Additional remarks phenotype | Mutant/mutation
The mutant lacks expression of G2 and expresses GFP under the control of the G2 promoter
Protein (function)
G2 (glycine at position 2) is predicted to be lipid modified on the glycine residue at position 2 through myristoylation, reflected by the presence of a strong canonical amino-terminal myristoylation motif. This protein is orthologous to the ILP1 of Toxoplasma (TGME49_313380).
Phenotype
Phenotype analyses indicate a role of G2 in morphology and motility of ookinetes and sporozoites, resulting in a loss of infectivity of both ookinetes (reduced oocyst formation) and sporozoites (no invasion of salivary glands).
This morphology phenotype in ookinetes is highly similar to that observed in parasite lines that lack the alveolins IMC1b or IMC1h, and in direct comparison G2-KO ookinetes were indistinguishable from IMC1b-KO or IMC1h-KO ookinetes (RMgm-147, RMgm-601). Null mutants of the alveolins IMC1a, IMC1b and IMC1h display markedly reduced tensile strength. Evidence is presented that the loss of infectivity of the G2 null mutants is unlikely to be caused by a loss of tensile strength.
Additional information
Transcript levels of g2 in P. berghei are over 7.5 times higher in wildtype gametocytes than in gametocytes of null mutants for the DDX6-class RNA helicase DOZI (development of zygote inhibited), which is a strong indicator of translational repression.
Northern analysis of purified asexual blood stages, gametocytes and ookinetes revealed the presence of g2-specific mRNA in ookinetes and gametocytes, with highest levels in the latter, an observation that is fully consistent with the predicted translational repression in the gametocyte.
Expression of the g2 gene product was studied using a transgenic parasite line expressing a full-length, carboxy-terminally GFP-tagged fusion protein from its endogenous promoter (see RMgm-908). These parasites developed normally in mice and mosquitoes and were readily transmitted by infected mosquito bites, demonstrating that the GFP fusion to the G2 protein did not adversely affect parasite development.
Examination of blood-stage parasites revealed very weak cytoplasmic GFP based fluorescence in female gametocytes. Fluorescence levels increased concomitant with ookinete development culminating in mature ookinetes with strong GFP signal concentrated both at the periphery of the cell and in an apically located cap-like structure with a pore in the centre. Retorts (i.e. developing, immature ookinetes) displayed cortical fluorescence only in the ‘ookinete’ portion that protrudes from the spherical zygote, indicating that G2 associates with the pellicle structure and not the plasma membrane. Indeed, immuno-gold labelling of ookinete ultrathin sections saw the large majority of gold particles associated with the pellicle. In young oocysts (4 days), GFP fluorescence had largely disappeared, except for the apical cap structure, and at six days the majority of oocysts lacked any discernible fluorescence. GFP-based fluorescence was again observed in oocysts from around 8-10 days, reaching peak levels during sporulation. The G2::GFP fusion protein concentrated at the periphery of the sporozoites, but in contrast to ookinetes was found associated with neither anterior nor posterior end. This is consistent with the observation that Plasmodium sporozoites, contrary to ookinetes, do not possess a cap-like structure.
Examination of the ultrastructure of ookinetes by electron microscopy showed the presence of an apparently normal pellicle structure and apical collar in the G2 null mutants. A normal looking pellicle structure was also present in G2 null mutant sporozoites. Wildtype P. berghei sporozoites typically possess 16-17 subpellicular microtubules that are arranged in an asymmetrical manner in which all but one closely spaced microtubules occupy approximately two thirds of the perimeter, while a solitary microtubule is positioned opposite. In contrast, the subpellicular microtubule organisation in G2 null mutant sporozoites was notably different with not one, but four equally spaced microtubules occupying approximately one half of the circumference, and the remaining closely-spaced microtubules in the opposite half. These observations indicate that knockout of G2 affects the organization of the subpellicular microtubules.
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