Summary

RMgm-920
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1008500; Gene model (P.falciparum): PF3D7_1436300; Gene product: translocon component PTEX150 (PTEX150)
Name tag: triple haemagluttinin (HA)/single streptactin II
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 1 August 2013, 20:12
  *RMgm-920
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 23869529
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/ResearcherMatthews, K; de Koning-Ward, T.F.
Name Group/DepartmentSchool of Medicine
Name InstituteDeakin University
CityWaurn Ponds, Victoria
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-920
Principal namePb150-HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stagePTEX150-HA expressed in blood stages
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot tested
OocystNot tested
SporozoitePTEX150-HA expressed in sporozoites
Liver stagePTEX150-HA expressed in liver stage merozoites
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal triple haemagluttinin (HA) and single Strep-II (Str) epitope tagged version of PTEX150

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.
These proteins are also expressed in early gametocytes, mosquito and liver stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in P. berghei (see RMgm-914, RMgm-915, RMgm-916, RMgm-917)  in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro (see RMgm-918).

Phenotype
PTEX150-HA expression in asexual blood stages, sporozoites and liver stage merozoites

Additional information
Immunofluorescence analyses of ring forms showed that HSP101-HA, PTEX88 and PTEX150-HA clearly co-localised with EXP2, showing a punctate labeling pattern outside the parasite periphery. These observations are in keeping with EXP2, HSP101 and PTEX150 associating at the PVM The localization of TRX2, however, was less clear and made difficult by the fact that expression of TRX2-HA was weak. In the early ring stages some co-labeling of TRX2-HA with EXP2 was observed but more pronounced internal parasite labeling with the anti-HA antibodies (Fig. 2A). As the parasites matured, the localization of TRX2-HA appeared more towards the parasite periphery  rather than PV/PVM and its localization within the parasite also more pronounced. By schizont stages TRX2-HA displayed punctate apical labeling, reminiscent of labeling pattern observed with other PTEX components in P. falciparum and Pb150-HA.

PTEX150-HA, HSP101-HA and PTEX88-HA expression was not observed at the PVM in gametocytes. Moreover, the expression of EXP2 at the PVM and punctate labeling of TRX2-HA within gametocytes was weak.

Immunofluorescence analyses showed that EXP2,HSP101 and PTEX150 were expressed in sporozoites and liver stage merozoites.

Other mutants
HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in P. berghei (see RMgm-914, RMgm-915, RMgm-916, RMgm-917
TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro (see RMgm-918).


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1008500
Gene Model P. falciparum ortholog PF3D7_1436300
Gene producttranslocon component PTEX150
Gene product: Alternative namePTEX150
Details of the genetic modification
Name of the tagtriple haemagluttinin (HA)/single streptactin II
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe targeting constructs were designed to integrate into the respective P. berghei PTEX locus by single cross-over recombination, such that the complete open reading frame was reconstituted while simultaneously introducing a combined C-terminal triple haemagluttinin (HA) and single Strep-II (Str) epitope tag.
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