Asexual blood stage | To expose any impact on export by ER retention of HSP101 masked during in vitro culture of P. falciparum blood stages, we tagged HSP101 and PTEX150 with kER in a transgenic P. berghei rodent malaria expressing DiCre and evaluated the impact of ER retrieval on parasite survival during mouse infection. Similar to P. falciparum knockER mutants, rapamycin treatment ex vivo produced efficient excision and ER retrieval of PbPTEX150 and PbHSP101. After overnight ex vivo culture to allow for tag switching, equal numbers of DMSO or rapamycin-treated parasites were IV injected into naïve mice. Strikingly, while DMSO controls became patent by day two post-injection, neither KDEL-fused PbPTEX150 or PbHSP101 parasites appeared over the course of 10 days before the experiment was terminated. |
Additional remarks phenotype | Mutant/mutation
The mutant expresses HSP101 tagged C-terminal with a KDEL ER-retrieval sequence. In addition it contains a diCre expression cassette containing the two genes Cre59-FKBP and Cre60-FRB placed on either side of the bidirectional eef1a promoter.
Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.10.02.510311
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.
These proteins are also expressed in early gametocytes, mosquito and liver stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in P. berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth.
HSP101 is only expressed in blood stages and not in liver stages. PTEX150 is expressed in both blood and liver stages.
Phenotype
From the paper:
Lack of observable phenotypes in vitro can sometimes be masked by the rich resources available in culturing conditions of P. falciparum. In addition, exported effectors critical for survival in the vertebrate host may be dispensable in culture. Indeed, export-related defects that escape detection in vitro can be exposed in vivo where parasites have to contend with host defenses and more limited nutrient access. To expose any impact on export by ER retention of HSP101 masked during in vitro culture of P. falciparum blood stages, we tagged HSP101 and PTEX150 with kER in a transgenic P. berghei rodent malaria expressing DiCre and evaluated the impact of ER retrieval on parasite survival during mouse infection. Similar to P. falciparum knockER mutants, rapamycin treatment ex vivo produced efficient excision and ER retrieval of PbPTEX150 and PbHSP101. After overnight ex vivo culture to allow for tag switching, equal numbers of DMSO or rapamycin treated parasites were IV injected into naïve mice. Strikingly, while DMSO controls became patent by day two post-injection, neither KDEL-fused PbPTEX150 or PbHSP101 parasites appeared over the course of 10 days before the experiment was terminated.
That ER-retrieval of PbHSP101 compromises function sufficiently to prohibit survival in the mammalian host reinforces the critical role of HSP101 in the PV and indicates that KDEL-mediated depletion of HSP101 from the P. falciparum PV does not sufficiently reduce PTEX activity to impact parasite fitness under less taxing in vitro culture conditions. Collectively, these results provide support for a distinct HSP101 activity in the ER upstream of its function in PVM translocation at the assembled 329 PTEX complex
Additional information
The rapamycin-inducible Cre recombinase (DiCre) established for use in P. falciparum uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves. In the DiCre system, Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin‐binding protein (either FKBP12 or FRB, the rapamycin‐binding domain of the FKBP12‐rapamycin‐associated protein mTOR).
Rapamycin‐induced heterodimerization of the two components restores recombinase activity (rapamycin‐mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn60‐343, called Cre60) each fused to a different rapamycin‐binding protein (FKBP12 and FRB respectively)results in the reconstitution of Cre recombinase activity).
This site–specific recombinase recognises short, 34 bp sequences called loxP sites and catalyses the excision or inversion of the floxed (flanked by loxP) DNA segment.
Other mutants
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