RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5098
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1033200; Gene model (P.falciparum): PF3D7_1409300; Gene product: DNA damage-inducible protein 1, putative (DDI1)
Details mutation: a fusion of P. falciparum DDIMyc with HA-tagged mutant E. coli DHFR (cDDHA)
Phenotype Asexual bloodstage;
Last modified: 4 November 2021, 13:33
  *RMgm-5098
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
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 Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherTanneru N, Sijwali PS
Name Group/DepartmentCSIR-Centre for Cellular and Molecular Biology
Name InstituteCSIR-Centre for Cellular and Molecular Biology
CityHyderabad
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-5098
Principal namePbKD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagePbKD parasites grew similar to wild type parasites in the presence of trimethoprim, but showed drastically reduced growth in the absence of trimethoprim and eventually disappeared. In the presence of trimethoprim, PbKD-infected mice had to be euthanized, whereas in the absence of trimethoprim, the parasitemia barely reached to 0.5% and the infection was self-limiting. Withdrawal of trimethoprim from PbKD-infected mice about 5% parasitemia also resulted in complete clearance of parasites.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mutated form of DDI1: a fusion of P. falciparum DDIMyc with HA-tagged mutant E. coli DHFR (cDDHA). cDD binds trimethoprim and is stable, but undergoes proteasomal degradation in the absence of trimethoprim

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.10.29.466443.


Protein (function)
PF3D7_1409300 DDI1 was first discovered as one of the over expressed proteins upon treatment of Saccharomyces cerevisiae with DNA damaging agents, hence, it was named as the DNA damage inducible 1 protein (DDI1). Deletion analysis demonstrated that both UBL and RVP domains of S. cerevisiae DDI1 (ScDDI1) are necessary for inhibition of protein secretion. DDI1 proteins are conserved in eukaryotes and involved in a variety of cellular processes, including proteasomal degradation of specific proteins and DNA-protein crosslink repair. All DDI1 proteins contain ubiquitin-like (UBL) and retroviral aspartyl protease (RVP) domains, and some also contain ubiquitin-associated (UBA) domain, which mediate distinct activities of these proteins. The DDI1 proteins of Plasmodium and other Apicomplexan parasites vary in domain architecture, share UBL and VP domains, and the majority of proteins contain the UBA domain.

Phenotype
DDI1 is essential for parasite development. Expression of DDI1 in all the major parasite stages suggests its importance for parasite development. Hence, we attempted to knock-out the PbDDI1 gene for investigation of its functions during parasite development. However, multiple knock-out attempts were unsuccessful.

We next employed a conditional knock-down approach in P. berghei by replacing the wild type PbDDI1 coding region with PfDDIMyc/cDDHA coding sequence, which would express fusion of PfDDIMyc with HA-tagged mutant E. coli DHFR (cDDHA). cDD binds trimethoprim and is stable, but undergoes proteasomal degradation in the absence of trimethoprim, thereby, causing a knock-down effect. The replacement of wild type PbDDI1 with PfDDIMyc/cDDHA and expression of DDIMyc/cDDHA fusion protein were successful, which confirmed that PbDDI1 and PfDDI1 are functionally conserved.

The knock-down parasites (PbKD) showed reduction in DDIMyc/cDDHA protein level in the absence of trimethoprim compared to that in the presence of trimethoprim, indicating the knock-down effect. PbKD parasites grew similar to wild type parasites in the presence of trimethoprim, but showed drastically reduced growth in the absence of trimethoprim and eventually disappeared. In the presence of trimethoprim, PbKD-infected mice had to be euthanized, whereas in the absence of trimethoprim, the parasitemia barely reached to 0.5% and the infection was self-limiting. Withdrawal of trimethoprim from PbKD-infected mice about 5% parasitemia also resulted in complete clearance of parasites.

Additional information
Evidence is shown that:
Plasmodium DDI1 is expressed in all major parasite stages
- DDI knock-down enhanced drug sensitivity of parasites
- DDI1 associates with chromatin and DNA-protein crosslinks
- DDI1 interacts with pre-mRNA-processing factor 19

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1033200
Gene Model P. falciparum ortholog PF3D7_1409300
Gene productDNA damage-inducible protein 1, putative
Gene product: Alternative nameDDI1
Details of the genetic modification
Short description of the mutationa fusion of P. falciparum DDIMyc with HA-tagged mutant E. coli DHFR (cDDHA)
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 parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe PbDDI1 gene was targeted for knock-out and knock-down using double cross-over homologous recombination approach. The 5'-UTR (flank 1) and 3’-UTR (flank 2) of PbDDI1 were amplified from P. berghei genomic DNA using PbDdi1-
Fl1F/ PbDdi1-Fl1R and PbDdi1-Fl2F/PbDdi1-Fl2R primer sets, respectively. The flank 1 and flank 2 were cloned into the HB-DJ1KO plasmid at NotI-KpnI and AvrII-KasI sites, respectively, to obtain HB-PbDDI-(FL1+FL2) plasmid. The GFP coding sequence was excised from pGTGFPbsc with KpnI-XhoI and subcloned into the similarly digested HB-PbDDI-(FL1+FL2) to obtain HB-pbDDIKO plasmid.
For construction of knock-down plasmid, the PfDDIMyc coding sequence was amplified from the P. falciparum genomic DNA using DDiexp-F/DDimyc Rep-R primers, digested with KpnI-XhoI and subcloned into the similarly digested HB-PbDDIKO plasmid in place of GFP to obtain HB-PfDDIKI plasmid. The E. coli mutant DHFR coding sequence with HA-tag (cDDHA) was amplified from the pPM2GDBvm plasmid (a kind gift from Dr. Praveen Balabaskaran Nina) using cDD-F/cDD-R primers, and cloned into the pGT-GFPbsc plasmid at KpnI/XhoI sites to obtain pGT-cDDHA plasmid. The PfDDIMyc coding sequence was amplified from HB-PfDDIKI plasmid using the PfDdi-reF/PfDdi-cDDR primers and cloned into the pGT-cDDHA plasmid at BglII-BamHI site to obtain the pGT-PfDDI-cDDHA plasmid. The pGT-PfDDIMyc/cDDHA plasmid was digested with BglII-XhoI to release the PfDDIMyc/cDDHA insert, which was cloned into the similarly digested HB-DDKI plasmid to obtain HB-pbDDIKD vector.
The HB-pbDDIKO and HB-pbDDIKD plasmids were purified using the NucleoBond® Xtra Midi plasmid DNA purification kit (MACHEREY-NAGEL), linearized with NotI-KasI, gel purified to obtain pbDDIKO and pbDDIKD transfection constructs, and used for transfection of P. berghei.
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