Mutant/mutation
The mutant lacks expression of APP and expresses the GFP-Luciferase fusion protein under control of the (schizont specific) ama-1 promoter.

Protein (function)
The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. P. falciparum and P. berghei expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor.
PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. Evidence has been presented for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol.

Phenotype
Blood stages show a strongly reduced growth rate in mice and produce strongly reduced levels of hemozoin compared to wild type parasites.

In a paper published in 2024 (https://doi.org/10.1101/2023.10.24.563045) mutant parasites lacking expression of APP were analyzed during mosquito development. Mosquito development was comparable to wild type parasites).

Additional information

Figure: Generation of P. berghei mutants ∆app (RMgm-813), ∆lap (RMgm-814) and ∆dap (RMgm-815).
(primer sequences can either be found in Lin et al. (2015). J. Exp. Med. or obtained from the Leiden Malaria Research Group)
(A) Schematic representation of the gene-deletion constructs targeting the ORF of genes expressing aminopeptidase P (app), leucyl aminopeptidase (lap) and aspartyl aminopeptidase (dap) by double cross-over homologous recombination and the wt gene loci before and after disruption. The constructs contain a drug-selectable marker cassette (SM; black box) and gene target regions (hatched boxes). Primer positions (arrows) for diagnostic PCRs are shown (see Table S4 for primer sequences and expected product sizes). (B) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm correct disruption of app in ∆app-a and ∆app-b. Northern analysis of blood-stage mRNA (right) confirms the absence of the app transcripts in the ∆app mutants. The following primers were used for diagnostic PCRs: 5' integration (5’ in): L7107/L4770; 3' integration (3’ in): L4771/L7108; SM (hdfhr::yfcuI): L4698/L4699; app ORF: L7109/L7110. Separated chromosomes of ∆app-a and ∆app-b were hybridized using a 3’UTR pbdhfr probe that recognizes the construct integrated into the app locus on chromosome 13, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. Northern blot was hybridized using a PCR probe recognizing the app ORF (primers L7109/L7110) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (C) Diagnostic PCR (left) and Southern analysis of separated chromosomes (center) confirm correct disruption of lap in mutant ∆lap. Northern analysis of blood-stage mRNA (right) confirms the absence of lap transcripts in the ∆lap mutant. The following primers were used for diagnostic PCRs: 5’ in, L6967/L4770; 3’ in, L4771/L6968; SM (hdfhr::yfcu), L4698/L4699; lap ORF, L6969/L6970. Separated chromosomes were hybridized using a 3’UTR pbdhfr probe that recognizes the construct integrated into lap on chromosome 13, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. Northern blot was hybridized using a PCR probe recognizing lap ORF (primers L6969/L6970) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (D) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm correct disruption of dap in ∆dap. Northern analysis of blood-stage mRNA (right) confirms the absence of the lap transcripts in the ∆dap mutant. The following primers were used for diagnostic PCRs: 5’ in, L6975/L4770; 3’ in, L4771/L6976; SM (hdfhr::yfcu), L4698/L4699; dap ORF, L6977/L6978. Separated chromosomes were hybridized using a 3’UTR pbdhfr probe that recognizes the construct integrated into dap on chromosome 8, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. For Northern blot was hybridized using a PCR probe recognizing dap ORF (primers L6977/L6978) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). See Table S4 for primers used for generation of probes.

Other mutants