| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene tagging,
Introduction of a transgene,
Introduction of a transgene
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30597708
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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 |
P. berghei ANKA 820cl1m1cl1 (RMgm-164)
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| Other information parent line | P. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517). |
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| The mutant parasite was generated by |
| Name PI/Researcher | Obrova K, Mair GR, Mueller AK |
| Name Group/Department | Center for Infectious Diseases, Parasitology Unit |
| Name Institute | Heidelberg University Hospital |
| City | Heidelberg |
| Country | Germany |
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| Name of the mutant parasite |
| RMgm number | RMgm-4578 |
| Principal name | FLP::HA (820) |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | The FLP::HA fusion protein was detected in both schizont- and gametocyte-enriched blood stage lysates revealing the fusion protein at the expected size of 186 kDa. Antibody staining of fixed parasites confirmed expression in schizonts, showing a speckled signal. High abundance of FLP::HA was observed in gametocytes, localizing to widespread, distinct intracellular speckles. Immuno-gold typically localized close to the vesicle membranes, which is in agreement with FLP being a predicted transmembrane protein. |
| Gametocyte/Gamete | The FLP::HA fusion protein was detected in both schizont- and gametocyte-enriched blood stage lysates revealing the fusion protein at the expected size of 186 kDa. Antibody staining of fixed parasites confirmed expression in schizonts, showing a speckled signal. High abundance of FLP::HA was observed in gametocytes, localizing to widespread, distinct intracellular speckles. Immuno-gold typically localized close to the vesicle membranes, which is in agreement with FLP being a predicted transmembrane protein.
Both male and female gametocytes showed a similar abundance and localization of FLP::HA. FLP-HA relocalizes to the cell peripery during activation of gametocytes. |
| Fertilization and ookinete | Not tested |
| Oocyst | Not tested |
| Sporozoite | Not tested |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal HA-tagged version of FLP and expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This mutant does not contain a drug-selectable marker. The hdhfr/yfcu selectable marker has been removed from the genome by negative selection
Protein (function)
The P. berghei ferlin-like protein (FLP, PBANKA_1224400) contains six predicted C2 domains, a C-terminal transmembrane domain and a Fer domain (a ferlin-specific motif of unknown function). These typical features cluster FLP together with ferlin (PBANKA_1319300) into the ferlin protein family.Ferlins are typically embedded in vesicular membranes via a single, C-terminal transmembrane domain with the N-terminus of the protein facing the cytosol.' Negative attempts to disrupt FLP (RMgm-4576) indicates an essential role during growth/multiplication of asexual stages.
Ferlins are established mediators of calcium-induced membrane fusion and regulated exocytosis in higher eukaryotes
Phenotype
FLP is essential for blood stage development/multiplication (see RMgm-4576) for unsuccessful attempts to disrupt the flp gene). In the 'promoter-swap' mutant RMgm-4577 the promoter of flp is replaced by an 'asexual blood stage’ promoter that is silent (or has a strongly reduced activity) in gametocytes (the promoter of clag, PBANKA_1400600). Analyses of this mutant provide evidence that FLP plays a role in egress of male and female gametocytes from the host red blood cell after activation.
Analyses of the mutant FLP::HA, expressing HA-tagged FLP shows that:
The FLP::HA fusion protein was detected in both schizont- and gametocyte-enriched blood stage lysates revealing the fusion protein at the expected size of 186 kDa. Antibody staining of fixed parasites confirmed expression in schizonts, showing a speckled signal. High abundance of FLP::HA was observed in gametocytes, localizing to widespread, distinct intracellular speckles. Immuno-gold typically localized close to the vesicle membranes, which is in agreement with FLP being a predicted transmembrane protein.
Both male and female gametocytes showed a similar abundance and localization of FLP::HA. FLP-HA relocalizes to the cell peripery during activation of gametocytes.
Additional information
From the paper:
'To assess whether FLP co-localizes with these previously published egress factors G377 (PBANKA_1463000) and PPLP2 (PBANKA_1432400), we generated flp::HA;g377::mCherry and flp::HA;pplp2::mCherry double mutants, in which FLP is endogenously tagged with HA while G377 and PPLP2 are endogenously tagged with mCherry, respectively. Neither of the lines showed co-localization between FLP and either of the two proteins, arguing against FLP localization to the G377-positive OBs (osmiophilic bodies) or to the PPLP2-positive egress vesicles.'
Other mutants |
*RMgm-4578
