression in gametocytes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of of PTEX88 and expresses GFP under the control of the HSP70 promoter

Protein (function)
Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex.
In this study HSP101, PTEX150, EXP2 could not be genetically deleted in P. berghei. In contrast, the putative thioredoxin-like protein TRX2 and PTEX88 could be deleted. See RMgm-942 for a mutant lacking expression of PTEX88. This mutant shows a reduced growth rate of asexual blood stages.

Phenotype
A mutant lacking expression of PTEX88 (RMgm-942) shows a reduced growth rate of asexual blood stages.
Analysis of the mutant expressing mCherry-tagged PTEX88 shows expression in all blood stgaes (see 'Additional information')

Additional information
PTEX88::mCherry bloodstages grew indistinguishable from wild type parasites, thus providing evidence for a functional complementation and simultaneously ruling out aberrant localization of the tagged protein.

Live imaging revealed that PTEX88::tag predominantly localizes to regions in the parasites cytoplasm of both asexual and sexual blood stages. A singular punctate structure was observed, which localizes to the periphery of the parasite in mature schizonts and free merozoites. In ring and trophozoite stages and gametocytes, the intraparasitic PTEX88::tag pattern is more diffuse. In the majority of the maturing trophozoites and young schizonts, it was observed that PTEX88::tag also localizes to thin, dynamic extensions, which reach from the parasite surface into the erythrocyte cytoplasm. These extensions only appeared when parasites had matured and were never evident in ring stages or sexual stages. Typically, a single extension emerges from one end of the parasite and extends far into the host cell, often following the shape of the red blood cell membrane towards the other end of the parasite. Sometimes two extensions emerged from opposite ends of a parasite. Infrequently, PTEX88::tag extensions emerged from opposite ends of a parasite. Infrequently, PTEX88::tag localized to multiple smaller extensions. The structures appeared dynamic in all cases, displaying waving- and folding-like motions.

See mutant RMgm-922 for the localization of PTEX88 in a mutant expressing HA-tagged version of PTEX88.

The standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgm-757 for details of this vector). The 3' fragment of PTEX88 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in pPTEX88-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.

Other mutants
See RMgm-917 for unsuccessful attempts to disrupt P. berghei PTEX88
See RMgm-942 for a mutant lacking expression of PTEX88
See mutant RMgm-922 for the localization of PTEX88 in a mutant expressing HA-tagged version of PTEX88.


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0941300
Gene Model P. falciparum ortholog PF3D7_1105600
Gene producttranslocon component PTEX88
Gene product: Alternative name
Details of the genetic modification
Name of the tagmCherry-3xMyc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgmDB-757 for details of this vector). The 3' fragment of PTEX88 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in pPTEX88-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.
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
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgmDB-757 for details of this vector). The 3' fragment of PTEX88 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in pPTEX88-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.
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_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 / InsertionInsertion locus
Gene Model of Parasite PBANKA_0941300
Gene producttranslocon component PTEX88
Gene product: Alternative name
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