RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5532
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1003300; Gene model (P.falciparum): PF3D7_0405600; Gene product: TMEM33 domain-containing protein, putative (TMEM33)
Name tag: mNeon-Green (containing a twin-strep epitope)
Phenotype Asexual bloodstage; Sporozoite;
Last modified: 15 July 2024, 22:57
  *RMgm-5532
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) : 38238886
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/ResearcherKamil M, Aly ASI
Name Group/DepartmentAly Lab, Department of Microbiology, Beykoz Institute of Life Sciences and Biotechnology
Name InstituteBezmialem Vakif University
CityIstanbul
CountryTurkey
Name of the mutant parasite
RMgm numberRMgm-5532
Principal namePbtmem33-mNG
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageA strong mNeonGreen signal was observed in asexual blood stages and sporozoites, with localization mostly appearing to be around the nucleus. Co-localization with ER-Tracker in sporozoites and with BIP in blood stages confirmed that TMEM33 is localized into the ER in both mosquito and blood stages of the parasite life cycle.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteA strong mNeonGreen signal was observed in asexual blood stages and sporozoites, with localization mostly appearing to be around the nucleus. Co-localization with ER-Tracker in sporozoites and with BIP in blood stages confirmed that TMEM33 is localized into the ER in both mosquito and blood stages of the parasite life cycle.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mNeon-Green-tagged TMEM33 (mNeon-Green contained a twin-strep epitope tag)

Protein (function)
Transmembrane protein 33 (TMEM33) protein family, conserved from zebrafish to mammals, contains three transmembrane domains and is localized to ER in all higher eukaryotes investigated. This protein was identified as an important regulator of intracellular calcium homeostasis through interaction with polycistine-2 that results in translocation of cathepsins and sensitization to apoptosis in renal tubular epithelial cells. TMEM33 plays important role in Vascular Endothelial Growth Factor-a (VEGFa) mediated calcium translocation and promotes angiogenesis in both human and zebrafish epithelial cells.
Domain analysis showed that P. falciparum and P. vivax, have four transmembrane domains like P. berghei; however, P. malariae, P. ovale, and P. knowlesi contain five transmembrane domains.

Phenotype
A strong mNeonGreen signal was observed throughout all life cycle stages analyzed, with localization mostly appearing to be around the nucleus. Co-localization with ER-Tracker in sporozoites and with BIP in blood stages confirmed that TMEM33 is localized into the ER in both mosquito and blood stages of the parasite life cycle.
Western blot analysis of the subcellular fractionation of blood stage parasites using anti-twin-strep monoclonal antibody confirmed its presence in the integral membrane fraction, which further confirmed the ER localization.

Additional information
Analysis of a mutant lacking expression of TMEM33 (RMgm-5531) showed the following:
Reduced growth/multiplication of asexual blood stages. Mice infected with Pbtmem33(−) parasites exhibited a peak average parasitemia of 2.57% on Day 8 pi with contrast to 19.64% for wild type parasites.
Normal gametocyte production and male gamete formation (exflagellation). Reduced formation of mature ookinetes (a roughly 50% reduction in ookinete numbers compared to wild type).
Strongly reduced production of oocysts, oocyst sporozoites, and salivary gland sporozoites, Strongly reduced sporozoite infectivity, as measured by injection of purified of wild type and Pbtmem33(−) sporozoites in mice (wild type infected mice showed parasitemia in thin blood smears on Day 4 pi, while Pbtmem33(−) infected mice did not show parasitemia in any of the thin blood smears till Day 16). Motility of Pbtmem33(−) sporozoites was not affected.

Evidence is presented that TMEM33 deficiency upregulates mRNA expression of autophagy-related genes.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1003300
Gene Model P. falciparum ortholog PF3D7_0405600
Gene productTMEM33 domain-containing protein, putative
Gene product: Alternative nameTMEM33
Details of the genetic modification
Name of the tagmNeon-Green (containing a twin-strep epitope)
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 modificationThe generation of Pbtmem33(−) and Pbtmem33-mNG strains was accomplished by double-crossover homologous recombination with modified donor plasmids that contain fluorescent protein and the hDHFR expression cassettes. The fluorescent protein markers are eGFP in the knockout donor plasmid (pAA20), and mNeonGreen (mNG) in the tagging plasmid (pAA32). PCR amplified fragments of 5′ UTR and 3′ UTR as homologous regions of the target loci were cloned into the knockout plasmids flanking the human DHFR cassette and the fluorescence protein marker cassettes to generate Pbp230p and Pbtmem33 constructs by using SacII/BamHI and HindIII/KpnI restriction enzymes, respectively. Similarly, downstream coding sequence and 3′ UTR sequences of TMEM33 were PCR amplified and cloned into the tagging plasmid to generate Pbtmem33-mNG construct by using SacII/EcoRI and HindIII/KpnI restriction enzymes, respectively. Transfection of P. berghei ANKA parasites were performed with SacII/KpnI linearized donor DNA plasmids, and the positive drug selection was done by oral pyrimethamine treatment.
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