| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) |
Gene tagging,
Gene tagging
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| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 18452513
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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 2.34
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| Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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| The mutant parasite was generated by |
| Name PI/Researcher | V. Carter; J.T. Dessens |
| Name Group/Department | Department of Infectious and Tropical Diseases |
| Name Institute | London School of Hygiene and Tropical Medicine |
| City | London |
| Country | United Kingdom |
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| Name of the mutant parasite |
| RMgm number | RMgm-116 |
| Principal name | mCHERRY/PbSR/EGFP |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Expression of the mCHERRY/PbSR/EGFP protein in female gametocytes (red and green fluorescence). No expression in blood stages and male gametocytes. Western blot analysis revealed that the C-terminal GFP tag was efficiently cleaved off the expressed PbSR protein (see also RMgm-117). mCherry (RFP) antibody detected full-length mCherry/PbSR protein (predicted to be 175 kDa in size). |
| Fertilization and ookinete | PbSR protein expression was analysed by western blot with anti-mCherry (RFP) antibody. This analysis confirmed that PbSR is present in gametocytes and is also present, at lower levels, in ookinetes. However, PbSR was not detected in oocysts and sporozoites with this assay. Confocal microscopic examination of mCherry/PbSR/EGFP parasites revealed green and red fluorescence throughout the cytoplasm of female gametocytes/macrogametes. This localization was not obviously associated with the periphery of the parasites, or with their host erythrocytes. A similar fluorescence pattern was observed in zygotes and early retorts (i.e. immature ookinetes). In contrast, in mature ookinetes fluorescence had become condensed into one to two cytoplasmic focal spots. Similar spots were observed in young oocysts on the basal side of mosquito midguts for up to 2 days post mosquito infection. PbSR-fluorescence was not co-localized with mitochondrial nor to lysosomal organelles but co-localized with hemozoin clusters. Fluorescence microscopy and immunoelectronmicroscopy show that the protein is associated with crystalloids in the ookinetes. Plasmodium crystalloids are cytoplasmic aggregations of closely packed spherical particles 25–35 nm in diameter. In P. berghei, haemozoin-containing vacuoles accumulate around the edges of the crystalloid inclusion bodies. |
| Oocyst | Not different from wild type |
| Sporozoite | Not different from wild type |
| Liver stage | Not different from wild type |
| Additional remarks phenotype | Mutant/mutation
The mutant expresses an mCherry- and GFP-tagged version of PbSR (PbSR (P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; LCCL domain containing protein CCp3). The endogenous pbsr gene is replaced with an mCherry-tagged (N-terminal) and gfp-tagged (C-terminal) version of pbsr (see also 'Additional information'). The mutant gave rise to large numbers of sporozoites in parasite-infected mosquitoes, and were efficiently transmitted to naïve mice by infected mosquito bites , demonstrating that the fluorescent protein tags did not affect the functionality of the PbSR protein.
Protein (function)
The pbsr gene is a member of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's. PbSR contains four LCCL domains; a LH2 (lipoxygenase homology 2) domain; two SR (scavenger receptor cysteine-rich) domains and a PTX/LamG (pentraxin/laminin-G) domain related to concanavalin A-like module.
Phenotype analyses of mutants lacking expression of PbSR (RMgm-113, RMgm-114) indicate a role of PbSR in the formation of sporozoites in the oocyst.
Phenotype
Analysis of the location of the tagged PbSR by fluorescence microscopy and immunoelectronmicroscopy shows that the protein is expressed in gametocytes and ookinetes and is associated with crystalloids in the ookinetes. Crystalloids are transient organelles that form in developing ookinetes and disappear after ookinete-to-oocyst transition. Plasmodium crystalloids are cytoplasmic aggregations of closely packed spherical particles 25–35 nm in diameter. In P. berghei, haemozoin-containing vacuoles accumulate around the edges of the crystalloid inclusion bodies.
Additional information
Western blot analysis of gametocyte proteins revealed that the C-terminal GFP tag was efficiently cleaved off the expressed PbSR protein (see also RMgm-117). mCherry (RFP) antibody detected full-length mCherry/PbSR protein (predicted to be 175 kDa in size).
Under nonreducing conditions the RFP/PbSR protein migrated as a smear of high-molecular-weight bands, indicating that PbSR forms protein complexes through disulphide bond formation.
Other mutants
RMgm-113: A mutant lacking PbSR
RMgm-114: A mutant lacking PbSR
RMgm-115: A mutant containing a mutated form of PbSR
RMgm-117: A mutant expressing a GFP- tagged version of PbSR
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*RMgm-116
