RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4866
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0932000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Details mutation: 'promoter-swap' mutant; the RON4 promoter replaced by the promoter of PBANKA_1443300 (msp9)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Sporozoite;
Last modified: 7 September 2020, 17:28
  *RMgm-4866
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) : 32817376
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherNozaki M, Ishino T
Name Group/DepartmentDivision of Molecular Parasitology, Proteo-Science Center
Name InstituteEhime University
CityMatsuyama, Ehime
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-4866
Principal nameP(msp9)-RON4
Alternative nameRON4-cKD
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteSporozoite numbers from midguts and hemolymph were not significantly different from those of control parasites indicating that RON4 is not critical for sporozoite formation, maturation inside oocysts, and release to the hemocoel. In contrast, the mean numbers of sporozoites residing in salivary glands were reduced 27-fold. Sporozoites showed reduced adhesion and gliding motility.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the 'promoter-swap' mutant the RON4 promoter replaced by the promoter of PBANKA_1443300 (msp9). This promoter is active in asexual blood stages (in schizonts) but is silent in mosquito stages, such as oocysts and sporozoites. In addition the mutant expresses GFP under the constitutive eef1a promoter.

Protein (function)
RON4 is a rhoptry neck protein expressed in rhoptries of merozoites and sporozoites. Unsuccessful attempts to knock-out the ron4 gene (RMgm-686) indicates an essential function in blood stages. A number of rhoptry neck proteins (RONs), including RON2, RON4, and RON5 are secreted and inserted into the target cellular membrane as a complex. 

Phenotype
Sporozoite numbers from midguts and hemolymph were not significantly different from those of control parasites indicating that RON4 is not critical for sporozoite formation, maturation inside oocysts, and release to the hemocoel. In contrast, the mean numbers of sporozoites residing in salivary glands were reduced 27-fold. Sporozoites showed reduced adhesion and gliding motility.

38% of control wild type hemolymph sporozoites start gliding, while 55% remain floating without attachment to the glass slide.  In contrast, approximately 85% of RON4-cKD hemolymph sporozoites drifted. These results indicate that RON4 is required for hemolymph sporozoite attachment to the glass slide. Accordingly, only 9% of RON4-cKD hemolymph sporozoites show gliding, which is 4-fold less than wild type gliding sporozoites, 

When embedded in Matrigel, 78% of control wild-type hemolymph sporozoites move continuously through the matrix, whereas only 15% of RON4-cKD hemolymph sporozoites display a circular mode of motility. 
These results indicate that RON4 is mainly required for sporozoite attachment and are also involved in the onset of sporozoite movement. 

Additional information
From the Abstract of the paper:
'Components of the merozoite rhoptry neck protein complex are also expressed in sporozoites, namely, RON2, RON4, and RON5, suggesting that invasion mechanism elements might be conserved between these infective stages. Recently, we demonstrated that RON2 is required for sporozoite invasion of mosquito salivary gland cells and mammalian hepatocytes, using a sporozoite stage-specific gene knockdown strategy in the rodent malaria parasite model, Plasmodium berghei. Here, we use a coimmunoprecipitation assay and oocyst-derived sporozoite extracts to demonstrate that RON2, RON4, and RON5 also form a complex in sporozoites. The sporozoite stage-specific gene knockdown strategy revealed that both RON4 and RON5 have crucial roles during sporozoite invasion of salivary glands, including a significantly reduced attachment ability required for the onset of gliding. Further analyses indicated that RON2 and RON4 reciprocally affect trafficking to rhoptries in developing sporozoites, while RON5 is independently transported.'

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0932000
Gene Model P. falciparum ortholog PF3D7_1116000
Gene productrhoptry neck protein 4
Gene product: Alternative nameRON4
Details of the genetic modification
Short description of the mutation'promoter-swap' mutant; the RON4 promoter replaced by the promoter of PBANKA_1443300 (msp9)
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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo repress ron4 or ron5 transcription only in sporozoites, their native promoter regions were replaced by homologous recombination with the promoter region of a merozoite-specific expressing gene (merozoite surface protein 9 [msp9]). DNA fragments for the homologous recombination cassettes (RON4-N, −17 to +869 bp from the start codon; ron4-upstream, −1047 to −459 bp; RON5-N, −65 to 602 bp; and ron5-upstream, −1344 to −595 bp) were amplified from genomic DNA of PbWT-GFP by PCR and ligated to the transgenic vectors containing an msp9 promoter region and a human dihydrofolate reductase (hDHFR)-expressing cassette to confer antimalarial drug resistance. The transgenic vectors were linearized with XhoI.
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 nameGFP (gfp-mu3)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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