Mutant/mutation
The mutant lacks expression of ATPase7
The mutant has been generated using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095

Protein (function)
A 1764–amino acid protein with 10 predicted transmembrane helixes with extensive identity to key P4-ATPase subdomains.
Asymmetric distribution of lipid molecules across the two leaflets of biological membranes is an important feature of eukaryotic cells. To establish asymmetry, eukaryotic organisms ranging from yeast to human encode a specific type of membrane transporters known as flippases that translocate phospholipids to the cytosolic side of membranes in a reaction driven by adenosine triphosphate (ATP). These flippases belong to the P4-ATPase subfamily of P-type adenosine triphosphatases (ATPases)), and CDC50 proteins act as essential P4-ATPase cofactors. Primate malaria parasites encode four putative canonical P4-ATPases, while rodent malaria parasites encode three.

Phenotype
Normal numbers of ookinetes are produced with wild-type gliding motility and morphology. ookinetes invade mosquito midgut epithelium. Evidence is presented that ookinetes are eliminated (within hours) during epithelium traversal.
No oocyst and sporozoite formation

Additional information
Evidence is presented that: 
- Elimination of Δatp7 ookinetes is independent of mosquito complement system 
- Ookinete microinjection into the mosquito hemocoel rescues the Δatp7 defects
- ATP7 is localized in the ookinete plasma membrane, and flippase activity is essential for its function (see below) 
- Phosphatidylcholine uptake defect of Δatp7 ookinetes 
- ATP7 colocalizes and interacts with the CDC50C cofactor in ookinetes
- CDC50C depletion in ookinetes phenocopies ATP7 deficiency during mosquito transmission

ATP7 was expressed in gametocytes, mosquito midgut oocysts, and salivary gland sporozoites but was not detected in asexual blood-stage parasites. ATP7 was detected in the cytoplasm of gametocytes and oocysts but displayed a peripheral localization in sporozoites. Costaining of the atp7::6HA gametocytes with -tubulin (male gametocyte specific) and HA antibodies showed that ATP7 was expressed only in female gametocytes. During zygote to ookinete differentiation in vitro, ATP7 was distributed in both the cytoplasm and the cell periphery from zygote to retort but was mostly localized to the cell periphery of mature ookinetes.

To test whether a conserved flippase motif within ATP7 is required for its function, we generated parasites carrying atp7 mutations that are predicted to compromise its flippase activity but not subcellular localization. It is known that the conserved motif Asp- Gly-Glu-Ser/Thr (DGES/T) in the actuator domain are critical for the catalytic activity of P4-ATPases and that E to Q mutation in these residues abolish the flippase activity. Accordingly, we replaced E210 with Q in the atp7::6HA strain in an attempt to generate an enzymatically inactive mutant designated as atp7m1. A control strain (atp7m2) was also generated with a silent mutation still encoding “DGES”. The E210Q substitution had no effect on the protein level or localization of ATP7 in atp7m1 ookinetes compared to the parental strain. The atp7m1 parasites produced comparable levels of ookinetes as the atp7::6HA strain but were eradicated during midgut traversal  and therefore developed no oocysts in the mosquitoes. These results suggest that ATP7 is a functional flippase and that its activity is required for midgut traversal of ookinetes.

To determine the female inheritance of ATP7 function,  genetic crosses of the Δatp7 with either Δmap2 (male gamete-deficient) or Δnek4 (female gamete- deficient) parasites were performed. Normal numbers of midgut oocysts were observed in mosquitoes on day 7 pi in the Δatp7 × Δmap2 but not the Δatp7 × Δnek4 cross, in agreement with the specific expression of ATP7 in female gametocytes.

From the Abstract:
'Disruption of ATP7 blocks the parasite infection of mosquitoes. ATP7 is localized on the ookinete plasma membrane. While ATP7-depleted ookinetes are capable of invading the midgut, they are eliminated within the epithelial cells by a process independent from the mosquito complement-like immunity. ATP7 colocalizes and interacts with the flippase cofactor CDC50C. Depletion of CDC50C phenocopies ATP7 deficiency. ATP7- depleted ookinetes fail to uptake phosphatidylcholine across the plasma membrane. Ookinete microinjection into the mosquito hemocoel reverses the ATP7 deficiency phenotype.'

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