Selective inhibition of apicoplast tryptophanyl-tRNA synthetase causes delayed death in Plasmodium falciparum

The malaria parasite Plasmodium falciparum relies on efficient protein translation. An essential component of translation is the tryptophanyl-tRNA synthetase (TrpRS) that charges tRNAtrp. Here we characterise two isoforms of TrpRS in Plasmodium; one eukaryotic type localises to the cytosol and a bacterial type localises to the remnant plastid (apicoplast). We show that the apicoplast TrpRS aminoacylates bacterial tRNAtrp while the cytosolic TrpRS charges eukaryotic tRNAtrp. An inhibitor of bacterial TrpRSs, indolmycin, specifically inhibits aminoacylation by the apicoplast TrpRS in vitro, and inhibits ex vivo Plasmodium parasite growth, killing parasites with a delayed death effect characteristic of apicoplast inhibitors. Indolmycin treatment ablates apicoplast inheritance and is rescuable by addition of the apicoplast metabolite isopentenyl pyrophosphate (IPP). These data establish that inhibition of an apicoplast housekeeping enzyme leads to loss of the apicoplast and this is sufficient for delayed death. Apicoplast TrpRS is essential for protein translation and is a promising, specific antimalarial target.

destinations 15,19 . Although translation in the mitochondrion is not well understood, it is thought that aminoacylated tRNAs are imported into the organelle as observed in some other protist parasites [20][21][22] .
The tryptophanyl-tRNA synthetase (TrpRS) is a class I aaRS characterised by a Rossman-fold catalytic domain containing canonical HIGH and KMSKS motifs 23,24 . The P. falciparum nuclear genome contains two putative TrpRS genes-we show here that one encodes an apicoplast targeted TrpRS with preferential activity for charging bacterial tRNA while the other transcribes a cytosolic TrpRS with preference for eukaryotic tRNA. Recently, the ligand-free and ligand-bound crystal structures of this latter, cytosolic TrpRS were solved 25,26 . Despite the high degree of structural similarity to the Homo sapiens orthologue, differences in conformational changes upon ligand binding and insertions within the parasite protein were observed which might allow selective inhibition 26 . In the current study, we investigated several putative inhibitors of TrpRS and show that one, indolmycin, specifically inhibits the apicoplast TrpRS and kills parasites in culture. Indolmycin produces a delayed death phenotype characteristic of apicoplast inhibitors, disrupts apicoplast segregation, and its growth inhibition is reversible by complementing apicoplast metabolism through exogenous addition of the apicoplast product isopentenyl pyrophosphate (IPP). These results confirm the apicoplast-specificity of the delayed death phenomenon, and highlight the potential of the apicoplast Pf TrpRS as a potential antimalarial drug target.

Results
Nuclear-encoded P. falciparum TrpRS isoforms are localised to the cytosol and the apicoplast.
Results of bioinformatics analyses revealed that the P. falciparum nuclear genome encodes two putative TrpRS genes. Located on chromosomes 12 and 13, the predicted mature coding sequences are 1680 bp (PlasmoDB ID: PF3D7_1251700) and 1899 bp (PlasmoDB ID: PF3D7_1336900). These genes, which we refer to hereafter as TrpRS api and TrpRS cyt respectively, were used in searches to identify further TrpRS genes that were used to construct multiple sequence alignments. Maximum likelihood phylogenetic trees inferred from these alignments revealed that the two Plasmodium TrpRSs have very different evolutionary origins (Fig. 1a). While the TrpRS cyt is grouped with other eukaryotic cytosolic TrpRS enzymes, the TrpRS api clusters with bacterial and other plastid TrpRS sequences (Fig. 1a). This bacterial origin is consistent with the presumed endosymbiotic origin of the apicoplast-localised TrpRS, but we cannot confidently determine whether the apicoplast TrpRS api is derived specifically from the original cyanobacterial ancestor of plastids. This alignment did not make for robust phylogenetic reconstruction, and other TrpRS phylogenies have previously been subject of conflicting interpretation. Our phylogenies were unstable based on species selection, tree-building methods, or on the inclusion or exclusion of regions of sequence alignment. Nonetheless, the TrpRS api consistently grouped with other bacterial TrpRSs, while the TrpRS cyt consistently grouped with other eukaryotic, cytosolic TrpRSs, consistent with their assumed evolutionary ancestry.
