Mutant/mutation
The mutant lacks expression of B9 (PBANKA_080810), P36 (PBANKA_100210) and P52 (PBANKA_100220). The genes p36 and p52 are disrupted using a single disruption construct in mutant RMgm-934. In this mutant the b9 gene is disrupted and is drug-selectable marker free.

Protein (function)
See mutant RMgm-932 for a description of the function of B9.
See mutant RMgm-597 for a description of P52 and P36.
All three proteins are 6-Cys family-related proteins and are expressed in sporozoites and/or (young) liver stages

Phenotype
The phenotype is comparable to mutants lacking only B9 (RMgm-932, RMgm-933, RMgm-934).
Gliding motility, hepatocyte traversal and hepatocyte invasion in vitro (HepG2) of mutant sporozoites is similar to wild type sporozoites. Liver stage development is strongly impaired and parasites do not develop into the schizont stage and most invaded parasites cannot be detected anymore at 24 hours after infection.
When Swiss or BALB/c mice were infected by intravenous inoculation of either 1 or 5x104 PbΔb9 sporozoites none of the mice developed blood-stage infections. When C57BL6 mice were infected with a high dose of 5x104 PbΔb9 sporozoites, 10-20% developed a blood-stage infection with a 3-4 days prolonged prepatent period.
These results indicate that that maturation of a few liver stages observed in PbΔb9 and PbΔp52Δp36 is not the result of compensation effects of these 6-Cys family-related proteins.

Additional information

Supporting Figure S7
Generation of P. berghei mutants lacking expression of B9, P52 and P36 (PbΔb9Δp52Δp36)
A. Schematic representation of the construct pL1164 targeting p52 and p36 for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the tgdhfr selection marker (SM). The p52/p36 locus is targeted for deletion in the Δb9 mutant which is selectable marker-free (PbΔb9-sm, line 1309cl1m0cl2; see Figure S8). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). B. Diagnostic PCRs (left) and Southern analyses of separated chromosomes (right) confirm the correct disruption of b9, p52 and p36 in Δb9Δp52Δp36 mutant. Primer pairs and amplicon sizes are shown in A. Selectable marker (M; primers 4501/4502); 5’-integration event (5’; primers L1389/L313); 3’-integration event (3’; primers L4239/4702); p52 and p36 ORF (O; primers L775/L1212); b9 (primers 4737/4738). See Table S1 for the sequence of the primers. For Southern analyses, pulsed field gel-separated chromosomes were hybridized with a 3’UTR pbdhfr probe. The PbΔb9Δp52Δp36 mutant has been generated in PbΔb9Δsm (1309cl1m0cl1). Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 9, the b9 disrupted locus on chromosome 8 and the endogenous dhfr-ts gene located on chromosome 7. C. RT-PCR analysis showing absence of p52, p36 and b9 transcripts in sporozoites of Δb9Δp52Δp36. PCR amplification using RNA of salivary gland sporozoites was performed either in the presence or absence of reverse transcriptase (RT+ or RT-, respectively) using the primers as shown in the left panel (see Table S1 for the sequence of the primers). The P. berghei csp gene was used as a positive control.

Other mutants
RMgm-597: double knock-out mutant lacking expression of p52 and p36
RMgm-932: mutant lacking expression of B9 in the WT background. B9 was disrupted by double cross-over homologous recombination using a plasmid DNA construct.
RMgm-933: mutant lacking expression of B9 in the GFP-Luciferase expressing background. B9 was disrupted by double cross-over homologous recombination using a linear construct generated by the 'Anchor-tagging' PCR-based method.
RMgm-934: mutant lacking expression of B9. The mutant does not contain a selectable marker.
RMgm-936: P.yoelii 17XNL mutant lacking expression of B9 in the GFP-Luciferase expressing background.