Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of Actin II in which His73 has a point mutation (his73Q) preventing methylation
Protein (function)
Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and have also nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the zygote. The protein is highly expressed in male gametes/gametocytes.
Phenotype
Unlike H74 in actin I, around 90% of actin II has a H73 methylation when expressed in insect cells. Methylation of this histidine stabilizes the filament. To confirm that H73 is indeed methylated in vivo, mass spectrometry was carried out on extracts from zygotes and this revealed a signal consistent with methylation.
To abolish the regulation by methylation, we introduced a point mutation in the actin II gene, obtaining the actIIH73Q mutant. This mutant was able to normally form male gametes, as exflagellation took place and ookinete conversion was comparable to the WT. Thus, a histidine at position 73 and its possible methylation are not important for the function of actin II in these early mosquito stages. Next, mice infected with WT and the mutant were offered to A. gambiae female mosquitoes. After 12-13 days, midguts were dissected and labeled for the oocyst capsule protein Cap380, and DNA was stained. All oocysts were counted, even those that were small, and this revealed only a minor decrease in the number of oocysts in the actIIH73Q strain comparing to the WT. However, the mutant oocysts were significantly smaller than the WT. In addition, 80% of WT oocysts contained DNA, while only 42% of the mutant oocysts had visible DNA staining. In the mutant oocysts, no sporozoites were formed
Additional information
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