Apicoplast-targeted products are characterised by an N-terminal trafficking sequence that consist of a signal peptide for trafficking to the ER and a transit peptide for post-translational protein routing to the apicoplast 27 . Manual inspection of the two TrpRS isoforms, as well as prediction using the PlasmoAP and PATS software revealed that TrpRS api bears an approximately 60 amino acid apicoplast trafficking leader sequence (Fig. 1b) which is not conserved with other TrpRSs outside the genus. Although TrpRS cyt also bears a divergent N-terminal sequence, this region has been previously shown to represent an unusual alanyl-tRNA synthetase editing domain (AlaX) followed by a eukaryote-specific extension 25,26 (Fig. 1b).
Subcellular localisation of TrpRS api and TrpRS cyt was verified by fusing the first 60 amino acids of each protein isoform to a C-terminal GFP and expressing these fusion-proteins in P. falciparum blood stage parasites. Immunoblot confirmed the expression of the fusion proteins. The TrpRS cyt protein had a motility consistent with the expected mass (33.6 kDa), while the TrpRS api exhibits a doublet band, consistent with the presence of products lacking the signal peptide but before (31.8 kDA) and after (29.4 kDA) processing of the transit peptide (Fig. 1c). Identification of organelle-specific protein localisation was carried out both via live cell fluorescence microscopy and immunofluorescence assay (IFA) using an antibody raised against an apicoplast-targeted protein, ACP 28 . Figure 1d shows colocalisation of GFP with endogenous ACP in the TrpRS api parasite line, validating an apicoplast localisation of the protein. This signal is distinct from the mitochondrial structure (Fig. 1d) that undergoes morphological transformations similar to the apicoplast throughout the parasite life cycle 29 . Analysis of the TrpRS cyt -GFP expressing parasite line revealed a cytosolic signal distribution with fluorescence excluded from the nucleus and the digestive vacuole (Fig. 1d), consistent with a previous report of localisation based on TrpRS cyt antiserum 25 . These data indicate that Plasmodium parasites possess two TrpRS enyzmes-one bacterial-like TrpRS targeted to the apicoplast and one eukaryotic-like TrpRS targeted to the cytosol. lacking the Plasmodium-specific N-terminal extension but with the eukaryote-specific sequence retained 26 was also expressed and purified. SDS-PAGE and mass spectrometry results confirm the production of soluble TrpRS api and TrpRS cyt proteins, isolated by Ni-NTA affinity chromatography ( Supplementary Fig. 1). TrpRS api and TrpRS cyt aminoacylation activities were determined by measuring the amount of 3 H-and 14 C-labelled tryptophan incorporated into the aminoacylated Trp-tRNA trp product. It is known that inorganic pyrophosphate (PPi) generated during the aminoacylation reaction inhibits catalysis by a number of aaRSs 30 . In this study, aminoacylation was carried out in the presence of PPiase to prevent build-up of PPi.

Characterisation of TrpRS api and TrpRS
The apparent kinetic parameters of P. falciparum TprRS enzymes were investigated for three substrates, ATP, tryptophan, and tRNA, by fitting the initial rate of aminoacylation as a function of substrate concentration to the Michealis-Menten equation (Table 1 and Supplementary Fig. 1). TrpRS cyt charged tryptophan and ATP with an apparent Km of 15.5 ± 0.6 and 622 ± 0.3 μ M, respectively. We then tested for TrpRS aminoacylation using tRNA Scientific RepoRts | 6:27531 | DOI: 10.1038/srep27531  from E. coli, S. cerevisiae, and P. falciparum. Charging of the eukaryotic-type Pf TrpRS cyt was more efficient using yeast tRNA (K m app = 0.23 ± 0.05 μ M) compared to E. coli tRNA, but enzyme activity was considerably increased when the assay was carried out using P. falciparum tRNA (K m app = 0.02 ± 0.004 μ M). Consistent with the bacterial origin of the TrpRS api , the reverse pattern was seen for this enzyme: it preferentially charged bacterial tRNA (K m app = 0.12 ± 0.03 μ M) over yeast tRNA but aminoacylated its natural substrate, Plasmodium tRNA, with 3-fold higher efficiency. Furthermore, the enzyme actively charged tryptophan (K m app = 5.3 ± 1.2 μ M) comparable with bacterial enzymes 31 . We also attempted to determine the K m app of TrpRS api for ATP, but inconsistency between biological replicates prevented us from establishing an accurate estimate.
