Mutant/mutation
The mutant expresses Exp2, C-terminally tagged with the glucosamine-activated glmS ribozyme, mRuby and 3xHA. Insertion of the glmS sequence into the exp2 3′-UTR enabled glucosamine-dependent depletion of EXP2 after hepatocyte invasion

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.10.02.510306

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. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro.

Phenotype
Addition of GlcN to ex vivo cultures for 18 hours substantially reduced EXP2 levels relative to untreated controls in the glmS line but did not reduce EXP2 levels in the control parasites lacking the ribozyme. Specifically, treatment with 0.5 mM GlcN reduced EXP2 levels by 23±12% while higher concentrations achieved ~50% knockdown (57±12% or 46±12% at 1 or 2 mM GlcN, respectively), similar to previous reports.
To determine the suitability of the glmS system for protein knockdown in cultured liver stage parasites, we first evaluated whether GlcN levels sufficient to mediate knockdown in blood stage parasites had any effect on proliferation of Huh7 or HepG2 hepatoma cell lines commonly used to cultivate the P. berghei liver stage. We assessed viability of hepatoma cells by quantifying metabolic activity using a resazurin assay and found that proliferation of both Huh7 and HepG2 cell types was not reduced at GlcN concentrations up to 1 mM. These results indicate GlcN can be supplied in hepatoma cultures at concentrations suitable for maximal blood-stage knockdown without host cell toxicity. As both HepG2 and Huh7 were similarly tolerant to GlcN levels, Huh7 cells were chosen for subsequent experiments due to their superior qualities for imaging intracellular parasites.

Additional information
We sought to develop a ligand-based conditional knockdown approach that would enable more precise control of the timing of knockdown. We choose the glmS strategy which involves introduction of a bacterial metabolite-responsive ribozyme sequence immediately downstream of the stop codon of the target gene. Knockdown is initiated by addition of glucosamine (GlcN) to the culture medium, which upon conversion to glucosamine-6-phosphate, activates the ribozyme resulting in cleavage of the mRNA 3’ UTR and transcript destabilization, reducing target protein levels. The glmS ribozyme has been widely used to study protein function in the P. falciparum blood stage where the ability to indefinitely culture the parasite in vitro allows GlcN to be delivered at sufficient concentration and duration to mediate robust knockdown. Indeed, the glmS approach is capable of producing lethal EXP2 knockdown in the P. falciparum blood stage The glmS system has also been shown to function in the P. berghei blood stage where ~2 mM GlcN was required to reduce the expression of a GFP reporter by 50% in culture, although difficulties in maintaining rodent malaria parasites ex vivo beyond a single developmental cycle limit the applicability of knockdown strategies requiring sustained presence of a ligand. Baseline GlcN serum concentrations are in the low nanomolar range in mice and can be increased to only ~2 μM by dietary supplementation, still far below the range observed to activate substantial glmS cleavage in Plasmodium. Thus while sufficient GlcN concentrations are unachievable for in vivo studies with glmS, endogenous GlcN levels are likely too low to significantly active the ribozyme in the rodent host so that tagging important or essential P. berghei genes with glmS should be tolerated. 

To evaluate whether glmS can control P. berghei EXP2 levels, we first introduced an mRuby3-3xHA tag followed by the glmS sequence at the endogenous P. berghei exp2 locus by double homologous recombination. The donor plasmid also contains a cassette for expression of GFP driven by the constitutive Pbhsp70 promoter. Addition of GlcN to ex vivo cultures for 18 hours substantially reduced EXP2 levels relative to untreated controls in the glmS line but did not reduce EXP2 levels in the control parasites lacking the ribozyme. Specifically, treatment with 0.5 mM GlcN reduced EXP2 levels by 23±12% while higher concentrations achieved ~50% knockdown (57±12% or 46±12% at 1 or 2 mM GlcN, respectively), similar to previous reports.  

To determine the suitability of the glmS system for protein knockdown in cultured liver stage parasites, we first evaluated whether GlcN levels sufficient to mediate knockdown in blood stage parasites had any effect on proliferation of Huh7 or HepG2 hepatoma cell lines commonly used to cultivate the P. berghei liver stage. We assessed viability of hepatoma cells by quantifying metabolic activity using a resazurin assay and found that proliferation of both Huh7 and HepG2 cell types was not reduced at GlcN concentrations up to 1 mM. These results indicate GlcN can be supplied in hepatoma cultures at concentrations suitable for maximal blood-stage knockdown without host cell toxicity. As both HepG2 and Huh7 were similarly tolerant to GlcN levels, Huh7 cells were chosen for subsequent experiments due to their superior qualities for imaging intracellular parasites.

To evaluate whether the glmS ribozyme can mediate control of parasite  protein levels in  the liver stage, we next generated a line bearing a 3xFLAG epitope tag followed by the glmS sequence (see RMgm-5258) at the endogenous P. berghei exp2 locus. This plasmid also contains a downstream cassette expressing nanoluciferase (NanoLuc) under the control of the Pbhsp70 promoter to provide a sensitive proxy for monitoring parasite development. Clonal lines  were derived and designated EXP2(glmS) (see RMgm-5258).

Other mutants