RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5275
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0931200; Gene model (P.falciparum): PF3D7_1116800; Gene product: heat shock protein 101 | chaperone protein ClpB2 (HSP101)
Name tag: KDEL ER-retrieval sequence
Phenotype Asexual bloodstage;
Last modified: 10 August 2023, 16:49
  *RMgm-5275
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 37552754
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4541
Other information parent lineThe mutant RMgm-4541 contains a diCre expression cassette containing the two genes Cre59-FKBP and Cre60-FRB placed on either side of the bidirectional eef1a promoter. This expression cassette has been introduced in the neutral p230p locus of the GIMOp230p line that has the yfcu/hdhfr positive/negative selectable marker integrated into the neutral p230p locus. This line was used to introduce the diCre expression cassette into the p230p locus using negative selection with 5-FC (see also RMgm-687). The mutant does not contain a drug-selectable marker.
The mutant parasite was generated by
Name PI/ResearcherFierro MA, Beck JR
Name Group/DepartmentDepartment of Biomedical Sciences
Name InstituteIowa State University
CityAmes, IA
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-5275
Principal namePbHSP101-kER
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageTo expose any impact on export by ER retention of HSP101 masked during in vitro culture of P. falciparum blood stages, we tagged HSP101 and PTEX150 with kER in a transgenic P. berghei rodent malaria expressing DiCre and evaluated the impact of ER retrieval on parasite survival during mouse infection. Similar to P. falciparum knockER mutants, rapamycin treatment ex vivo produced efficient excision and ER retrieval of PbPTEX150 and PbHSP101. After overnight ex vivo culture to allow for tag switching, equal numbers of DMSO or rapamycin-treated parasites were IV injected into naïve mice. Strikingly, while DMSO controls became patent by day two post-injection, neither KDEL-fused PbPTEX150 or PbHSP101 parasites appeared over the course of 10 days before the experiment was terminated.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses HSP101 tagged C-terminal with a KDEL ER-retrieval sequence. In addition it contains  a diCre expression cassette containing the two genes Cre59-FKBP and Cre60-FRB placed on either side of the bidirectional eef1a 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.
These proteins are also expressed in early gametocytes, mosquito and liver stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in P. berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth.
HSP101 is only expressed in blood stages and not in liver stages.  PTEX150 is expressed in both blood and liver stages.

Phenotype
From the paper:
Lack of observable phenotypes in vitro can sometimes be masked by the rich resources available in culturing conditions of P. falciparum. In addition, exported effectors critical for survival in the vertebrate host may be dispensable in culture. Indeed, export-related defects that escape detection in vitro can be exposed in vivo where parasites have to contend with host defenses and more limited nutrient access. To expose any impact on export by ER retention of HSP101 masked during in vitro culture of P. falciparum blood stages, we tagged HSP101 and PTEX150 with kER in a transgenic P. berghei rodent malaria expressing DiCre and evaluated the impact of ER retrieval on parasite survival during mouse infection. Similar to P. falciparum knockER mutants, rapamycin treatment ex vivo produced efficient excision and ER retrieval of PbPTEX150 and PbHSP101. After overnight ex vivo culture to allow for tag switching, equal numbers of DMSO or rapamycin treated parasites were IV injected into naïve mice. Strikingly, while DMSO controls became patent by day two post-injection, neither KDEL-fused PbPTEX150 or PbHSP101 parasites appeared over the course of 10 days before the experiment was terminated.
That ER-retrieval of PbHSP101 compromises function sufficiently to prohibit survival in the mammalian host reinforces the critical role of HSP101 in the PV and indicates that KDEL-mediated depletion of HSP101 from the P. falciparum PV does not sufficiently reduce PTEX activity to impact parasite  fitness under less taxing in vitro culture conditions. Collectively, these results provide support for a distinct HSP101 activity in the ER upstream of its function in PVM translocation at the assembled 329 PTEX complex

Additional information
The rapamycin-inducible Cre recombinase (DiCre) established for use in P. falciparum uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves. In the DiCre system, Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin‐binding protein (either FKBP12 or FRB, the rapamycin‐binding domain of the FKBP12‐rapamycin‐associated protein mTOR).
Rapamycin‐induced heterodimerization of the two components restores recombinase activity (rapamycin‐mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn60‐343, called Cre60) each fused to a different rapamycin‐binding protein (FKBP12 and FRB respectively)results in the reconstitution of Cre recombinase activity). 
This site–specific recombinase recognises short, 34 bp sequences called loxP sites and catalyses the excision or inversion of the floxed (flanked by loxP) DNA segment.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0931200
Gene Model P. falciparum ortholog PF3D7_1116800
Gene productheat shock protein 101 | chaperone protein ClpB2
Gene product: Alternative nameHSP101
Details of the genetic modification
Name of the tagKDEL ER-retrieval sequence
Details of taggingC-terminal
Additional remarks: taggingProtein secretion is a fundamental biological process of all living cells. In eukaryotes this is accomplished by a dedicated secretory pathway that begins with import into the Endoplasmic Reticulum (ER) for initial folding and post-translational modification, followed by sorting in the Golgi and post-Golgi compartments. ER-resident proteins that escape the ER via bulk flow in COPII vesicles are returned to the ER in COPI vesicles by ERD2 surveillance, which binds a C-terminal retrieval sequence (“KDEL” and variants) in the cis Golgi and releases it in the ER in a pH-dependent manner. In this present work, we developed and characterized a novel DiCre-based knock-sideways tool called knockER that enables conditional fusion of KDEL to the C-terminus of secreted proteins, subjecting the target protein to ERD2 surveillance in the cis Golgi to cause its retrieval to the ER.
Commercial source of tag-antibodies
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor generation of PTEX150-kER and HSP101-kER lines in P. berghei, the plasmid pBAT was digested with MluI/XhoI and the primer P56 was used as an insert to remove the intervening AvrII, SnaBI and XhoI sites from the MCS. Next, the mRuby3 coding sequence fused to the PbEXP2 signal peptide was amplified with P57/58 and inserted between SwaI/BamHI, replacing the GFP. The mNG-kER tagging sequence was amplified from plasmid JRB508 using P59/60 and inserted between SacII/SphI, replacing the mCherry-3xMYC. Finally, 5’ flanks to tag P. berghei hsp101 or ptex150 with mNG-kER were amplified from P. berghei genomic DNA using P61/62 or P63/64, respectively, and 3’ flanks were amplified using P65/66 or P67/68, respectively. The corresponding 5’ and 3’ flank amplicons were assembled with an intervening AflII site in a second PCR reaction using P65/62 or P67/64, respectively, and inserted at SacII, resulting in plasmids pPbHSP101-kER and pPbPTEX150-kER. The plasmids were linearized at AflII and transfected into the marker-free HP DiCre line (RMgm-4541).
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