Comparison of the kinetic values between the two enzymes showed a profound difference when charging P. falciparum tRNA, with the rate of aminoacylation of TrpRS cyt for P. falciparum tRNA observed to be 15-fold greater than that of TrpRS api .
These findings show that the two Pf TrpRS enzymes efficiently catalyse the aminoacylation of tryptophan onto its substrates. Furthermore, consistent with the substrate they encounter within the cell, the TrpRS api preferentially charges tRNA from a bacterial source, whereas TrpRS cyt favours tRNA from a eukaryotic source.

TrpRS api complements a TrpRS mutant E. coli. Functional complementation of an E. coli TrpRS mutant
was carried out to further validate the activity of recombinant P. falciparum TrpRSs. The E. coli KY4040 strain harbours an unstable TrpRS that requires high concentrations of L-tryptophan and ATP to function 32,33 . KY4040 transformed with an empty expression vector was viable in permissive growth conditions containing 0.05 μ g/μ L tryptophan (Fig. 2a), but not in repressive conditions lacking tryptophan (Fig. 2b). This defect in KY4040 was not restored by expression of the eukaryotic-type Pf TrpRS cyt , but was successfully complemented by expression of Pf TrpRS api , which restored bacterial growth in medium lacking tryptophan (Fig. 2b). These findings are in agreement with our kinetic data on aminoacylation, indicating that the Pf TrpRS api but not Pf TrpRS cyt can efficiently recognise and charge bacterial tRNA. [34][35][36][37][38] were identified via the literature and screened according to their pharmacokinetics properties for inhibition studies in P. falciparum (Supplementary Table 1). The tryptophan analogue and natural product, indolmycin, is a well-characterised TrpRS inhibitor and was selected for testing. To find additional compounds with similar structures, analogues of indolmycin were identified via compound similarity searches-two additional compounds STK505786 and PH000586 were also selected for testing. A previously characterised bacterial TrpRS inhibitor, referred to as SPECS_C1 38 was also selected for testing against P. falciparum growth ( Table 2). Both indolmycin and SPECS_C1 displayed low cytotoxicity to mammalian cells [36][37][38] . The compounds were tested against P. falciparum according to an inhibition assay described previously 39 .

Bacterial-type TrpRS inhibitors arrest intraerythrocytic P. falciparum. TrpRS inhibitors
Although indolmycin treatment of P. falciparum 3D7 and W2mef did not result in growth inhibition during the first life cycle, substantial growth arrest was observed at the succeeding replicative cycle (IC 50 = 1.7 ± 0.5 μ M; Fig. 3a-c and Table 2). This inhibition of parasite growth only in the second cycle after treatment is characteristic of apicoplast inhibition 8 . The delayed death phenotype was confirmed from microscopic analysis of Giemsa-stained parasites treated with indolmycin. Even at the highest concentration of indolmycin assayed, parasites were able to progress through the first life cycle exhibited through normal cell division. In the next life cycle, an apparent morphological defect manifested as pale and vacuolated parasite forms were observed (Fig. 3c). These findings suggest that the bacterial-TrpRS inhibitor, indolmycin, kills P. falciparum by targeting the apicoplast housekeeping function. In an effort to understand what structural features of indolmycin might be important for biological activity, commercially available analogues were sourced and tested against malaria parasites. It was determined that replacing the oxazolone side chain of indolmycin with an α -hydroxyester resulted in a very large  Table 2). Due to the paucity of available, close analogues of indolmycin, finer details on structure-activity relationship requirements for optimum activity would require a synthetic investigation.
