Mutant/mutation
The mutant lacks expression of PbPl. In addition, it expresses mCherry under the control of the  P. berghei hsp70 promoter.

Protein (function)
The protein contains a predicted signal sequence and a carboxyl terminus that is 32% identical to the human lecithin:cholesterol acyltransferase, a secreted phospholipase.

Phenotype
Oocyst/sporozoite phenotype: Normal oocyst production, normal sporozoite formation inside oocysts. Reduced number of hemolymph and salivary gland sporozoites despite the production of normal numbers of sporozoites within oocysts. These observations indicate a defect in sporozoite egress
Liver stage phenotype: Normal liver parasite load after injection of sporozoites. Normal development of liver stage parasites into maturing liver schizonts. PbPL-KO parasites produced approximately 60% fewer detached cells compared to WT parasites. A significant proportion of detached cells in the KO population showed an aberrant morphology; merozoites were not released into the hepatocyte cytoplasm but remained clustered together. At 54 hpi, before PVM rupture, no significant difference in the number of hepatocytes containing merozoites existed between WT, PbPL-KO and complemented parasites, indicating normal merozoite formation up to this time point. In contrast, at 65 hpi, a time point where the PVM is disrupted in normally developed WT parasites, a significantly higher number of attached hepatocytes containing merozoites was seen for PbPL-KO parasites compared to WT parasites, indicating impaired merozoite release. PbPL-KO parasites that developed to the merozoite stage either did not rupture the PVM at all or this process was significantly delayed (see Additional Information for a method measuring PVM disruption).

Additional information
Immunofluorescence assays (IFA) with  anti-PbPL antiserum revealed that PbPL colocalizes with the parasitophorous vacuole membrane (PVM) resident protein exported protein I (ExpI, PBANKA_092670) in infected hepatocytes 30 and 54 hours post-infection (hpi). At 30 hpi, PbPL was also observed in vesicular structures within the parasite cytoplasm, which may be newly synthesized PbPL located in secretory vesicles being transported to the PVM. The PVM localization was conformed by generating parasites expressing a PbPL-GFP fusion protein under the liver stage specific lisp2 (PBANKA_100300) promoter (RMgm-1226) in which PbPL-GFP also localized to the PVM.

To validate that any potential mutant phenotype is the result of the absence of PbPL, the pbpl gene was introduced into PbPL-KO parasites, taking advantage of the fact that the vector used for generation of the PbPL-KO contains a fusion of the positive drug selectable marker hdhfr (human dihydrofolate reductase) and the negative marker yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase) under the control of the P. berghei eef1α promoter. To allow complementation, the selectablemarker was first removed by treating KO parasites with 5-Fluorocytosine (5-FC), selecting for marker-free PbPL-KO parasites that had undergone homologous recombination between the two 3’dhfr untranslated regions present in the targeting vector flanking the hdhfr::yfcu cassette (Fig. 2A). Successful removal of the selectable marker in a clonal KO parasite line was confirmed by diagnostic PCR. In a next step,  these marker-free PbPL-KO parasites were complemented by transfection of a plasmid, in which expression of a V5-tagged PbPL is under the control of the endogenous pbpl promoter. The correct complementation was confirmed in three clonal lines (CMP1–3) by diagnostic PCR. In addition, PbPL expression during liver stage infection in these complemented lines was demonstrated by IFA using either our anti-PbPL antiserum or an anti-V5 antibody. Evidence is presented thatt the complemented parasites had a wild type phenotype with respect to egress from the hepatocytes.

To further analyze impaired merozoite release in PbPL-KO parasites and a potential role of PbPL in PVM disruption,  GFP-expressing HepG2 cells were infected with WT and PbPL-KO parasites and analyzed their intrahepatic development from the cytomere stage to cell detachment by live-cell time-lapse microscopy. An intact PVM is impermeable to host cell-derived GFP, whereas PVM rupture leads to a rapid GFP influx into the PV. Analysis of GFP influx by live-cell time-lapse microscopy therefore allows the determination of the percentage of merozoite-forming parasites with a disrupted PVM and quantification of the speed of PVM disintegration. Nearly all WT parasites that developed to the merozoite stage were able to disrupt the PVM, with an average time of 70 minutes. In contrast, PbPL-KO parasites that developed to the merozoite stage either did not rupture the PVM at all or this process was significantly delayed.

Other mutants
See RMgm-202 and RMgm-203 for other mutants with a disrupted/mutated PL gene