RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4693
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0311700; Gene model (P.falciparum): PF3D7_0214900; Gene product: rhoptry neck protein 6 (RON6)
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; Sporozoite;
Last modified: 30 November 2019, 21:15
  *RMgm-4693
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) : 31552198
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/ResearcherTokunaga N, Ishino T
Name Group/DepartmentDivision of Molecular Parasitology
Name InstituteProteo-Science Center, Ehime University
CityToon
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-4693
Principal name-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageSee below for detailed information of absence/presence of expression in merozoites and sporozoites
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteSee below for detailed information of absence/presence of expression in merozoites and sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal cmyc-tagged version of the 'rhoptry' protein and expresses GFP under control of the constitutive eef1a promoter.

Protein (function)
See below more details about the different rhoptry proteins

Phenotype
See below for detailed information of absence/presence of expression in merozoites and sporozoites

Additional information
In this study the following rhoptry proteins of P. falciparum were analysed for expression in P. berghei merozoites and sporozoites. The following localisation was detected in sporozoites:

ron2, PBANKA_1315700; rhoptry
ron4, PBANKA_0932000; rhoptry
ron5, PBANKA_0713100; rhoptry
ron6, PBANKA_0311700; apical end
ralp1, PBANKA_0619700; rhoptry
asp/ron1, PBANKA_1003600; rhoptry
rap1, PBANKA_1032100; rhoptry
ron3, PBANKA_1464900; rhoptry
rama, PBANKA_0804500; rhoptry
rhoph1a, PBANKA_1400600; not detected
rhoph2, PBANKA_0830200; not detected
rhoph3, PBANKA_0416000; not detected

Transgenic parasite lines were generated by single-crossover homologous recombination to express each rhoptry protein fused with a C-terminal c-Myc tag. Ten transgenic parasite lines were successfully isolated. Specific antibodies against recombinant protein were prepared for ASP/RON1 and RAMA, since they are predicted to have C-terminal glycosylphosphatidylinositol (GPI) anchored domains and therefore the modification of their C-terminal structure might disrupt their function. Specific antibodies against the middle region of RON3 were also prepared, because it was demonstrated that a 40 kDa fragment of C-terminal RON3 is cleaved during schizont maturation in P. falciparum
Protein lysates of 1.5 × 105 schizonts and oocyst-derived sporozoites of each transgenic parasite line expressing c-Myc tagged rhoptry protein or WT-GFP were analyzed by western blotting using anti-c-Myc antibodies or specific antibodies against ASP/RON1, RAMA, and RON3. In schizonts all examined c-Myc tagged rhoptry proteins, except for RON3, were detected at the expected size of full-length, demonstrating that c-Myc fused rhoptry proteins are successfully expressed. In addition, the processed forms of RON4 and RALP1 were detected at ∼60 and 40 kDa. In the case of RON3, anti-c-Myc antibodies detected ∼40 kDa fragment as reported in P.falciparum, while anti-RON3 antibodies recognized two bands, near 250 kDa, corresponding to the full-length and processed RON3. It was confirmed that aroughly 40kDa fragment of the C-terminal region in RON3 is cleaved in P.berghei mature schizonts as well as in P. falciparum. Antibodies against ASP/RON1 and RAMA recognized corresponding proteins at the size of expected full- and processed-proteins, confirming the specificity of these antibodies. RhopH1A and RhopH3 proteins were not detected in sporozoites, while RhopH2 was detected as a far weaker band compared to that in schizonts, which is in good agreement with the transcriptional data. In addition, RON2 production in sporozoites was significantly less than in schizonts.

To determine the precise localization of rhoptry proteins, immuno-electron microscopy (IEM) was performed using schizont stage merozoites and oocyst sporozoites. 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. This is consistent with the observation that the sub-localization of RON11 in rhoptries differs between merozoites and sporozoites. Only ASP/RON1 tends to accumulate in the thinner part in rhoptries near the tip of sporozoites.

RON2, RON4, RON5, RALP1, RAP1, and RAMA 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. ASP/RON1 and RON3 could not be detected by anti-ASP/RON1 or anti-c-Myc antibodies, which might due to less target or c-Myc tagged protein amounts in salivary gland sporozoites.

Other mutants
Tokunaga N, Ishino T
 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0311700
Gene Model P. falciparum ortholog PF3D7_0214900
Gene productrhoptry neck protein 6
Gene product: Alternative nameRON6
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) PCR construct 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 markereef1a
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 (Ishino et al., 2019). 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 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 modification
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