One of the analysed compounds, SPECS_C1, killed parasites at a low μ M range (IC 50 = 14.3 ± 3.3 μ M Table 2), but resulted in lysis of erythrocytes at similar concentrations, so its specificity for TrpRS in this system is doubtful. SPECS_C1 was identified as a TrpRS inhibitor using in silico structure-based virtual screens, and aside from analysis of its effects on bacterial culture turbidity, the compound has not been characterised against a wide range of eukaryotic culture conditions 38 .

Increasing tryptophan concentration rescues indolmycin-induced inhibition of P. falciparum.
Given the structural similarity between indolmycin and tryptophan, we hypothesise that the compound interferes with tryptophan processing in the parasites. To test this hypothesis, proliferation of parasites treated with indolmycin and cultured at different concentrations of tryptophan were determined from growth-response curves. While parasite viability was not affected in a tryptophan-depleted media source, the addition of indolmycin severely affected parasite growth, resulting in an 8-fold reduction in IC 50 (Fig. 3d). However, an increase in tolerance to indolmycin toxicity that is directly proportional to the concentration of tryptophan supplemented in the media was observed. The addition of tryptophan to concentration 10x and 100x greater than that found in normal medium resulted in a 1.7-and a 22-fold increase, respectively, in IC 50 (Fig. 3d). Negative controls with changed glutamine concentrations in the growth medium had no effect on P. falciparum sensitivity to indolmycin. (Fig.  S2). These findings are consistent with indolmycin competing with tryptophan incorporation in the parasites.
Indolmycin-treatment abolishes the parasite apicoplast. The delayed death inhibition described above suggests that indolmycin affects growth of P. falciparum by targeting the apicoplast. Recently, it has been shown that apicoplast inhibition can be rescued by supplying parasites with abundant exogenous source of an essential apicoplast metabolite, IPP 3 . In this study, IPP rescue was used as a tool to characterise indolmycin-induced parasite toxicity and to validate the target of the compound.
A SYBR Green assay was first used to assess the viability of indolmycin-treated 3D7 P. falciparum following IPP supplementation. Based on nuclear replication, we observe a complete rescue of parasite growth when challenged with indolmycin and cultured in the presence of IPP ( Fig. 3a and Table 2) even at concentrations of indolmycin well above those normally required to kill all parasites in the absence of IPP. Another indication of apicoplast destruction is the loss of the organellar genome. Genomic analysis was performed to further validate the loss of the apicoplast by indolmycin-treatment. gDNA was extracted at various time course post-drug challenge and genes encoded in the nucleus (GAPDH), mitochondria (cytb3), and apicoplast (tufA) were amplified via PCR. While parasites were able to grow and proliferate normally for at least ten replicative cycles, the apicoplast genome of indolmcyin-challenged parasites was gradually but completely abolished whereas nuclear-and mitochondrial-encoded DNA remained abundant (Fig. 4a).
Apicoplast loss after indolmycin treatment was then visualised via fluorescence microscopy of a transgenic parasite line expressing DsRed and GFP fused to the leader sequences of apicoplast and mitochondrion markers acyl carrier protein (ACP) and citrate synthase (CS), respectively. Parasites treated with both indolmycin (100 μ M) and 200 μ M IPP proliferated as well as untreated controls, indicating that an apicoplast process is the sole target of indolmycin at these concentrations. Figure 4b shows that, in contrast to untreated samples that exhibit distinctive apicoplast morphology, indolmycin treatment results in apicoplast disruption during the second cycle, with apicoplast proteins dispersing into numerous puncta in the cytoplasm. The mitochondrion maintains its morphological transformation throughout the assay, suggesting that the parasites were viable and replicating normally.
Taken together, these findings show that the parasites were able to bypass the indolmycin-induced toxicity in the absence of a functional apicoplast by using exogenous IPP to maintain isoprenoid synthesis. Together these data indicate that indolmycin affects parasite growth by specifically targeting tryptophan utilisation in the apicoplast.

Indolmycin inhibition of TrpRS aminoacylation.
