Mutant/mutation
The mutant lacks expression of PBANKA_1229000 (PHIST protein)

Protein (function)
In the P. berghei genome sequence  3 phist genes (namely, PBANKA_1145400, PBANKA_1229000 and PBANKA_0700800) were identified via BLAST analysis using PHIST domains and pssm profiles as queries. This result differs from the single P. berghei phist gene described in the original annotation. Orthologs of PBANKA_1145400 and PBANKA_1229000 were identified in the genome nucleotide sequence databases for other rodent malaria parasites; namely, P. yoelii (PY00289 and PY01786, respectively) and P. chabaudi (PCAS_1144900 and PCAS_1229700, respectively). Additional phist genes were not identified in rodent malaria parasites. BLAST analyses using the PHIST domain as queries, identify as reciprocal best hits PBANKA_1229000, the P. vivax PHIST protein PvPHIST/CVC-8195 (PVX_093680), and P. falciparum PF3D7_0801000. This indicates possible orthologous (vertically inherited) relationships and is supported by the observed synteny of adjacent genes, including the ookinete-expressed gene warp, together composing a locus which is conserved across the Plasmodium genus. PBANKA_1229000 is internally localized in the P. berghei genome, but the locus appears to be sub-telomeric in species other than rodent malaria parasites.
The P. berghei PHIST proteins PBANKA_1145400 and PBANKA_1229000 possess similar features to P. falciparum PHIST proteins; namely, signal peptides and PEXEL/HT trafficking motifs; and single PHIST domains, which are divergent in aa sequence with respect to each other. Within the single predicted ORF of PBANKA_0700800 we were unable to identify a signal peptide sequence, and there were no attractive upstream ORFs suggestive of an erroneous gene model. We thus propose that PBANKA_0700800 is a pseudogene. The remaining P. berghei phist genes possess the typical 2-exon gene structure, in which the signal peptide is encoded on the first exon and the PEXEL/HT motif is located within the second exon.


Phenotype
To describe the development of the asexual blood stages of PBANKA_1229000 KO lines we determined the course of infection in Swiss Webster and C57Bl/6 mice following either inoculation with sporozoites or intraerythrocytic stages. An apparent trend (but not statistically different) towards long survival was observed in Swiss Webster mice or C57Bl/6 mice that were intravenously (i.v.) injected with 10(4) and 10(5) schizonts (Swiss Webster mice) or 10(3) or 10(4) mixed asexual stages (C57Bl/6 mice) of PBANKA_1229000 KO parasites. When we monitored the survival of mice after i.v. inoculation of 1,000 P. berghei ANKA wt or PBANKA_1229000 KO sporozoites, we observed a longer survival time of mice infected with PBANKA_122900 KO than those infected with wt parasites, which is in support of the above mild growth delay of asexual blood stages. We did not detect differences in reticulocyte versus normocyte preference or a lower production of merozoites per schizonts

No differences were observed in gametocyte-, oocyst-  and sporozoite-production and inhepatocyte invasion and liver stage development when compared to wild type parasites.

Additional information
RT-PCR was performed using cDNA that was prepared from synchronized asexual stages, gametocytes and mosquito stage parasites. PBANKA_1145400 and PBANKA_1229000 are predominantly transcribed in mature schizont stage parasites, and the transcript levels in schizonts were comparable to the levels of ama1 transcripts (PBANKA_0915000), which was used as a positive marker for schizont stage transcription in addition to the control housekeeping gene, hsp70 (PBANKA_0914410). Low expression of PBANKA_114540 was observed in gametocytes and no transcripts were detected in mosquito stages. Lack of detectable PBANKA_0700800 transcripts supports the above annotation as a pseudogene.

Immunolocalization studies using affinity purified rabbit polyclonal anti-sera against PBANKA_1145400 and PBANKA_1229000 revealed that both proteins are exported to the erythrocyte cytoplasm and observed throughout the intraerythrocytic life cycle. In early asexual stages, both proteins exhibited a punctate, vesicle-like localization in the erythrocyte cytoplasm while in more mature stages the protein distribution appeared more diffuse. Immunofluorescence analysis also suggests that PBANKA_1145400 and PBANKA_1229000 associate with vesicle-like structures, or aggregations, in the erythrocyte cytoplasm. In addition, the proteins co-localize, suggesting they share a common export system, or aggregation, in the rodent malaria parasites. Staining of non-permeabilized cells was negative, indicating that PBANKA_1145400 and PBANKA_1229000 are not exposed on the surface of the infected erythrocyte (data not shown). We did not observe association of either PHIST protein with the erythrocyte membrane.

Phenotype analyses of the blood stages of a mutant expressing an mCherry-tagged version of this protein (RMgm-708) by fluorescence microscopy did not reveal any fluorescence. However, IFA analysis using antibodies against mCherry and against the protein showed export into the host erythrocyte with a punctate localisation pattern. ((Moreira C. et al., 2016; Plos One 11(3):e0152510)).

Other mutants
RMgm-708: mutant expressing an mCherry-tagged version of PBANKA_1229000