Summary

RMgm-1102
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0931200; Gene model (P.falciparum): PF3D7_1116800; Gene product: heat shock protein 101 | chaperone protein ClpB2 (HSP101)
Details mutation: The hsp101 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Details conditional mutagenesis: Downregulation of HSP101 expression by addition of the tetracycline derivative ATc
Phenotype Asexual bloodstage;
Last modified: 27 July 2014, 15:13
  *RMgm-1102
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) : 25043043
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherElsworth, B.; de Koning-Ward, T.F.
Name Group/DepartmentDeakin University
Name InstituteDeakin University
CityWaurn Ponds
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-1102
Principal namePbi101KD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageStrongly reduced growth (parasitemia) in mice treated with ATc. Evidence is presented that treated parasites are able to invade and develop into ring forms but are affected in the development of trophozoite and schizont stage. Evidence is presented that export of proteins into the host RBC is affected in ATc-treated Pbi101KD blood stages.
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 hsp101 locus was mutated using construct pPRF-TRAD4-Tet07-HAPRFhDHFR (RMgm-797) for introducing a tet-inducible promoter and TRAD4 activating domain

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.
The unsuccessful attempts to disrupt the hsp101 gene (RMgm-914, RMgm-945) indicate an essential role of HSP101 in blood stages.

Phenotype
HSP101 expression in Pbi101KD blood stages was downregulated by addition of the tetracycline derivative anhydrotetracycline (ATc). Strongly reduced growth (parasitemia) in mice treated with ATc. Evidence is presented that treated parasites are able to invade and develop into ring forms but are affected in the development of trophozoite and schizont stage. Evidence is presented that export of proteins into the host RBC is affected in ATc-treated Pbi101KD blood stages.

Additional information
To examine the growth effect in more detail, purified ring-stagePbi101 KD parasites were injected into mice pre-exposed to ATc, then isolated 29 h later and cultured in vitro with ATc. Parasites invaded erythrocytes in the mice and developed normally into ring stages. However, parasites appeared morphologically abnormal by the 34 h time point and were incapable of developing into schizonts by the 46 h time point, unlike Pbi101KD parasites not exposed to ATc. Asynchronous Pbi101KD ring-stage parasites cultured in vitro for 16 hin the presence ofATc demonstrated a threefold to sixfold decrease in hsp101 messenger RNA in schizont stages and a 85–90% knockdown of HSP101 protein by the 29 h time point.

Other mutants


  Mutated: Mutant parasite with a mutated 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
Short description of the mutationThe hsp101 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Inducable system usedTET-based (TRAD4)
Short description of the conditional mutagenesisDownregulation of HSP101 expression by addition of the tetracycline derivative ATc
Additional remarks inducable system
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Conditional expression system for stage-specific, tetracycline-dependent (ATc) gene regulation.
Tetracycline derivative anhydrotetracycline(ATc).
For tetracyclin (ATc)-dependent gene regulation the hsp101 locus was mutated using construct pPRF-TRAD4-Tet07-HAPRFhDHFR (RMgm-797) for introducing a tet-inducible promoter and TRAD4 activating domain
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 tetracyclin (ATc)-dependent gene regulation the hsp101 locus was mutated using construct pPRF-TRAD4-Tet07-HAPRFhDHFR (RMgm-797) for introducing a tet-inducible promoter and TRAD4 activating domain

The constructpTg-ranTRAD4-iHSP101 was used to generate the P. berghei i101 KD. This plasmid was based on pPRF-TRAD4-Tet07-HAPRFhDHFR but modified to include a BsiWI restriction site downstream of the profilin coding sequence and a BssHII restriction enzyme site between the profilin 5' untranslated region and the TRAD4 sequence. This enabled cloning of the first 1.7 kilobases (kb) of theHSP101 coding sequence (PbANKA_09312;amplified with DO390F, 5'-caccctgcagATGGTACGGAACATTGCTAAAAATT-3', and DO414R, 5'-gtatcgtacgccatggCTATAACTCTTGGTTTACCCG-3', the latter containing an internal NcoI site) into the PstI and BsiWI cloning sites and 0.85 kb of the HSP101 5' untranslated region (amplified using oligonucleotides DO392F, 5'-gtaccatggCGTACGGTATGCAATTGCTCTTAATGCATTTGC-3', and DO394R, 5'-tatgcgcgc TTTCTACTAAATTTATAGTAAATATAGATATA-3') into the NcoI and BssHII sites. Before transfection, DNA was linearized with NcoI.
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