| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene tagging
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30154793
Reference 2 (PMID number) : 29515528
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| 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 | Fernandes P, Mueller AK |
| Name Group/Department | Centre for Infectious Diseases, Parasitology Unit |
| Name Institute | University Hospital Heidelberg |
| City | Heidelberg |
| Country | Germany |
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| Name of the mutant parasite |
| RMgm number | RMgm-4482 |
| Principal name | PbmaLS_05 CT EG FP |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | Through live imaging of PbmaLS_05 EGFP parasites, we observed expression of the full-length PbmaLS_05 in rings, blood-stage- and liver-stage schizonts. The localization was branched in intra-hepatic parasites during the cytomere stage of development and distinctly more punctuate in blood-stage schizonts, similar to what has previously been observed for parasite mitochondria and apicoplast. Co-staining of PbmaLS_05 EGFP liver stage schizonts, merosomes and blood-stage schizonts with a mitochondrial marker, showed partial co-localization of the PbmaLS_05 protein with the mitochondria.
However, treatment of PbmaLS_05 EGFP liver stages with azithromycin, a drug that inhibits biogenesis of the apicoplast abolished the branched structure observed for PbmaLS_05, even though the mitochondrial structure was retained, suggesting that PbmaLS_05 localizes to the parasite apicoplast. To confirm that PbmaLS_05 localized to the apicoplast of blood-stage parasites, we co-stained PbmaLS_05 EGFP blood-stage schizonts with antibodies against GFP and ACP, an apicoplast-specific protein. Co-localization of both antibodies as observed by the yellow signal in PbmaLS_05 EGFP but not PbANKA WT schizonts confirmed that PbmaLS_05 indeed localizes to the apicoplast of blood-stage schizonts. |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | Not tested |
| Sporozoite | Not tested |
| Liver stage | Through live imaging of PbmaLS_05 EGFP parasites, we observed expression of the full-length PbmaLS_05 in rings, blood-stage- and liver-stage schizonts. The localization was branched in intra-hepatic parasites during the cytomere stage of development and distinctly more punctuate in blood-stage schizonts, similar to what has previously been observed for parasite mitochondria and apicoplast. Co-staining of PbmaLS_05 EGFP liver stage schizonts, merosomes and blood-stage schizonts with a mitochondrial marker, showed partial co-localization of the PbmaLS_05 protein with the mitochondria.
However, treatment of PbmaLS_05 EGFP liver stages with azithromycin, a drug that inhibits biogenesis of the apicoplast abolished the branched structure observed for PbmaLS_05, even though the mitochondrial structure was retained, suggesting that PbmaLS_05 localizes to the parasite apicoplast. To confirm that PbmaLS_05 localized to the apicoplast of blood-stage parasites, we co-stained PbmaLS_05 EGFP blood-stage schizonts with antibodies against GFP and ACP, an apicoplast-specific protein. Co-localization of both antibodies as observed by the yellow signal in PbmaLS_05 EGFP but not PbANKA WT schizonts confirmed that PbmaLS_05 indeed localizes to the apicoplast of blood-stage schizonts. |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of PbmaLS_05.
Protein (function)
PbmaLS_05 has a predicted protein size of 306 kDa that contains two predicted transmembrane domains and one predicted P loop containing nucleoside triphosphate hydrolase domain.
The ortholog of PBANKA_140100 (PbmaLS_05), i.e., PF3D7_1302500, was initially identified as a putative antigen that is differentially expressed in intra-hepatic stages of P. falciparum radiation-attenuated sporozoites in comparison to Pf wild-type.
Phenotype
RT-PCR evidence of expression throughout the complete life cycle (in all stages).
Analyses of blood- and liver-stages expressing the GFP-tagged PbmaLS_05: evidence presented for an apicoplast location (see below).
See also RMgm-4481 for a mutant lacking expression of PbmaLS_05. Parasites lacking PbmaLS_05 showed a decreased ability to infect RBC (and immature reticulocytes), Reduced mortality due to experimental cerebral malaria (ECM). Evidence is presented for normal liver stage development.
Additional information
Through live imaging of PbmaLS_05 EGFP parasites, we observed expression of the full-length PbmaLS_05 in rings, blood-stage- and liver-stage schizonts. The localization was branched in intra-hepatic parasites during the cytomere stage of development and distinctly more punctuate in blood-stage schizonts, similar to what has previously been observed for parasite mitochondria and apicoplast. Co-staining of PbmaLS_05 EGFP liver stage schizonts, merosomes and blood-stage schizonts with a mitochondrial marker, showed partial co-localization of the PbmaLS_05 protein with the mitochondria.
However, treatment of PbmaLS_05 EGFP liver stages with azithromycin, a drug that inhibits biogenesis of the apicoplast abolished the branched structure observed for PbmaLS_05, even though the mitochondrial structure was retained, suggesting that PbmaLS_05 localizes to the parasite apicoplast. To confirm that PbmaLS_05 localized to the apicoplast of blood-stage parasites, we co-stained PbmaLS_05 EGFP blood-stage schizonts with antibodies against GFP and ACP, an apicoplast-specific protein. Co-localization of both antibodies as observed by the yellow signal in PbmaLS_05 EGFP but not PbANKA WT schizonts confirmed that PbmaLS_05 indeed localizes to the apicoplast of blood-stage schizonts.
Other mutants
See also RMgm-4481 for a mutant lacking expression of PbmaLS_05.
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*RMgm-4482
