Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of GAMA and expresses the reporter GFP-Luciferase under the constitutive eefia promoter.
Protein (function)
GAMA is a micronemal protein with a C-terminal GPI-anchor. P. berghei GAMA consists of 625 aa with a predicted molecular mass of ~71 kDa, (signal peptide cleavage results in a 69 kDa mass). In P. falciparum, two additional asparagine rich repeat regions result in a larger 738 aa protein (predicted molecular weight ~85 kDa, 83 kDa after cleavage of the signal peptide). For P. berghei, SignalP-5.0 predicts a signal peptide with a cleavage site at S21/L22. For P.falciparum, cleavage is predicted at the same locus, A21/L22.
P. berghei and P. falciparum share 44.8% identity across the full-length sequence, with areas of higher conservation found towards the N-terminus (residues 64-347 = 60% identity) and C-terminus (residues 501-700 = 60% identity) split by a central repeat region of low shared identity (residues 348-500, with predicted disorderly structure. The C-terminal transmembrane domain is predicted to be cleaved and appended with a GPI anchor. Downstream of signal peptide cleavage, P. falciparum contains seven cysteine residues prior to the central repeat region and one downstream which are conserved across all Plasmodium species investigated. P. berghei and other rodent infective species of Plasmodium bear an additional cysteine at position 152 in the alignment. GAMA is present in all Plasmodium species and indeed all other intracellular apicomplexans.
Phenotype
Imaging of mature blood-stage schizonts revealed mCherry signal predominantly localized in puncta toward the exterior facing end of merozoites, typical of an apical localization.
Internal localization of mCherry staining throughout ookinetes, frequently accumulated towards the wider apical end.
In oocysts, at day seven, using a long exposure time, weak mCherry signal was visible . No significant increase in mCherry signal was observed until day 11, when a subset of oocysts became brightly mCherry positive. mCherry was first localized within the nascent apical tip of developing sporozoites inside oocysts. mCherry localization becomes less restricted to the apical tip as sporozoites matured replicating the tip-backwards maturation of micronemes within developing sporozoites.
Live-imaging of midgut sporozoites recovered from mechanically ruptured mosquito midguts found GAMA-mCherry to be localized within sporozoites, occupying the same space as cytoplasmic GFP, with a somewhat punctate localization.
Salivary gland sporozoites exhibited a localization of GAMA-mCherry similar to that observed in midgut sporozoites; however, there was an increase in the abundance of protein.
Evidence is presented that GAMA is concentrated at the apical tip of sporozoites where it is then translocated to the sporozoite surface and shed during the process of gliding motility.
Additional information
Further phenotypic analysis of GAMA-mCherry revealed no detectable changes in the number of oocysts per midgut, the number of sporozoites per midgut, the number of sporozoites per salivary gland, and the infectiousness of these sporozoites to mice. In light of the strong phenotypes observed across these life cycle stages in a mutant lacking GAMA expression (ΔGAMA; RMgm-5188) parasites we concluded that the tagging strategy did not interfere with the function of GAMA
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