Mutant/mutation
The mutant lacks expression of B9 (PY17X_0811300; PY00153) and expresses the fusion gene GFP-Luciferase under control of the P. berghei eef1a promoter

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. B9 transcripts are found in sporozoites. Evidence is presented for translational repression of b9 transcripts in P. berghei sporozoites. The B9 protein is found in (young) liver stages. For P. falciparum B9 evidence is presented for a location at the plasmalemma membrane of liver stages. P. falciparum and P. berghei mutants lacking expression of B9 abort development during development of young liver stages.

Phenotype
See RMgm-932, RMgm-933 and RMgm-934 for RMgm-P. berghei mutants lacking expression of B9. These mutants have been analysed in more detail than the P. yoelii mutant described here.
Development of PyΔb9 was like WT parasites in the blood (as they develop a 0.5-2% parasitemia after 8 days from an infection initiated by a single parasite) and in mosquito-stage development (normal production of oocysts and sporozoites). Liver-stage development was analysed in BALB/c mice after inoculation of 104 or 2x105 sporozoites by in vivo imaging and analysis of subsequent blood-stage infections. In vivo imaging of mice infected with 2x105 PyΔb9 sporozoites showed faint luminescent signals in the liver at 3hpi that were significantly higher than background values of uninfected mice. At 40hpi none of the infected mice showed luminescence signals above background. A blood-stage infection was only detected in 1 out of 8 mice infected with 2x105 sporozoites with a long prepatent period of 10 days. These results indicate that PyΔb9 parasites, like P. berghei Δb9, are severely compromised in liver stage development.

Additional information

Supporting Figure S8
Generation of P. yoelii mutants lacking expression of B9 (PyΔb9)
A. Schematic representation of the construct pL1438 targeting b9 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 hdhfr::yfcu selection marker (SM). 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 integration of the construct in Δb9. Primer pairs and amplicon sizes are shown in A. Selectable marker (M; primers 4698/4699); 3’-integration event (3’; primers 4239/7260); ORF (O; primers 7261/7262). 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. PyΔb9 mutant has been generated in the reference PyGFP-Luccon which has a gfp-luciferase gene integrated into the silent 230p locus (PY03857 and PY04774) on chromosome 3. Hybridization with the 3’UTR dhfr-ts probe recognizes the integrated construct on chromosome 8, the reporter GFP-Luccon construct on chromosome 3, and the endogenous dhfr-ts gene located on chromosome 2.

Other mutants
RMgm-932: P. berghei 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: P. berghei 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: P. berghei mutant lacking expression of B9. The mutant does not contain a selectable marker.
RMgm-935: P. berghei triple knock-out mutant lacking expression of B9, P52 and P36