Mutant/mutation
The mutant lacks expression of Plasmepsin 4 (PM4; plasmepsin IV) and expresses the reporter fusion protein GFP-luciferase under the control of the schizont-specific ama-1 promoter.

Protein (function)
Plasmepsin4 is an aspartic protease of the digestive vacuole and is involved in hemoglobin degradation. P. falciparum has four genes encoding digestive vacuole plasmepsins. Most other Plasmodium species, including P. berghei, have only a single gene encoding a digestive vacuole plasmepsin, plasmepsin4.

Phenotype
Asexual blood stages show a slightly reduced growth rate. A prolonged, less synchronized cell cycle in combination with a delayed and somewhat reduced schizont maturation process results in a slightly reduced multiplication rate in vivo.

The mutant shows a virulence-attenuated phenotype in mice. Blood stages of the mutant are significantly less virulent than wild type P. berghei parasites. Mutant parasites failed to induce experimental cerebral malaria (ECM) in ECM-susceptible mice (i.e. C57Bl/6) and ECM-resistant mice (i.e. BALB/c) were able to clear infections.

In vivo imaging of CD36-mediated sequestration of the schizont stage indicates that CD36-mediated sequestration is comparable to wild type (see also RMgm-32 for the method of in vivo imaging by measuring bioluminescence of the schizonts).

Additional information
After a single infection all convalescent mice were protected against subsequent parasite challenge for at least one year. Real-time in vivo parasite imaging and splenectomy experiments demonstrated that protective immunity acted through antibody-mediated parasite clearance in the spleen. This study shows that a single  gene disruption can generate virulence-attenuated parasites that do not induce cerebral complications and, moreover, are able to stimulate strong protective immunity against subsequent challenge with wild type parasites.

Other mutants
RMgm-314: An independent mutant lacking expression of Plasmepsin4
RMgm-315: An independent mutant lacking expression of Plasmepsin4