Mutant/mutation
The mutant expresses a mutated form of MyoA with the serine at position 19 replaced by phosphorylation incompatible alanine (S19A)

published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.03.29.437488

Protein (function)
Myosin A is the founding member of unconventional class XIV myosins. These myosins are remarkably small, essentially composed of the actin-interacting ATPase domain, followed by a very short tail domain. Class XIV myosins are exclusively found in the Alveolata branch of eukaryotic organisms, including the Apicomplexa. Plasmodium Myosin A is considered vital for merozoite invasion of erythrocytes. Myosin A is a component of the glideosome of apicomplexan parasites, which is an actin- and myosin-based machine located at the pellicle, between the plasma membrane (PM) and inner membrane complex (IMC), that powers parasite motility, migration, and host cell invasion and egress. It is composed of myosin A, its light chain MLC1, and two gliding-associated proteins, GAP50 and GAP45.
The unusual myosin A of Plasmodium spp. has a unique N-terminal extension which is important for red blood cell invasion by P. falciparum merozoites in vitro and harbors a phosphorylation site at serine 19.
Myosin A is vital for blood stage development/multiplication (merozoite invasion of erythrocytes).

Phenotype
Sporozoite numbers in the hemolymph were slightly enhanced in the S19A parasite line, resulting in an increase in the ratio of hemolymph to midgut sporozoites.
Far fewer sporozoites were found in salivary glands of this parasite line than in the other lines. Reduced motility and migration of sporozoites. Reduced ' force production' by sporozoites.
The S19A parasite line only infected 60% of mice (5 out of 8) via natural transmission by mosquito bite (with a 1 day prolonged prepatent period).

Additional information
Evidence is presented that
- Exchange of 3’myoA by 3’dhfs leads to reduced myoA expression levels resulting in a defect in salivary gland invasion

In order to leave the 3’UTR within the myoA locus intact, we next used a cloning strategy that resulted in selection marker free parasite lines (GIMO transfection; Lin et al., 2011). In a first step, we generated a clonal ama1 promoter swap parasite line (selected by positive selection). In a second step, we replaced the ama1 promoter by the endogenous myoA promoter and the myoA ORF carrying the mutation of interest at the 5’ end of myoA (using negative selection).
 

To assess the role of the unique N-terminus of MyoA on parasite motility, we focused on two amino acids, E6 and S19, which are thought to be especially important as they interact with switch I/II and the converter, respectively and are required to transport actin filaments at maximum speed.
In the paper the following (other) mutants are described:
' we introduced a reverse charge at position 6 (mutant E6R) and replaced serine at position 19 with either a phosphorylation incompatible alanine (mutant S19A) or a phosphomimetic aspartate (mutant S19D)'
' To study the overall importance of the N-terminal extension, we also generated a construct aiming to delete the first 19 amino acids from the N-terminus of MyoA. While we obtained parasites with the point mutations after transfection, we failed to delete the N-terminal extension in four attempts, suggesting that this part of the protein is essential. Interestingly, E6R and S19A mutants grew as well as wild type while S19D parasites grew significantly slower in the blood. In contrast, mutagenesis had no effect on mosquito infection as assessed by the numbers of oocysts present in mosquitoes fed on infected mice (Fig 3D). This indicates that the mutations had no major effect on ookinete formation and migration. Oocysts produced normal numbers of sporozoites in all lines, which were able to exit into the hemolymph.
The S19A parasite line only infected 60% of mice (5 out of 8) via natural transmission by mosquito bite, while all other parasite lines infected 100% of mice'

Other mutants