Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) |
Gene disruption,
Introduction of a transgene
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Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 38238886 |
MR4 number |
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Parent parasite used to introduce the genetic modification |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone |
Not applicable
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Other information parent line | |
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The mutant parasite was generated by |
Name PI/Researcher | Kamil M, Aly ASI |
Name Group/Department | Aly Lab, Department of Microbiology, Beykoz Institute of Life Sciences and Biotechnology |
Name Institute | Bezmialem Vakif University |
City | Istanbul |
Country | Turkey |
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Name of the mutant parasite |
RMgm number | RMgm-5531 |
Principal name | Pbtmem33(−) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype |
Asexual blood stage | 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. |
Gametocyte/Gamete | Normal gametocyte production and male gamete formation (exflagellation) |
Fertilization and ookinete | Reduced formation of mature ookinetes (a roughly 50% reduction in ookinete numbers compared to wild type). |
Oocyst | Strongly reduced production of oocysts, oocyst sporozoites, and salivary gland sporozoites. |
Sporozoite | 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. |
Liver stage | 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). |
Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of TMEM33 and expresses eGFP
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
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.
Additional information
Analysis of a mutant expressing C-terminal mNeon-Green-tagged TMEM33 (RMgm-5532) showed the following (mNeon-Green contained a twin-strep epitope tag):
- 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.
Evidence is presented that TMEM33 deficiency upregulates mRNA expression of autophagy-related genes.
Other mutants |