RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4134
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PbANKA_0830200; Gene model (P.falciparum): PF3D7_0929400; Gene product: high molecular weight rhoptry protein 2 (RhopH2)
Details mutation: The rhoph2 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Details conditional mutagenesis: Downregulation of RhopH2 by addition of the tetracycline derivative anhydrotetracycline(ATc)
Phenotype Asexual bloodstage;
Last modified: 16 April 2017, 12:39
  *RMgm-4134
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28252383
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/ResearcherCounihan NA; de Koning-Ward TF
Name Group/DepartmentSchool of Medicine
Name InstituteDeakin University
CityWaurn Ponds
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4134
Principal namePbRhopH2 iKD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageIn this study blood stage parasites were analysed with knock-down of RhopH2. Knockdown of RhopH2 in PbRhopH2 iKD blood stages was performed by addition of the tetracycline derivative anhydrotetracycline (ATc) to drinking water of infected mice and by addition of ATc to (short-term) cultures of ringforms. Parasites of these cultures were analysed directly or after infection of mice and during the course of infection. Analyses of the mutant showed the following:
- Parasites grew poorly in mice that had been pre-exposed to ATc 24 h prior to infection
- Parasites in which RhopH2 had been depleted, exhibited delayed progression to trophozoite stage and the schizont stages displayed aberrant morphology, often appearing vacuolated and containing fewer merozoites
- Parasites depleted of RhopH2 could invade erythrocytes but exhibited a delay in the transition from the early to more mature trophozoite forms
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
For tetracyclin (ATc)-dependent gene regulation the rhoph2 locus was modified to contain a tet-inducible promoter and TRAD4 activating domain (the rhoph2 locus was modified to insert an anhydrotetracycline (ATc)-regulated transactivator element (TRAD) downstream of the endogenous rhoph2 promoter and a minimal promoter with TRAD binding sites upstream of the rhoph2 coding sequence)

Protein (function)
RhopH2 is one of ~15 known proteins that localize to the rhoptry bulb in Plasmodium merozoites. It is found in a high molecular weight complex with RhopH1 and RhopH3 that is discharged from merozoites, associating with the erythrocyte surface upon merozoite contact. The localization of RhopH proteins in the newly-infected erythrocyte is less clear as multiple localizations, including the PV membrane (PVM), Maurer’s clefts and the cytosolic face of the erythrocyte membrane have been described for its constituents using different experimental approaches
RhopH2 and RhopH3 are each encoded by a single gene. In contrast, RhopH1 in P. falciparum is encoded by a multi-gene family comprising five variant genes termed clag 2, 3.1, 3.2, 8 and 9

Phenotype

In this study blood stage parasites were analysed with knock-down of RhopH2. Knockdown of RhopH2 in PbRhopH2 iKD blood stages was performed by addition of the tetracycline derivative anhydrotetracycline (ATc) to drinking water of infected mice and by addition of ATc to (short-term) cultures of ringforms. Parasites of these cultures were analysed directly or after infection of mice and during the course of infection. Analyses of the mutant showed the following:
- Parasites grew poorly in mice that had been pre-exposed to ATc 24 h prior to infection
- Parasites in which RhopH2 had been depleted, exhibited delayed progression to trophozoite stage and the schizont stages displayed aberrant morphology, often appearing vacuolated and containing fewer merozoites
- Parasites depleted of RhopH2 could invade erythrocytes but exhibited a delay in the transition from the early to more mature trophozoite forms

See also the mutants RMgm-2439 and RMgm-1915 generated in the 'Plasmogem screen' where evidence is presented for reduced growth of mutants lacking RhopH2 and the essential nature of RhopH3 for blood stage growth, respectively.

Additional information
In the same paper RhopH2 is analysed in P. falciparum. The data presented provide evidence for a role of RhopH2 in new permeability pathways (NPPs)
From the abstract:
'Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity

Other mutants
See also the mutants RMgm-2439 and RMgm-1915 generated in the 'Plasmogem screen' where evidence is presented for reduced growth of mutants lacking RhopH2 and the essential nature of RhopH3 for blood stage growth, respectively.


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PbANKA_0830200
Gene Model P. falciparum ortholog PF3D7_0929400
Gene producthigh molecular weight rhoptry protein 2
Gene product: Alternative nameRhopH2
Details of the genetic modification
Short description of the mutationThe rhoph2 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Inducable system usedTET-based (TRAD4)
Short description of the conditional mutagenesisDownregulation of RhopH2 by addition of the tetracycline derivative anhydrotetracycline(ATc)
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo engineer the PbRhopH2 inducible knockdown (iKD) line, the first 1477 bp of the PbRhopH2 coding sequence (PbANKA_0830200) that had been PCR amplified with the primers DO291F and DO67R was cloned into the PstI and NheI sites of the modified pPRF-TRAD4-Tet07583 HAPRF-hDHFR (Pino et al., 2012) described in Elsworth et al (Elsworth et al., 2014). Also cloned into the NheI and BssHII sites of this vector were 1279 bp of the rhoph2 5' UTR sequence immediately upstream of the RhopH2 start codon, which had been PCR amplified using the primers DO62F and DO63R. Before transfection into P. berghei ANKA parasites, pTRAD4-iRhopH2ss was linearized with NheI.
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