RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5218
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1409100; Gene model (P.falciparum): PF3D7_1310600; Gene product: ras-related protein Rab-5B (RAB5b; secretory complex protein 61 alpha; Sec61-alpha; Rab GTPase 5b)
Name tag: HA
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
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 Liver stage;
Last modified: 12 July 2022, 13:23
  *RMgm-5218
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) : 35649358
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 507cl1 (RMgm-7)
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherLahree A, Mota MM
Name Group/DepartmentInstituto de Medicina Molecular- João Lobo Antunes (iMM-JLA), Faculdade de Medicina
Name InstituteUniversidade de Lisboa
CityLisboa
CountryPortugal
Name of the mutant parasite
RMgm numberRMgm-5218
Principal namePbRab5b_HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageThe cellular localization of P. berghei Rab5b during the liver stage was investigated using parasite lines expressing this protein with a C-terminal monomeric Azami-Green (PbRab5b_mAG; 1460) tag or 2x C-terminal hemagglutinin (HA)-tag (PbRab5b_HA). The majority of the PbRab5b signal was in the parasite cytosol with a small peripheral fraction detected in close proximity to the PVM and in the tubovesicular network (TVN). Mature EEFs presented a higher peripheral PbRab5b signal, and the overall signal also increased with EEF development.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a 2xHA C-terminal tagged form of Rab5b and expresses GFP under the constititutive eef1a promoter

Protein (function)
P. falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport.
This family contains three Rab5 isoforms (Rab5a,b and c)

Phenotype
The cellular localization of P. berghei Rab5b  during the liver stage was investigated using parasite lines expressing this protein with a C-terminal monomeric Azami-Green (PbRab5b_mAG; 1460) tag or 2x C-terminal hemagglutinin (HA)-tag (PbRab5b_HA). The majority of the PbRab5b signal was in the parasite cytosol with a small peripheral fraction detected in close proximity to the PVM and in the tubovesicular network (TVN). Mature EEFs presented a higher peripheral PbRab5b signal, and the overall signal also increased with EEF development. 

Additional information

Evidence is presented for the following: 
Hepatocyte expressed endosomal; protein APPL1 deposition at the P. berghei PVM occurs independently of host cell (hepatocyte) Rab5 but depends on a parasite Rab5 isoform. Ectopic expression of a host Rab5 mutant (Q79L, lacking GTPase activity) results in the retention of APPL1 on host endosomes, leading to a stripping of its signal at the PVM together with a reduction in parasite size, both of which were rescued in parasites expressing the Plasmodium Rab5b Q91L isoform (also lacking its GTPase function; RMgm-5219).

Live imaging of PbRab5b_mAG schizonts (45–48 hpi) in mRFP-APPL1-expressing HepG2 cells led to the identification of peripheral PbRab5b-positive structures engaging with APPL1-positive vesicles in the host cell. The same could be confirmed in PbRab5b_HA EEFs (48 hpi) via immunofluorescence (IF), where the peripheral PbRab5b signal was highly proximal to APPL1 signal along the PVM/TVN at that resolution. Upon quantification, we observed that only a small fraction of PbRab5b colocalized with APPL1 at the parasite periphery in late schizonts. Mature schizonts had clearer peripheral PbRab5b- APPL1 signal proximity. While we could not conclude that PbRab5b always faces the host cytosol, a partial-permeabilization-based IF in HepG2 cells infected with PbRab5b_HA parasites was used to identify a PVM-proximal PbRab5b pool closely accessible from the host cytosol. Acetone-methanol- permeabilized (full permeabilization) EEFs displayed intracellular PbRab5b and cytosolic GFP staining, While 0.01% saponin (partial permeabilization)-treated EEFs lacked these cytosolic signals. However, in partially permeabilized cells, we noted a signal of PbRab5b around the PVM/TVN, which was discontinuous but present even in regions with low PbUIS4 signal. Overall, there appeared to be a fraction of PbRab5b in close proximity to the PVM, providing for its peripheral pool between the PVM and PPM, which was close to the APPL1 signal there. 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1409100
Gene Model P. falciparum ortholog PF3D7_1310600
Gene productras-related protein Rab-5B
Gene product: Alternative nameRAB5b; secretory complex protein 61 alpha; Sec61-alpha; Rab GTPase 5b
Details of the genetic modification
Name of the tagHA
Details of taggingC-terminal
Additional remarks: tagging
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor the generation of HA-tagged Rab5b expressing P.berghei parasite line, a double homologous recombination strategy was employed. The P.berghei rab5b (Pbrab5b) gene was amplified with a high fidelity polymerase (Phusion polymerase, Thermo Scientific) using the primers 1 and 2 to insert the 2x-HA tag on the Pbrab5b amplicon. 3′ÚTR of Pbrab5b gene was amplified using the primers 3 and 4. Blunt-ended PCR products were purified with QIAquick PCR purification kit (Qiagen, Hilden, Germany) and used for insertion in pJet-1.2 cloning vector (CloneJET PCR cloning kit) according to manufacturer's instructions. The ligation mixture was used for bacterial transformation of E. coli DH5α chemically competent cells (produced in-house according to the manufacturer's protocol, NZYCompetent Cells Preparation Buffer, NZYtech). Transformed cells were spread on LB-Ampicillin (100 μg/mL, Sigma) agar and incubated at 37°C for 16–24 h, for selection of transformants.
Pbrab5b-HA fragment was released from pJet1.2 backbone through digestion with HindIII (New England biolabs, Massachusetts, USA) and NotI (New England Biolabs), as per manufacturer's instructions. The Plasmodium expression vector (PEV) (Setua et al., 2020) was also digested with HindIII and NotI. The digests were resolved on 1% agarose gel and the band corresponding to Pbrab5b-HA (1.3 kb) and the linearized PEV (4.9 kb) were excised and purified using the QIAquick Gel Extraction Kit (Qiagen). The sticky-ended DNA fragments were ligated using T4-ligase (New England Biolabs) in a molar ration of 10:1 (insert to vector) as per manufacturer's instructions. The ligation mixture was used to perform bacterial transformation. Transformants were screened by colony PCR (NZYTaq II master mix, Nzytech) and sub-cultured in LB-Ampicillin (100 μg/mL) broth at 37°C, 220 rpm for 16–24 h. Plasmid DNA from transformants were extracted and purified using the NZYprep kit (Nzytech). The Pbrab5b-HA-PEV construct and Pbrab5b_3ÚTR-pJet1.2 construct were digested with HindIII and KpnI followed by resolution on 1% agarose and purification of PbRab5b_3ÚTR band (1.02 kb) and the linearized PbRab5b-HA-PEV vector (6.2 kb). These digested fragments were ligated and transformed as described above. The final cloning vector (7.2 kb) was linearized with HindIII prior to the transfection of P. berghei merozoites.
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 (gfp-mu3)
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 gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP 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