RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5126
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: P.berghei ddi1 gene replaced with P. falciparum ddi1 gene, C-terminally tagged with E. coli dhfr
Phenotype Asexual bloodstage;
Last modified: 13 December 2021, 14:05
  *RMgm-5126
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-5126
Principal namePbKD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageThe 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 knockdown 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 at 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
In the mutant the P.berghei ddi1 gene (open reading frame) has been replaced with the P. falciparum ddi1 gene, C-terminally tagged with E. coli dhfr (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.  

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

Protein (function)
Several non-proteasomal ubiquitin-binding proteins contribute to the proteasome function. One such class of proteins are the shuttle proteins, which include Rad23, Dsk2 and  DDI1. These proteins contain a ubiquitin-like (UBL) domain and a ubiquitin-associated  (UBA) domain, which mediate their interactions with the proteasome and ubiquitin chains of the  ubiquitinated protein, respectively. DDI1 also contains an aspartyl protease domain similar to the  retroviral aspartic protease (RVP) of HIV protease. S. cerevisiae DDD1 (ScDDI1) is the most studied among all DDI1 proteins. An P. falciparum ortholog was termed as the  DNA damage-inducible  protein 1 (PfDDI1), as it shares 33% identity with ScDDI1, contains putative UBL and RVP domains, and has a putative aspartyl protease motif (FVDSGA) in the RVP domain.

Phenotype
The unsuccessful attempts to disrupt this gene (see RMgm-5125) indicate an essential function during asexual blood stage growth/multiplication.

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 knockdown 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 at about 5% parasitemia also resulted in complete clearance of parasites.

Additional information

Evaluation of PbKD parasites: The PbKD parasites were assessed for the effect of knock-down on PfDDIMyc/cDDHA protein level, erythrocytic growth and virulence. For the effect on PfDDIMyc/cDDHA protein level, a 2-3 months old naïve BALB/c mouse was infected intraperitoneally with a fresh stock of PbKD parasites, maintained under 70 µg/ml pyrimethamine + 40 µg/ml trimethoprim in drinking water, and the blood collected at 8-10% parasitemia. The blood was washed with FBS-RPMI medium, resuspended in FBS-RPMI medium at 2% haematocrit without or with 50 µM trimethoprim, gassed, and incubated for 12 hours at 37o C. Parasites were purified by saponin lysis and processed for western blotting using mouse anti-HA and mouse anti-β actin (as a loading control), followed by HRP-conjugated goat anti-mouse IgG as described in the western blotting section. 

For the effect of growth and virulence, 2-3 months old female BALB/c mice were infected intraperitoneally (2x105  parasites/mouse) with wild type P. berghei ANKA (5 mice) or PbKD parasites (10 mice). The PbKD-infected mice were divided into 2 groups of 5 mice each; one group was given 70 µg/ml pyrimethamine in drinking water (-TMP) and another group was given 70 µg/ml pyrimethamine + 40 µg/ml trimethoprim in drinking water (+TMP). Blood was collected from each mouse one day before the infection (pre-immune sera) and during later time points (days 14 and 28 of post-infection). Each mouse was monitored for infection by making Giemsa stained blood smears to determine parasitemia.

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 mutationP.berghei ddi1 gene replaced with P. falciparum ddi1 gene, C-terminally tagged with E. coli dhfr
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.

For the knock-out constructs, 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 pGT-GFPbsc with KpnI-XhoI and subcloned into the similarly digested HB-PbDDI- (FL1+FL2) to obtain HB-pbDDIKO plasmid.

For the construction of knock-down plasmid, the PfDDIMyc coding sequence was amplified from the P. falciparum genomic DNA using DDiexp[1]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. All the flanks and coding regions were sequenced to ensure that they were free of undesired mutations, and the presence of different regions was confirmed by digestion with region-specific restriction enzymes. 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