The effect of indolmycin on the formation of charged tRNA trp by TrpRS api and TrpRS cyt was determined using the established functional assay for TrpRS activity. Figure 5a reveals that at higher concentrations of indolmycin, tRNA trp formation by TrpRS api was reduced to background levels. Aminoacylation of TrpRS cyt on the other hand was only slightly inhibited. These results are consistent with the ex vivo proliferation assays which support an apicoplast target for indolmycin.
The nature of TrpRS inhibition by indolmycin was explored using the recently determined crystal structure of Pf TrpRS api homologue (5DK4; 33% sequence homology) from Bacillus stearothermophilus with bound indolmycin, ATP, and magnesium 40 superimposed with the structure of the cytosolic Plasmodium TrpRS (4J75; Fig. 5b-d) with bound charged tryptophan 26 . A structural alignment was performed by overlaying the indole moiety of indolmycin with that of tryptophan. Comparing the tryptophan binding pockets of BsTrpRS and the (indolmycin-insensitive) cytosolic Pf TrpRS in this way reveals significant structural differences. In the case of BsTrpRS, the indole nitrogen of indolmycin forms a hydrogen bond with D132, while in Pf TrpRS the indole nitrogen of the tryptophan derivative is hydrogen bonded to the sidechain hydroxyl groups of Y306 and Q341 (Fig. 5d). These amino acids reside on separate structural elements surrounding the binding site in each enzyme, with (Bs)D132 part of the α -helix directly below the binding pocket and (Pf )Y306 and Q341 part of the β -sheet adjacent to the binding site. Inspection of the binding mode of indolmycin in this alignment provides some clues as to why Pf TrpRS cyt is not sensitive to inhibition by the compound. In particular, the alignment shows potential steric clashes between the methyl group of indolmycin and the Pf TrpRS cyt protein backbone in the region of the R309, and between the oxazolinone ring and the sidechain of S343 (Fig. 5d).
The program I-TASSER was used to create a three dimensional structure of the apicoplast Pf TrpRS, using the crystal structure of BsTrpRS as a template (Fig. 5e-g). Predicting the Pf TrpRS api structure in this way resulted in a model with an iTasser C-score = − 2.42 which is lower than the score that correlates with ~90% prediction accuracy for global topology 41 . Nonetheless, the predicted structure shows residues that serve as determinants for indolmycin binding in BsTrpRS to be a similar position in the parasite apicoplast protein-with (Bs)H43, D132, and Q147 in equivalent positions to (Pf )H57, D232, and Q247, respectively (Fig. 5g). Taken together, these results suggest that whereas the cytosolic Pf TrpRS generates steric clashes that preclude indolmycin binding, the Pf TrpRS api shares the BsTrpRS indolmycin-binding residues, allowing inhibition of tRNA trp aminoacylation and, therefore a block in apicoplast protein translation.

Discussion
The ongoing emergence and spread of antimalarial drug resistance creates a serious need for the identification of new molecular targets and compounds with distinct antimalarial activities. Inhibitors of protein translation fulfil this requirement, and several tRNA synthetases have been advanced as targets of potential antimalarial compounds 11-16 . In the current study, we have shown that intraerythrocytic stage malaria parasites have two TrpRS isoforms; TrpRS cyt localises to the cytosol and preferentially aminoacylates eukaryotic tRNA while TrpRS api is targeted to the apicoplast and efficiently aminoacylates bacterial tRNA. Performing aminoacylation reactions with tRNA isolated from P. falciparum increased the catalytic efficiency of TrpRS cyt and TrpRS api enzymes. However, because the translation machinery differs between prokaryotic and eukaryotic enzymes, the assay was limited by our inability to specifically isolate apicoplast tRNA. Apicoplast tRNA represents a minor fraction of total purified tRNA and this is likely reflected in the kinetic values obtained for the TrpRS api enzyme. It is known that eukaryotic and prokaryotic tRNAs have different identity elements 42 that promote substrate-specific binding. This is consistent with the differential activity observed for these substrate using both the apicoplast and cytosolic TrpRSs. The poor cross-recognition between the eukaryotic and bacterial enzymes and substrates explains the persistence of two TrpRS isoforms in Plasmodium spp. Though some dual-localised aaRSs have adapted to recognise both organellar and nuclear-encoded tRNAs 15,19 the Pf TrpRS cyt recognises the bacterial type tRNA trp poorly, necessitating retention of compartment-specific TrpRS enzymes in Plasmodium.
