RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4188
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0614300; Gene model (P.falciparum): PF3D7_0716600; Gene product: cysteine desulfurase (SufS)
Details mutation: The mutant contains a FRTed 3'UTR of the SufS locus
Details conditional mutagenesis: The 3'UTR of the SufS locus is removed in the oocyst/sporozoite stage by the Flp/FRT system
Transgene
Transgene not Plasmodium: FlpL recombinase of yeast (FlpL)
Promoter: Gene model: PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (TRAP; SSP2; SSP-2)
3'UTR: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite;
Last modified: 15 January 2020, 15:52
  *RMgm-4188
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28695709
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4186
Other information parent lineThis parasite expresses the yeast FlpL recombinase under the control of the promoter of trap. The mutant does not contain a selectable marker.
The mutant parasite was generated by
Name PI/ResearcherCharan M; Habib S
Name Group/DepartmentDivision of Molecular and Structural Biology, CSIR-Central Drug Research Institute
Name Institute, CSIR-Central Drug Research Institute
CityLucknow
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-4188
Principal nameSufScKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNormal numbers of oocysts. Reduced sporulation in oocysts, strong reduction of the number of salivary gland sporozoites
SporozoiteReduced sporulation in oocysts, strong reduction of the number of salivary gland sporozoites and reduced 'infectivity' of sporozoites.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of SufS. The mutant expresses the yeast FlpL recombinase under the control of the trap promoter  and contains a FRTed  3'-UTR of the SufS locus.

This mutant has been generated by replacement of the endogenous 3'-UTR of the SufS locus with a FRTed 3'-UTR in the mutant RMgm-4186 that expresses FlpL. This mutant expresses the yeast FlpL recombinase under the control of the promoter of trap and does not contain a selectable marker. The flpl gene is integrated into the silent 230p locus by double cross-over integration.

The 3'-UTR of SufS is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269, RMgm-747). Removal of the FRTed 3'-UTR of SufS has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in oocysts/sporozoites that resulted in the excision of the 3'-UTR of SufS which was flanked by FRT sequences.

Protein (function)
Iron-sulfur [Fe-S] clusters are inorganic cofactors that are found on proteins from a range of biological processes and are present in most organisms. The assembly of [Fe-S] clusters on apoproteins is not a spontaneous process but is mediated by distinct pathways that mobilise sulfur atoms from L-cysteines, and assemble them onto scaffold components. The scaffold also receives iron from iron donors and the assembled [Fe-S] clusters are transferred to the target apoprotein via carrier protein(s). In bacteria, three distinct sets of factors/enzymes encoded as operons assemble [Fe-S] clusters; the bacterial isc, suf and nif operons encode proteins that are components of the ISC (iron-sulfur cluster formation), SUF (sulfur mobilization) and NIF (nitrogen fixation) assembly systems. The ISC system is the major [Fe-S] biogenesis system with the SUF system being activated under iron starvation or oxidative stress.
In eukaryotes, [Fe-S] assembly systems are thought to have evolved from bacterial endosymbionts. The SUF pathway is found in plastid-containing protozoa and plants. The e apicoplast, the relict plastid of the malaria parasite, harbours a functional SUF pathway. Except apicoplast-encoded SufB in Plasmodium spp., all constituent proteins of the SUF pathway are nuclear-encoded and targeted to the apicoplast. These include the cysteine desulfurase SufS and its interacting partner SufE [18], SufC and SufD that are predicted to constitute the scaffold complex with SufB, and putative carrier proteins SufA and Nfu. Components of the SUF pathway in P. berghei have also been reported to be refractory to gene deletion. Apicoplast proteins that likely require [Fe-S] clusters include ferredoxin, two enzymes (IspG and IspH) of the DOXP pathway of isoprenoid biosynthesis, LipA (lipoic acid synthase), and MiaB (tRNA methylthiotransferase).

Phenotype
In the SufScKO line, the 3'-UTR of SufS is excised during development in the mosquito midgut.  Evidence is presented for normal numbers of oocysts, reduced sporulation in oocysts, strong reduction of the number of salivary gland sporozoites and reduced 'infectivity' of sporozoites

Additional information
Since SufS is essential for blood stage develepment/multiplication a stage-specific knockout (cKO) mutant using the FlpL/FRT system was generated.
The mutant was used to determine the role of SufS in oocysts/sporozoites. Evidence is presented that SufS plays a role in the formation of (infectious) sporozoites (inside oocysts).

Other mutants
 


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0614300
Gene Model P. falciparum ortholog PF3D7_0716600
Gene productcysteine desulfurase
Gene product: Alternative nameSufS
Details of the genetic modification
Short description of the mutationThe mutant contains a FRTed 3'UTR of the SufS locus
Inducable system usedFlp/FRT
Short description of the conditional mutagenesisThe 3'UTR of the SufS locus is removed in the oocyst/sporozoite stage by the Flp/FRT system
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 modificationThe SufS targeting plasmid pSufS/FRT consisted of the C-terminus of the SufS ORF followed by the first FRT site, 3’-UTR of TRAP, hDHFR cassette, the second FRT site and 3’-UTR of SufS
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 nameFlpL recombinase of yeast (FlpL)
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe FlpL coding sequence was flanked by 1.58 kb and 0.55 kb of 5′ and 3′ regulatory sequences of trap, respectively.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1349800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative nameTRAP; SSP2; SSP-2
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_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
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