Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of UIS2. The mutant expresses the yeast Flp recombinase under the control of the trap promoter  and contains a mutated uis2 gene that contains two FRT sites. In the mutant the 3’UTR of TRAP together with DHFR flanked by 2 FRT sites were inserted after the stop codon of uis2. This mutant has been generated by replacement of the endogenous uis2 gene by an 'FRTed' uis2 gene in mutant RMgm-268 that expresses Flp.

The uis2 locus locus is disrupted by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-268). Removal of the FRTed uis2 sequence has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the  uis2 sequence which was flanked by FRT sequences.

Protein (function)
UIS2 is a phosphatase, putative; serine/threonine protein phosphatase, putative.
UIS2 contains a predicted metallo-phosphatase domain enclosed by large N and C-terminal domains.

Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). In this study evidence is presented that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2.

Phenotype
Unsuccessful attempts to disrupt the uis2 gene using standard methods of gene disruption (see RMgm-1364) indicate an essential role of UIS2 during blood stage development.

In the mutant described here the Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence UIS2 expression specifically in sporozoites/liver stages. Removal of the FRTed uis2 sequence has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the  uis2 sequence which was flanked by FRT sequences.

The uis2 cKO parasite developed normally in the mosquito vector. The number of sporozoites (Ssp) in uis2 cKO and wt parasites was very similar. Next, we compared the phosphorylation levels of eIF2α in Ssp obtained from uis2 cKO and from wt parasites by immunoblot using specific antibodies. We found that phosphorylation of eIF2α was greatly enhanced in the absence of the uis2 phosphatase and the mutant sporozoites invaded HepG2 cells as effectively as wt. Nevertheless, the development of the uis2 cKO Ssp in HepG2 cells was profoundly inhibited two days post-infection: the mutants maintained the sporozoite shape while the wt parasites rounded up and developed into spherical liver stages. In addition, the number of  exo-erythrocytic forms (EEFs) and the P. berghei 18S rRNA of the uis2 cKO parasites were profoundly decreased in the liver stages as compared to the wt parasites When the uis2 cKO sporozoites were injected into mice either by mosquito bite or by intravenous injection, only 4 out of 11 mice were infected as detected by Giemsa staining of blood smears. In addition, the pre-patent day was delayed for 3 days in the 4 mice infected with the uis2 cKO sporozoites as compared to wt. We then examined the genotype of the blood and liver stage parasites originated by uis2 cKO sporozoites. The parasites were uis2 (+) and expressed the UIS2 antigen as the wt, indicating that the uis2 locus had not been completely excised by the recombinase FlpL. The incomplete excision in the FlpL/FRT-mediated conditional mutagenesis system has been previously reported. The overall conclusion of these experiments is that uis2 is essential for liver stage development.

Additional information
Evidence is presented that UIS2 phophatase domain dephosphorylated eIF2α-P and has an activity similar to that of the PP2C/PPM family of phosphatases.

Other mutants
 RMgm-1364: Unsuccessful attempts to disrupt the uis2 gene