The retention of a bacterial TrpRS api in Plasmodium spp. creates opportunities for parasite-specific inhibition. We explored this susceptibility by testing inhibitors of bacterial TrpRSs against Plasmodium growth-indolmycin was the most promising of these. Indolmycin is a natural product and tryptophan analogue isolated from Streptomyces griseus 43,44 , that affects growth of various gram-positive and -negative bacteria 31,37 . One of the tested compounds, SPECS_C1 was discovered from a high-throughput virtual screening of the binding affinity of a compound library against a predicted structural model of Staphylococcus epidermis TrpRS, followed by biochemical assays assessing the effect of hit compounds on enzymatic activities 38 . While SPECS_C1 was shown to display dose-dependent inhibition of S. epidermis and S. aureus TrpRS activity in vitro, the observed lack of inhibition against E. coli suggests that the effect is species-specific. The apparent lysis of human erythrocytes in our assays at parasite-killing concentrations suggests that further variation to this compound would be necessary before any parasite-specific activity could be investigated.
Previous studies have reported that indolmycin kills bacteria by competitive inhibition of the tryptophanbinding pocket of TrpRS 31,37 . Furthermore, two independent studies comparing aminoacylation of the indolmycin-resistant TrpRS to the sensitive gene from S. coelicolor and B. stearothermophilus showed that indolmycin affects tRNA trp formation as demonstrated by the kinetics of competitive inhibition of tryptophan 31,37 . Given the structural similarity between indolmycin and tryptophan, competitive inhibition can also be deduced as the underlying antimalarial mode of action of the compound. This is supported by our data showing that levels of exogenous tryptophan modulate P. falciparum sensitivity to indolmycin.
In previous reports exploring the structural basis for the selectivity of indolmycin for bacterial but not eukaryotic TrpRSs, (Bs)H43 was implicated in indolmycin sensitivity. Replacement of this amino acid with asparagine resulted in resistance to indolmycin 31,35 , potentially by disruption of the hydrogen bond with the oxazolinone ring. Our structural alignment of Pf TrpRS cyt with BsTrpRS shows structural differences surrounding the tryptophan-binding pocket that may underlie the differential binding of indolmycin by each enzyme. The combination of our phylogenetic analysis and homology modelling results suggests that the apicoplast TrpRS may be structurally homologous to bacterial TrpRSs and therefore shares their sensitivity to indolmycin.
Consistent with the relationship of the Pf TrpRS api to bacterial TrpRSs we found that indolmycin specifically inhibits Pf TrpRS api but not Pf TrpRS cyt , and specifically ablates apicoplast function. The only essential product of the apicoplast in blood stage P. falciparum is the isoprenoid precursor IPP 3 . Complete rescue of parasite growth inhibition by indolmycin using the apicoplast metabolite IPP suggests that apicoplast protein translation is the only important target of indolmycin in these blood stage parasites. The disappearance of the apicoplast genome and the disruption of apicoplast morphology in these indolmycin treated parasites is further proof of the apicoplast target of this compound.

Materials and Methods
P. falciparum culture. 3D7 and W2mef P. falciparum were maintained in a continuous culture consisting of human erythrocytes (O + , 2% haematocrit) resuspended in RPMI 1640 with 3.6% sodium bicarbonate and 5% Albumax (complete media), and incubated in a gas mixture consisting of 5% CO 2 , 1% O 2 , and 94% N 2 at 37 °C 45 . Parasitemia was determined every 48 hours through microscopic examination of blood smears fixed in absolute methanol and stained with 10% Giemsa solution.
Transfection of P. falciparum was carried out by electroporation as described previously 46 . Briefly, 100 μ g of purified plasmid DNA was resuspended in warm TE buffer and cytomix (120 mM KCl, 0.15 mM CaCl 2 , 2 mM EGTA, 5 mM MgCl 2 , 10 mM K 2 HPO 4 /KH 2 PO 4 pH 7.6, 25 mM HEPES pH 7.6). Synchronous ring-stage parasites (5-10% parasitemia) were added to the plasmid and electroporated at 0.31 kV and 950 μ F in a 0.2 cm cuvette. Transfectants were cultured in complete media with 20 nm WR92210, and viable parasites were observed in cultures after three weeks.
