Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal GFP-Strep tagged version of Cap380
Protein (function)
Cap380 (Plasmodium oocyst capsule protein 380) localises to the P. berghei oocyst capsule, and its depletion leads to oocysts that are gradually eliminated
Phenotype
To investigate sporozoite egress in P. berghei by fuorescence microscopy, we used our Cap380/GFP-Strep parasite line to visualise the capsule.
Assessment of GFP expression by confocal fluorescence microscopy of live Cap380/GFP parasites revealed its expression in oocysts from young oocyst through to sporulation. Fluorescence localised predominantly at the oocyst periphery.
To also visualise sporozoites, we used our parasite line IMC1c/mCherry (RMgm-1122) that stably expresses the sporozoite-expressed alveolin IMC1c fused to mCherry and displays red fuorescence in mature sporozoites. To combine these two fuorescent markers in the same oocyst, the two lines were genetically crossed and the resulting ookinete cultures presented to naïve mosquitoes in membrane feeders, as described resulting in the selection of mutant RMgm-5519.
Additional information
From the paper: 'To study the oocyst capsule during sporozoite egress, we examined Cap380/GFP-Strep oocyst-infected midguts at two weeks post-infected blood feed, when oocyst sporulation is considered to be at its peak. In addition to intact oocysts, live fluorescence microscopy revealed the presence of flattened capsules presumably resulting from oocysts vacated, or being vacated, by their sporozoites. Notably, these empty capsule structures were not visible when midguts were examined by phase contrast (i.e. without capturing GFP fluorescence). This demonstrates that the oocyst does not need to be intact or alive for the capsule to fluoresce, consistent with the capsule constituting a three-dimensional, supramolecular, extracellular matrix that retains water and homeostatic balance.
Other mutants
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