Mutant/mutation
The mutant lacks expression of both Plasmepsin 4 (PM4) and berghepain-­2 (BP2).

Protein (function)
P. falciparum parasites catabolize more than half of the hemoglobin (Hb)  in the RBC. The amino acids derived from Hb proteolysis are used for  protein synthesis and energy metabolism. The proteolysis of Hb is accompanied by the  release of free heme, which is cytotoxic for the parasite and is rapidly detoxified and  converted into hemozoin (Hz). Therefore, both Hb degradation and heme detoxification are  considered to be essential for P. falciparum survival. The digestion of Hb is a  conserved and semi-ordered process, which principally occurs within the acidified digestive  vacuole (DV). After the initial cleavage by by aspartic and papain-like cysteine endoproteases, Hb unfolds and becomes accessible to further proteolysis by downstream proteases. In the P.  falciparum DV there are four aspartic proteases (plasmepsins I-IV) and two papain-like cysteine  proteases (falcipains 2a,b and 3) capable of hydrolyzing native Hb. Most other Plasmodium species, including P. berghei, have only a single gene encoding a digestive vacuole plasmepsin, plasmepsin4.


Berghepain-2 (BP2) is equivalent to the three P. falciparum digestive vacuole falcipains (papain-like cysteine endoproteases), FP-2 (FP2a, PF3D7_1115700; FP2b, PF3D7_1115300) and -3 (PF3D7_1115400). The gene encoding BP2 has a syntenic location to the three falcipain genes of P. falciparum and also the the single copy vivapain-2 of P. Vivax (PVX_086040).

Both P. falciparum and P. berghei have an additional papain-like cysteine protease encoded by a syntenic gene (falcipain 1, FP1, PF3D7_1458000 and berghepain 1, BP1, PBANKA_132170). Evidence has been presented that this protease is located (and active) in the (apical end) of merozoites and not in the digestive vacuole, suggesting that it has no role in hemoglobin digestion.

Gene disruption studies of hemoglobinases  demonstrated that P. falciparum has developed redundant and overlapping Hb degradation  pathways demonstrating the importance of Hb digestion for the parasite.

Since in P. berghei PM4 is the only DV aspartic protease and BP2 is the single syntenic  ortholog of the 2 P. falciparum DV cysteine endoproteases (FP-2 and -3), the absence of  these enzymes is expected to result in the failure to proteolyse Hb.

Phenotype
Blood stages show strongly reduced growth/multiplication. Blood stages show a strongly reduced hemoglobin digestion and hemozoin formation and parasites can grow and multiply in reticulocytes without the formation of hemozoin.
Gametocyte production is normal. Gametocytes show a strongly reduced hemozoin formation and gametocytes can be formed without detectable hemozoin. Normal ookinete production. Ookinetes show strongly reduced (or absent) clusters of hemozoin (originating from female gametocytes).
Mutant blood stages shows a reduced sensititivity to chloroquine, whereas sensitivity to artesunate is similar to wild type blood stages.

Additional information
Blood-stages of Δpm4Δbp2 have a severely reduced multiplication rate  (2.2–4.6x per 24 hour compared to wild type (wt) rates of 10x per 24 hour). After an initial slow rise of parasite numbers  in Δpm4Δbp2-infected mice, parasitemia can reach levels of up to 50% when mice became  anemic and the RBCs were principally reticulocytes. At high parasitemias mature schizonts (Sz)  were present in the blood, most of which contained 8–12 merozoites. Even though  ring forms of Δpm4Δbp2 were observed in both mature RBCs and reticulocytes, mature trophozoites (Tz)  and Sz were exclusively found in reticulocytes, indicating that Δpm4Δbp2  parasites, while retaining their ability to invade all RBCs, are unable to develop in  normocytes. Schizonts are smaller in size and produce fewer merozoites. Most Δpm4Δbp2-Tz have an ‘amoeboid-like’ appearance with translucent vesicles inside  their cytoplasm and their Tz and Sz have little or no visible Hz While wt-Tz contained large numbers of Hz crystals, more than 40% of  Δpm4Δbp2-Tz had no visible Hz crystals.