To monitor organellar morphology in indolmycin treated parasites, we used a double transfectant parasite line (3D7) expressing the apicoplast acyl-carrier protein (ACP) fused to DsRed and mitochondrial citrate synthase (CS) fused to eYFP 29 . These parasites were a kind gift from Professor Geoffrey McFadden (The University of Melbourne). The deleterious effect of drug treatment was overcome by maintaining the parasites in complete media supplemented with 200 μ M IPP following a method described previously 3 .

Bioinformatic analyses. Full-length sequences of putative TrpRS api (PlasmoDB ID: PF3D7_1251700) and
TrpRS cyt (PlasmoDB ID: PF3D7_1336900) were obtained from PlasmoDB 47 . Prediction of an apicoplast-trafficking presequence was performed using PlasmoAP 47,48 and Predict Apicoplast-Targeted Sequences (PATS) 49 . Multiple sequence comparison of the TrpRS protein in Plasmodium species and other organisms was carried out using clustalOmega 50 and were manually adjusted and edited using Jalview 51 . Maxiumum likelihood trees were inferred from this alignment using PhyML 52 with 1,000 bootstrap replicates performed.

Cloning of plasmids. N-terminal fragments of TrpRS api
1-180bp and TrpRS cyt 1-180bp were synthesised with XhoI and XmaI restriction sites (BioBasic Inc), and cloned into pGlux for episomal expression in 3D7 P. falciparum. This vector contains a green fluorescence protein (GFP) and the human dihydrofolate reductase (hDHFR) that confers resistance to WR99210. Plasmid sequences were confirmed by Sanger sequencing (Australian Genome Research Facility Ltd).
A version of TrpRS api 180-1683bp that lacks the N-terminal trafficking sequence was codon optimised, synthesised with BamHI and HindIII restriction sites (BioBasic Inc), and cloned into and pColdIV for complementation in E. coli KY4040 and pET-21a(+) that allow in-frame fusion of a C-terminal polyhistidine tag for bacterial protein expression. Construct sequences were confirmed by Sanger sequencing. For comparison of enzymatic activity, a glycerol stock of TrpRS cyt 687-1896bp was obtained from Wim G. Hol at the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and Wes Van Voorhis at the Center for Emerging and Re-emerging Infectious Diseases (CERID).
Microscopy. Imaging of live cells was performed by staining infected erythrocytes with 20 nM MitoTracker ® (Thermo Fisher Scientific) which stains the mitochondrion and 0.5 μ g/mL of DAPI to visualise the nucleus.
Briefly, 500 μ L of parasite culture was pelleted, resuspended in MitoTracker ® , and washed twice with PBS (1x), before final staining with DAPI. For visualisation of ACP-DsRed/CS-eYFP 3D7 P. falciparum, nuclear staining was carried out prior to fluorescence microscopy.
Immunofluorescence assay (IFA) in solution was carried out to analyse protein subcellular localisation in intraerythrocytic parasites. Infected erythrocytes (8-10% parasitemia) were fixed in PBS (1x) containing 4% (v/v) paraformaldehyde and 0.0075% (v/v) glutaraldehyde for 30 min, permeabilised with 0.1% (v/v) Triton X-100 for 10 min, and blocked with 3% (w/v) BSA for 30 min. Cells were pelleted and incubated for one hr in blocking solution with mouse anti-GFP and rabbit anti-ACP as primary antibodies. Subsequent incubation was carried out in Alexa Fluor ® 488 and 594-conjugated anti-mouse and anti-rabbit as secondary antibodies. Cells were washed with 500 mg/mL of DAPI before final resuspension in DABCO and PBS (1x).
