Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of PP1. The mutant expresses the yeast Flp recombinase under the control of the trap promoter  and contains a mutated pp1 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 pp1. This mutant has been generated by replacement of the endogenous pp1 gene by an 'FRTed' pp1 gene in mutant RMgm-268 that expresses Flp.

The pp1 locus locus is disrupted by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-268). Removal of the FRTed pp1 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  pp1 sequence which was flanked by FRT sequences.

Protein (function)
PP1 is a serine/threonine protein phosphatase.

Phenotype
Unsuccessful attempts to disrupt the pp1 gene using standard methods of gene disruption (see RMgm-1038, RMgm-1363) indicate an essential role of PP1 during blood stage development.

In the mutant described here the Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence PP1 expression specifically in sporozoites/liver stages. Removal of the FRTed pp1 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  pp1 sequence which was flanked by FRT sequences.

The pp1 mRNA level was significantly decreased in these mutants and PP1 protein was not detected in Pbpp1 cKO sporozoites. The pp1 cKO parasites completed the life cycle in Anopheles mosquitoes and produced similar numbers of midgut and salivary gland sporozoites as the wild type (wt). This is consistent with the low level of Pbpp1 mRNA in the mosquito vector. The mutant sporozoites then developed into liver stages and produced hepatic merozoites in vitro as shown by IFA. PP1 was disrupted in 96.3% (SD +/- 4.6%) of the hepatic schizonts. Quantitative PCR analysis showed that the liver stage development of Pbpp1 cKO Ssp in HepG2 cells and in mice was indistinguishable from the wild type TRAP/FlpL(-) sporozoites. However, the pp1 cKO merozoites exiting the hepatocytes did not infect the mouse blood. Thus, these findings corroborate the essential role of pp1 in the parasite´s erythrocytic cycle. In addition, the levels of phosphorylation of eIF2α in wt and pp1 cKO sporozoites were indistinguishable by immunoblot. In sum, PP1 is not the enzyme that de-phosphorylates eIF2α-P, which is required for liver stage transformation of sporozoites.

Additional information


Other mutants
RMgm-1038; RMgm-1363:Unsuccessful attempts to disrupt the pp1g gene
RMgm-1039: A mutant expressing a GFP-tagged (C-terminal) form of PP1G