This mutant was generated by disruption of pm4 in a mutant lacking the bp2 gene (∆bp2-b; RMgm-809). The Δbp2-b mutant was generated using construct pL1602, which contains the hdhfr::yfcu  SM flanked by two identical 3’UTR dhfr sequences. A recombination event  between the two 3’UTR dhfr sequences results in the removal of the hdhfr::yfcu selectable marker (SM).  Negative selection with 5-fluorocytosine (5-FC) was used to select for parasites that have  removed the SM cassette. Mice infected with Δbp2-b parasites were treated with a daily  single dose of 0.5 ml of 20 mg/ml drug/day for a period of 4 d starting at a peripheral  parasitemia of 0.1–0.5%. Resistant parasites were collected between day 5 and 7 after  initiation of the 5-FC treatment, and cloned parasites were obtained by the method of  limiting dilution. The genotype of mutant Δbp2-b^-sm (line 1619cl1m1cl1) was analyzed by diagnostic Southern  analysis to confirm removal of the hdhfr::yfcu SM. The gene encoding PM4 was  subsequently targeted in this line by standard transfection and drug selection procedures.



Figure: Generation of two independent P. berghei ∆pm4∆bp2 mutants.
(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 wt berghepain 2 (bp2) gene locus, the disrupted bp2 locus in mutant deletion ∆bp2-b and the locus of ∆bp2-b-sm. In the ∆bp2-b-sm the drug selectable marker (SM, black) has been removed by negative selection using 5-FC. Construct pL1602, used to generate mutant ∆bp2-(B) targets bp2 by double cross-over homologous recombination at the target regions (hatched boxes) and contains a positive-negative SM (hdhfr::yfcu) flanked on both sides by 3’pbdhfr sequences (blue boxes). The application of negative selection (5-FC) on ∆bp2-b parasites permits the selection of ∆bp2-b-sm parasites without the SM that has been excised from the genome as a result of a recombination event between the two 3'pbdhfr sequences. Restriction sites and size of the expected fragments in Southern analysis (see B) are shown. (B) Southern analysis of EcoRI digested DNA of wt, ∆bp2-b and ∆bp2-b-sm parasites, confirming correct integration of construct pL1602 in ∆bp2-b and the subsequent removal of the SM cassette in ∆bp2-b-sm after negative selection (see A for the expected sizes of the EcoRI fragments). Hybridization was performed using a probe recognizing 3’UTR of bp2 (primers L5460/L5461). (C) Schematic representation of the genedeletion construct targeting the ORF of plasmepsin 4 (pm4) by double cross-over homologous recombination and the wt gene locus 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. (D) Diagnostic PCR (left) and Southern analysis of separated chromosomes (center) confirm correct disruption of pm4 and bp2 in ∆pm4∆bp2-a and ∆pm4∆bp2-b. Northern analysis of blood-stage mRNA (right) confirms the absence of transcripts of pm4 and bp2 in the mutants. The following primers were used for diagnostic PCRs: 5' integration (5’ in): L5516/L4096; 3' integration (3’ in): L1662/L5517; SM (hdfhr::yfcu): L4698/L4699; pm4 ORF: L5518/L5519; bp2 ORF: L5026/L5027. Separated chromosomes were hybridized using an hdhfr probe that recognizes the construct integrated into pm4 on chromosome 10. Northern blots were hybridized using a PCR probe recognizing the bp2 ORF (primers L5026/L5027) or the pm4 ORF (primers L5518/L5519) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). See Table S4 for primers used for generation of the probes.

Other mutants
RMgm-808: A mutant lacking expression of PM4
RMgm-809: A mutant lacking expression of BP2
See the link for other 'plasmepsin 4' mutants