RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_0820200; Gene model (P.falciparum): PF3D7_0919300; Gene product: thioredoxin-like protein 1 (TrxL1)
Name tag: GFP
Phenotype Sporozoite;
Last modified: 14 March 2023, 15:23
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) : 36869034
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherFerreira JL, Grünewald K
Name Group/DepartmentCentre for Structural Systems Biology
Name InstituteCentre for Structural Systems Biology
Name of the mutant parasite
RMgm numberRMgm-5317
Principal namePbTrxL1-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteHigh expression in P. berghei sporozoites of PbTrxL1-GFP
Liver stageNot tested
Additional remarks phenotype

The mutant expresses C-terminal GFP-tagged version of TrxL1.

Protein (function)
The eukaryotic protozoan parasites of Plasmodium spp., rely on a scaffold of ordered subpellicular microtubules (SPMTs) as their main structural cytoskeletal components. SPMTs lie below and interact with a double membrane known as the inner membrane complex (IMC) located beneath the plasma membrane. Together these structures are referred to as the pellicle which is found across Apicomplexa, including, for example, Toxoplasma gondii. Although eukaryotic tubulins are highly conserved, there are appreciable differences which make apicomplexan microtubules stand out. 
A feature unique to apicomplexan tubulin is its formation of L-shaped semi-tubules which, with accessory proteins, form a helical structure termed the conoid. The conoid is a specialised structure involved in invasion. Although characterised in Toxoplasma, homologues of some conoid components are expressed in Plasmodium spp. In the  invasive forms of apicomplexan parasites, a structurally diverse MTOC located at their apical end, the Apical Polar Ring (APR), coordinates the higher order spatial control of SPMTs, but the specific mechanism remains unknown. 

Sporozoites and ookinetes are motile, elongated forms with a dense SPMT scaffold  underneath the IMC. In the paper it is shown that SPMT's of these stages consist of 13- protofilament microtubules with twice-interrupted luminal helices. Similar structures, named Interrupted Luminal Helices (ILH), were first observed in flagellar ends of human spermatozoa, and more recently in equine and porcine spermatozoa, and tachyzoites of Toxoplasma gondii. The T. gondii ILH consists of thioredoxin-like proteins 1 and 2 (TrxL1, TrxL2) and subpellicular microtubule protein 1 (SPM1). Plasmodium spp. have homologues of TrxL1 (PfTrxL1; PBANKA_0820200; PF3D7_0919300) and SPM1 (PfSPM1; PBANKA_0810700; PF3D7_0909500) but lack TrxL2, which in the Toxoplasma ILH is present in both. Compatible with the ILH being composed of SPM1 and TrxL1 in Plasmodium, high expression in Plasmodium berghei (Pb) of PbSPM1-GFP and PbTrxL1-GFP was seen in the endogenously-tagged sporozoite lines, PbSPM1-GFP and PbTrxL1-GFP (see phenotype below).

High expression in P. berghei sporozoites of PbTrxL1-GFP (and PbSPM1-GFP; see mutant RMgm-5316)

Additional information

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0820200
Gene Model P. falciparum ortholog PF3D7_0919300
Gene productthioredoxin-like protein 1
Gene product: Alternative nameTrxL1
Details of the genetic modification
Name of the tagGFP
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 modificationEndogenous tagging was performed essentially as described before using single-crossover integration. As both SPM1 and TrxL1 show an overall gene length of less than 1 kilobase, the entire open-reading frame was amplified from PbANKA wildtype genomic DNA. The resulting DNA fragment was cloned into the pL18 vector using EcoRI and BamHI restriction sites. The reverse primer encoded for six alanines that were used as a linker. The pL18 vector harbours the hDHFR gene for positive selection using the drug pyrimethamine. Prior to transfection, the vector was linearized using SwaI (SPM1) and BsmI (TrxL1), respectively, followed by ethanol precipitation. The linearized pL18-SPM1-GFP/pL18-TrxL1-GFP vectors were each transfected into an unmodified P. berghei ANKA strain using standard protocols
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