Mutant/mutation
The mutant lacks expression of P52 and SPECT2 (and expresses GFP under the constitutive hsp70 promoter).

P52(-)GFP parasites were generated by insertion of a GFP expression cassette into the P52 gene locus of wild-type parasites by homologous recombination. P52(-)GFP parasites were sorted using flow cytometry. The selected parasites were further separated from wild-type parasites by limiting dilution. Double knockout parasites were prepared from these parasites by additional disruption of SPECT2 using a pyrimethamine resistant gene as a selectable marker. 

Protein (function)
P52 (P36p) and P36 are members of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins . The proteins are characterised by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organisation throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.
Both genes have been shown to play a role in invasion/development of sporozoites in hepatocytes (see PF3D7_0404400 and PF3D7_0404500).

The spect2/pplp1 gene is a member of a small, conserved family of proteins encoding perforin-like proteins containing membrane-attack complex/perforin domains (MACPF). The P. berghei genome contains 5 PLP proteins. SPECT2/PPLP1 is specifically expressed in salivary gland sporozoites (not in midgut sporozoites) and has a micronemal location (micronemes)

Phenotype
From analysis of B9(-) parasites (RMgm-5266) that have the same phenotype as P52(-) parasites::
Normal numbers of motile salivary gland sporozoites are formed. While 30 wild-type sporozoites were sufficient to establish infection in all rats, even 1 × 10 B9(-) sporozoites were insufficient to establish infection in all rats.B9(-) parasites formed abnormal liver stages (LS) and displayed increased cell traversal activity. The number of B9(-) LS parasites formed in HepG2 cells was 6% that of wild-type parasites at 24 and 48 h post-inoculation (hpi) of sporozoites. The average diameter of the B9(-) LS parasites decreased to approximately 80% that of wild-type LSs at 48 hpi. There was a decreased number of sporozoites entering the hepatocytes by productive invasion and reciprocally, an increased number of sporozoites that continued migration through the hepatocytes. Evidence is presented that B9(-) LS parasites develop in the nucleus of hepatocytes. Evidence is presented that sporozoites lose the capacity to switch invasion modes by disruption of either B9 or P52 (see also mutants RMgm-5267 and RMgm-5268). 
From analysis of B9/SPECT2 DKO parasites (RMgm-5267) that have the same phenotype as P52/SPECT2 DKO parasites: : 
With t he disruption of SPECT2 in B9(-) parasites, the cell traversal activity of sporozoites disappeared. In HepG2 cell culture no LSs of B9/SPECT2 DKO parasites were identified using fluorescence microscopy, demonstrating that all LS parasites inside the nucleus 5 were derived from sporozoites that invaded Hep G2 cells for cell traversal.
Sporozoites in side these cells were still observed in B9 SPECT 2 DKO parasites and the number increased following SPECT2 disruption to a level similar to that of wild type. This result means that sporozoites lacking both cell invasion modes still enter ed HepG2 cells.

Additional information
To demonstrate that B9 is expressed in sporozoites, parasites expressing this gene as a FLAG tagged protein were prepared using a centromere plasmid, followed by immunofluorescent staining of the salivary gland sporozoites. The tag was inserted near the N-terminus of the protein following the predicted signal peptide (because the C-terminus portion is predicted to be cleaved by modification of the glycosylphosphatidylinositol (GPI) anchor. The B9 protein was detected in small particles in the cytoplasm and co localized with P52, suggesting that it is targeted to micronemes as other members of this family that are expressed at this stage.

From the paper analysing B9(-) parasites (RMgm-5266): We also performed cell wounding and membrane repair assays to evaluate the cell traversal activity of B9(-) sporozoites . The number of HepG2 cells damaged by invasion of B9(-) sporozoites was approximately 3 fold higher than that of wild type parasites. These results suggest that the disruption of B9 impaired the switching capacity of sporozoites . More specifically, there was a decreased number of sporozoites entering the hepatocytes by productive invasion and reciprocally, an increased number of sporozoites that continued migration through the hepatocytes . This phenotype of B9(-). is similar to the previously reported phenotypes of P52 disrupted P52(-) and P36 disrupted P36(-) parasites

At 8 hpi wild type sporozoites were observed near the nucleus and inside the PVM, extending towards the nucleus of HepG2 cells. In clear contrast, very few B9(-) sporozoites were observed in the cytoplasm and UIS3 was not detected around them, suggesting that no PVMs were formed around them. Most B9(-) sporozoites were located in the nucleus and were in the process of transform ing in to the LS. UIS 3 was not detected around them. At 24 hpi wild type LS parasites developed into LSs near the nucleus of HepG2 cells At the same time, B9(-) parasites had transformed into LSs inside the nucleus of HepG2 cells and had undergone multiple nuclear divisions. At 48 hpi B9(-) LSs significantly increased in size compared w ith that at 24 hpi, but the progress of their nuclear division varied and their development was obviously delayed compared with that of wild type LSs. No LSs were identified in the cytoplasm at 24 and 48 hpi. An insignificant number of wild type LS parasites were located in side the nucleus of HepG2 cells and, similar to B9 (-) LS parasites, they had no PVMs and exhibited retarded nuclear division. 

Other mutants