| Additional remarks phenotype | Mutant/mutation
The mutant expresses an mCherrey tagged version of serine repeat antigen 2(SERA2; papain family cysteine protease, putative) and expresses GFP under the control control of the eef1a promoter.
Protein (function)
Plasmodium species possesses SERAs of two major groups, specified as 'cysteine-type SERAs' and 'serine-type SERAs'. Serine-type SERAs contain an active site serine residue instead of the canonical cysteine residue. The genomes of different Plasmodium species contain different numbers of SERA genes. P. falciparum possesses nine SERA protease genes whereas the P. berghei genome contains five. P. falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. In P. falciparum mutants have been generated lacking expression of SERA1, 2, 3, 4 , 7, 8 and 9 without a distinct phenotype in the blood stages (MvCoubrie J.E. et al, 2007, Infection and Immunity, 75:5565-74).
SERA genes of P. berghei are arranged in a tandem cluster that contains two serine-type SERAs (PbSERA1, -2) and three cysteine-type SERAs (PbSERA3, -4 and -5).
Sequence homology and syntenic organisation indicate that PbSERA3, -4 (PB000352.01.0) and -5 (PB000649.01.0) are orthologs of P. falciparum SERA6, -7, -8.
P. berghei encodes two members of the SERAser subfamily, PbSERA1 and PbSERA2. Direct sequencing of cDNA from asynchronous blood stages permitted identification of the complete coding sequences (Genbank accession numbers: EU917224 and EU917225 for PbSERA1 and PbSERA2, respectively). Comparison of the P. berghei orthologs with the founding member PfSERA5 (PFB0340c) illustrates the overall amino acid sequence similarity of ~35% to the human malaria protein.
Phenotype
PbSERA2/mCherry became detectable only at the end of liver stage development, with an intracellular distribution in the parasite. SERA2/mCherry gave a strong signal associated with individual merozoites inside merosomes.
See also mutants RMgm-367, RMgm-368 and RMgm-369 for mutants lacking expression SERA1, SERA2 or both SERAs. The phenotype analyses indicate that SERA1 does not have an essential role throughout the complete life cycle.
Additional information
SERA1 and SERA2 are transcribed during formation of liver stage and blood stage merozoites. No transcription was detected in oocysts/sporozoites.
In mutant RMgm-365 PbSERA1/mCherry was detected in mid and late liver stages, and localized predominantly to the parasitophorous vacuole. In contrast, SERA2/mCherry became detectable only at the end of liver stage development, with an intracellular distribution in the parasite. SERA1/mCherry was not detected in merosomes, in contrast to SERA2/mCherry, which gave a strong signal associated with individual merozoites inside merosomes.
To get further insights into the distribution of PbSERAser proteins, immunofluorescence analysis of late liver stages, using antibodies generated against the C185 terminus of PbSERA1 (anti-SERA1C) or the central domain of PbSERA2 (anti- SERA2M) was performed. In late liver stages, staining with anti-SERA1C antibodies was mostly restricted to the PV membrane (PVM), as shown by co-localization with the PVM marker 1exported protein 1 (EXP1). This pattern was also observed at more advanced stages of development, in cytomeres and fully differentiated merozoite- containing parasites. This distribution is reminiscent of the fluorescence pattern observed in parasites harboring a mCherry tag at the C-terminus of PbSERA1. In contrast, anti-SERA2M antibodies showed a more complex distribution in late liver schizonts and cytomeres, staining both the PVM and more internal structures.
Other mutants
RMgm-102: A mutant lacking expression of SERA8 (PB000649.01.0; PFB0325c), a cysteine-type SERA homologue (serine repeat antigen 8, SERA-8; ECP1, egress cysteine protease 1; PbSERA5)
RMgm-271: A mutant expressing GFP under the control of the 5'-UTR of PbSERA3 (an ortholog of P. falciparum SERA6)
RMgm-272: The mutant expresses in addition to the endogenous SERA3 (the ortholog of P. falciparum SERA6) a TAP-tagged form of SERA3 under control of its own 5'-UTR and the dhfr/ts 3'-UTR
RMgm-365: A mutant expressing the endogenous SERA1 protein fused to the mCherry red fluorescent protein
RMgm-367: A knock-out mutant lacking expression of SERA1
RMgm-368: A knock-out mutant lacking expression of SERA2
RMgm-369: A double knock-out mutant lacking expression of SERA1 and SERA2 |
*RMgm-366
