Additional remarks phenotype | Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of of PTEX88 and expresses GFP under the control of the HSP70 promoter
Protein (function)
Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex.
In this study HSP101, PTEX150, EXP2 could not be genetically deleted in P. berghei. In contrast, the putative thioredoxin-like protein TRX2 and PTEX88 could be deleted. See RMgm-942 for a mutant lacking expression of PTEX88. This mutant shows a reduced growth rate of asexual blood stages.
Phenotype
A mutant lacking expression of PTEX88 (RMgm-942) shows a reduced growth rate of asexual blood stages.
Analysis of the mutant expressing mCherry-tagged PTEX88 shows expression in all blood stgaes (see 'Additional information')
Additional information
PTEX88::mCherry bloodstages grew indistinguishable from wild type parasites, thus providing evidence for a functional complementation and simultaneously ruling out aberrant localization of the tagged protein.
Live imaging revealed that PTEX88::tag predominantly localizes to regions in the parasites cytoplasm of both asexual and sexual blood stages. A singular punctate structure was observed, which localizes to the periphery of the parasite in mature schizonts and free merozoites. In ring and trophozoite stages and gametocytes, the intraparasitic PTEX88::tag pattern is more diffuse. In the majority of the maturing trophozoites and young schizonts, it was observed that PTEX88::tag also localizes to thin, dynamic extensions, which reach from the parasite surface into the erythrocyte cytoplasm. These extensions only appeared when parasites had matured and were never evident in ring stages or sexual stages. Typically, a single extension emerges from one end of the parasite and extends far into the host cell, often following the shape of the red blood cell membrane towards the other end of the parasite. Sometimes two extensions emerged from opposite ends of a parasite. Infrequently, PTEX88::tag extensions emerged from opposite ends of a parasite. Infrequently, PTEX88::tag localized to multiple smaller extensions. The structures appeared dynamic in all cases, displaying waving- and folding-like motions.
See mutant RMgm-922 for the localization of PTEX88 in a mutant expressing HA-tagged version of PTEX88.
The standard pBAT vector was used to generate the gene deletion constructs. This vector contains: (i) a drug-selectable cassette, (ii) a high expressing GFP cassette, (iii) a C-terminal red fluorescent (mCherry) and triple epitope (3xMyc) tag, (iv) two extensive multiple cloning sites (see RMgm-757 for details of this vector). The 3' fragment of PTEX88 was amplified from gDNA using XhoI and KpnI to generate the intermediate construct pPTEX-IM.
A fragment of the carboxy-terminal coding region was amplified from gDNA and fused in frame to the mCherry-3xMyc tag in pPTEX88-IM using SacII and HpaI. The carboxy-terminal coding sequences cloned into the tagging plasmids were verified by commercial Sanger sequencing. All vectors targeting the putative PTEX translocon components were linearized with AhdI and ApaLI before transfection into wild-type P. berghei strain ANKA parasites.
Other mutants
See RMgm-917 for unsuccessful attempts to disrupt P. berghei PTEX88
See RMgm-942 for a mutant lacking expression of PTEX88
See mutant RMgm-922 for the localization of PTEX88 in a mutant expressing HA-tagged version of PTEX88.
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