RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4713
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0932000; Gene model (P.falciparum): PF3D7_1116000; Gene product: rhoptry neck protein 4 (RON4)
Name tag: c-myc
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 Asexual bloodstage; Oocyst; Sporozoite;
Last modified: 4 February 2020, 13:28
  *RMgm-4713
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31981605
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone 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/ResearcherIshino T, Torii M
Name Group/DepartmentDivision of Molecular Parasitology, Proteo-Science Center
Name InstituteEhime University
CityToon, Ehime
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-4713
Principal nameron4-cmyc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagesee below for description of expression in rhoptries of merozoites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
Oocystsee below for description of expression in rhoptries of sporozoites in oocysts
Sporozoitesee below for description of expression in rhoptries of salivary gland sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal cmyc-tagged version of ron4, PBANKA_0932000

Protein (function)
From the Abstract:

Recently we demonstrated that rhoptry neck protein 2 (RON2), which is crucial for tight junction formation in merozoites, is also important for sporozoite invasion of both target cells. With the aim of comprehensively describing the mechanisms of sporozoite invasion, the expression and localization profiles of rhoptry proteins were investigated in Plasmodium berghei sporozoites. Of 12 genes representing merozoite rhoptry molecules, nine are transcribed in oocyst-derived sporozoites at a similar or higher level compared to those in blood-stage schizonts. Immuno-electron microscopy demonstrates that eight proteins, namely RON2, RON4, RON5, ASP/RON1, RALP1, RON3, RAP1, and RAMA, localize to rhoptries in sporozoites. It is noteworthy that most rhoptry neck proteins in merozoites are localized throughout rhoptries in sporozoites. This study demonstrates that most rhoptry proteins, except components of the high-molecular mass rhoptry protein complex (rhoph1a, PBANKA_1400600; rhoph2, BANKA_0830200; rhoph3, PBANKA_0416000), are commonly expressed in merozoites and sporozoites in Plasmodium spp., which suggests that components of the invasion mechanisms are basically conserved between infective forms independently of their target cells

Phenotype
Ten mutants expressing 10 different c-myc tagged rhoptry proteins were analysed. Immuno-electron microscopy (IEM) analysis showed:
In P. berghei merozoites, RON2, RON4, RON5, RALP1, and ASP/RON1, which are categorized as rhoptry neck proteins in Pf merozoites, were confirmed to localize to the rhoptry neck region. In addition, RAP1, RON3, RhopH1A, RhopH2, and RhopH3 are observed in the rhoptry bulb region, as reported for Pf merozoites. RAMA is observed on the rhoptry membrane at the bulb region. RON6 could not be detected by anti-c-Myc antibodies, possibly because its protein amount in merozoites is not sufficient to be observed by IEM.

In sporozoites formed inside oocysts, it was confirmed that three components for the RhopH complex do not accumulate in rhoptries, as expected from the observation of far less amounts of transcripts and proteins in sporozoites compared to merozoites. Other than the RhopH complex, all proteins examined are localized to rhoptries in sporozoites as well as in merozoites. However, most proteins are distributed throughout rhoptries in sporozoites, despite their sub-localization in merozoites, suggesting that sub-compartmentation in rhoptries might be different between merozoites and sporozoites.

In salivary gland sporozoites RON2, RON4, RON5, RALP1 and RAP1 were detected in rhoptries of sporozoites residing in salivary glands, indicating that rhoptry proteins reside in rhoptries after sporozoite invasion of salivary glands, presumably available for subsequent invasion of hepatocytes in mammalian hosts. RON3 could not be detected, which might due to less target or c-Myc tagged protein amounts in salivary gland sporozoites.

Additional information
ron2, PBANKA_1315700;
ron4, PBANKA_0932000;
ron5, PBANKA_0713100;
ron6, PBANKA_0311700;
ralp1, PBANKA_0619700;
asp/ron1, PBANKA_1003600; (not tagged with c-myc) 
rap1, PBANKA_1032100;
ron3, PBANKA_1464900;
rama, PBANKA_0804500; (not tagged with c-myc)
rhoph1a, PBANKA_1400600;
rhoph2, PBANKA_0830200;
rhoph3, PBANKA_0416000.

Transcript analysis showed:
Six molecules categorized as encoding rhoptry neck proteins in merozoites (ron2, ron4, ron5, ron6, rhoptry-associated leucine zipper-like protein 1 (ralp1), and apical sushi protein (asp)/ron1) are also transcribed in sporozoites. Among six genes encoding rhoptry proteins localized to the bulb region in merozoites, rhoptry-associated protein 1 (rap1), ron3, and rhoptry associated membrane antigen (rama) are also transcribed in sporozoites; while the other three, encoding the components of the high-molecular mass rhoptry protein complex (RhopH complex), are transcribed far less in sporozoites than in schizonts. This data raises the possibility that RhopH1A, RhopH2, and RhopH3 may play roles predominantly in merozoites. In contrast, ron5, ron6, asp/ron1, and ron3 are predominantly transcribed in sporozoites vs. schizonts. The transcripts of rhoptry molecules expressed in sporozoites increase during sporozoite maturation in oocysts. After sporozoite invasion of salivary glands, the transcript amounts of ron2, ron4, rap1, ron3, and rama are significantly decreased, while transcripts of ron5, ron6, ralp1, and asp1/ron1 remain high or increase. In salivary gland sporozoites, ron5 and ron6 are the highest transcribed among rhoptry molecules; however, their amounts remain ∼200-fold less than that of a micronemal molecule, sporozoite protein essential for cell traversal 2 (spect2), whose transcription is strongly enhanced after sporozoites invade salivary glands.

Other mutants


  Tagged: Mutant parasite with a tagged 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
Name of the tagc-myc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 modificationTo generate transgenic parasites expressing a rhoptry protein tagged with c-Myc at its C-terminus, the native locus of the targeted rhoptry molecule in the WT-GFP genome was replaced by single crossover homologous recombination with an expression cassette of the C-terminus of the rhoptry protein fused with a c-Myc tag, similar to the generation of RON2-c-Myc expressing parasites. Approximately 1,000–2,000 base pair of DNA fragments including the C-terminus of each rhoptry protein were amplified with specific primers by PCR from genomic DNA of WTGFP. Amplified PCR fragments of RAP1, RhopH1A, RhopH2, and RhopH3 were inserted into the pL0033 plasmid (BEI Resources, Manassas, VA, USA) at SacII and NcoI sites just before the c-Myc tag coding region, and these plasmids were then linearized at endogenous HpaI, SpeI, and XbaI sites, respectively. RON5 and RALP1 fragments were inserted into an NdeI site disrupted pL0033 vector, which was linearized at an endogenous NdeI site. To introduce XbaI recognition sites for linearization into the PCR fragments of RON3 and RON4, site directed mutagenesis was performed to introduce mutations without amino acid substitution as follows: RON3, 5716A > T and 5717G > C; and RON4, 1711T > C. Using the same strategy, the endogenous NcoI site in the amplified RON6 fragment was disrupted, to avoid interference with ligation into the SacII and NcoI sites of pL0033 (1878C > A). These DNA fragments were inserted into the pL0033 plasmid at SacII and NcoI sites, which were linearized at an introduced XbaI site for RON3 and RON4, and at an endogenous BamHI site for RON6.
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 procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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