Samples were loaded onto Mini-PROTEAN ® TGX ™ Precast Gels (Bio-Rad) in standard Tris-glycine buffer, and polyacrylamide gel electrophoresis separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 10% (w/v) skim milk in TBS/Tween. Membrane-bound proteins were incubated in mouse anti-GFP and rabbit anti-mouse HRP-conjugated primary and secondary antibodies, respectively.
Following a 5-minute incubation in SuperSignal ® West Pico Chemiluminescent (Thermo Fisher Scientific) substrate, protein blots were analysed using the Gel Pro Analyzer 4.0.
Protein expression and purification. E. coli Rosetta that contains the pRARE plasmid was transformed with 5 ng of TrpRS api _pET-21a(+) and TrpRS cyt _AVA0421 plasmids and plated on LB agar plates supplemented with ampicillin and chloramphenicol. A single colony was expanded in 0.5 L of ZYP-5052 auto-induction media 54 and incubated at 37 °C for 18 hours with proper aeration. Isopropyl β -D-1-thiogalactopyranoside (IPTG; 1 mM) was added to TrpRS api _pET-21a(+) and the culture was incubated for two more hours.
Cells were harvested by centrifugation at 6,000 × g for 30 mins. Pelleted cells were resuspended in BugBuster ® Master Mix (Merck Millipore) with complete ™ , EDTA-free Protease Inhibitor (Roche) and passed through a French press for complete lysis of cell membrane. TrpRS proteins were purified by nickel-NTA chromatography using an imidazole gradient. Fractions were concentrated and dialysed against 100 mM HEPES using Amicon ® Ultra Centrifugal Filters (Merck Millipore) and stored in 10% glycerol at 80 °C. Fractions collected from batch purification and concentrated protein were analysed via SDS-PAGE.
Protein mass spectrometry. In-gel trypsin digestion was carried out to validate protein expression by mass spectrometry. Purified TrpRS api and TrpRS cyt separated from a protein mixture by SDS-PAGE was gel-excised, destained overnight with 50 mM triethylammonium bicarbonate (TEAB) in acetonitrile, and incubated in 10 mM tris(2-carboxyethyl)phosphine (TCEP) to reduce disulfide bonds and iodoacetamide to alkylate free cysteines. Proteins were digested with proteomic-grade trypsin (Sigma-Aldrich) and peptides were analysed on the Thermo Scientific Orbitrap Elite ™ mass spectrometer. Data analysis was carried out using MASCOT v2.4 55 using a Plasmodium subset of UNIPROT as the database with a mass tolerance of 20 ppm and 0.6 Da, 3 possible missed cleavage events, and one variable modification allowing for oxidized Methionine.
In vitro assay for Pf TrpRS activity. Total RNA was extracted from P. falciparum 3D7 wild type parasites by TRIzol (Sigma-Aldrich) treatment and tRNA-containing small RNA species were isolated using the PureLink ™ miRNA Isolation Kit (Thermo Fisher Scientific). To determine the kinetics of Pf TrpRS inhibition, an intermediate concentration of tRNA substrate was used to create progress curves in the presence of varying concentrations of the inhibitors (0.1-100 μ M indolmycin) following a method described previously 37 . Purified proteins were incubated with the inhibitor for 1 hr at 37 °C and aminoacylation was carried out as described above.
Assessment of parasite viability following indolmycin-treatment and IPP supplementation was carried out by performing a proliferation assay with the metabolite added in the media in the second replicative cycle (48-96 hours) of the parasites.
Tryptophan rescue assays. Synchronous early ring-stage P. falciparum 3D7 parasites were adapted to RPMI 1640 devoid of tryptophan. Tryptophan was added at 0.02, 0.2, and 2 mM corresponding to 1x, 10x, and 100x the concentration of the amino acid in complete media, respectively.
Proliferation rate of tryptophan-starved and -fed parasites treated with different concentrations of indolmycin was assessed using the SYBR Green assay described above. Growth of glutamine-starved and -fed parasites were analysed as controls. Pf TrpRS api 61-559aa without the presequence region was modeled by I-TASSER 41,58 using PDB 5DK4 as a preferred template without sequence alignment. The predicted protein structure (C-score = − 2.42) in complex with ATP• Mg 2+ • indolmycin from PDB 5DK4 was then visualised with PyMol.