Prefoldins are novel regulators of the unfolded protein response in artemisinin resistant Plasmodium falciparum malaria

Abstract

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug “Biperiden” that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13^R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13^R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites.

Malaria is one of the biggest threats to global health, which has existed alongside people for more than 40 centuries. Artemisinin-based combination therapies (ACTs) are the first line of recommended treatment for the malaria burden. However, resistance to artemisinin and ACTs in Plasmodium falciparum is spreading rapidly throughout Southeast Asia, posing a threat to control worldwide malaria eradication and elimination (1). Several mechanisms are proposed to play a role in the development of artemisinin resistance in the Plasmodium parasite. Clinical artemisinin resistance is strongly linked to polymorphism (Y493H, R539T, I543T, and C580Y) in the β-propeller domain of the Kelch13 (K13) protein (PF3D7_1343700) (2). This specific domain is entailed to bind with interacting substrates and allow them for ubiquitination. The function of Kelch13 is not well understood in Plasmodium, however, it shows similarity to Kelch/BTB/POZ ubiquitination adapters. K13 is hypothesized to function as a ubiquitin-ligase adapter and targets the ubiquitin-mediated degradation of proteins (3, 4).

A recent study carried out in vivo transcriptomic analysis of 1048 P. falciparum isolates from patients with acute malaria and reported that artemisinin resistance is related to enhanced expression of unfolded protein response pathways involving the main Plasmodium reactive oxidative stress complex and Tailless complex polypeptide 1 Ring Complex (TRiC) chaperone complexes. The same report investigated the expression profile of genes linked to artemisinin resistance and found overexpression of one of the genes “Prefoldin” in the artemisinin-resistant patient sample (5). Another report on the Leishmania parasite revealed that FAZP (Prefoldin homolog of Plasmodium) is overexpressed by 3.5-fold in ART-resistant parasites in comparison to wild-type parasites (6). In light of the above facts, we selected Prefoldin (PFD) family of proteins to explore their functions in malaria parasite and further attempted to investigate their relevance with respect to artemisinin resistance. Prefoldin is an evolutionarily conserved hetero-hexameric molecular co-chaperone, vital for maintaining cellular homeostasis in both physiological and pathological conditions. PFD complexes are ubiquitously present in all archaea and eukaryotes, and their main function is to capture nascent polypeptides and deliver them to Chaperonin Containing Tailless complex polypeptide 1 (CCT) or TRiC for proper folding. As of now, mainly Prefoldin’s roles have been identified in the folding of cytoskeletal proteins actin and tubulin. However, our present understanding of Prefoldin's possible function in protein homeostasis maintenance is restricted. Surprisingly, each of the single subunits can bind to independent unique interactors, allowing them to execute diverse tasks (7).

Since the emergence and spread of parasites resistant to ACTs have made the strategies more difficult to control the disease, a comprehensive understanding of parasite factors that govern the success of the pathogen in making resistant to current antimalarials is strongly required for therapeutic intervention and disease management. In line with this, lack of understanding of the PFD complex in the Plasmodium parasite represents a significant void in our functional understanding of chaperones and co-chaperones and demands to explore their role in relevance to artemisinin resistance. In the present study, we delineated the role of Prefoldins in the artemisinin-mediated resistance mechanism by demonstrating its interaction with the K13 protein in ART-sensitive Pf3D7 and resistant Pf3D7k13^R539T strains. We identified and characterized the orchestrated assembly of the PFD co-chaperone heteroprotein complex in P. falciparum and elucidated its functional significance in the unerring folding of crucial cytoskeletal and invasion-related parasite proteins, specifically Pfα-tubulin-I and Merozoite Surface Protein (MSP-1), respectively. Through an examination of FDA-approved chemotypes targeting PfPFD, this study unveils 1-{bicyclo[2.2.1]hept-5-en-2-yl}-1-phenyl-3-(piperidin-1-yl)propan-1-ol (commonly referred to as Biperiden or BPD) as a potential inhibitor of the PfPFD complex, exhibiting substantial anti-plasmodial efficacy. The functional complementation assay conducted in the orthologous system Saccharomyces cerevisiae demonstrates rescued yeast growth and affirms the reduced sensitivity of artemisinin in the PFD6 complemented strain. Using a clinically relevant ring survival assay, we demonstrate that PfPFDs inhibitor BPD reduces the number of parasite survivors following artemisinin treatment in resistant parasites. Overall, our study contributes to understanding the role of PfPFDs in providing artemisinin resistance and present BPD as a promising candidate for further exploration as an antimalarial agent. These findings offer a unique opportunity to tackle artemisinin resistance management and the development of novel antimalarial chemotherapeutics.

Results

Prefoldins identified as interacting partners of PfKelch13

A previous study reported that artemisinin resistance is linked with up-regulated expression of unfolded protein response pathways involving Prefoldins of malaria parasite (5). Also, artemisinin resistance is strongly associated with the polymorphism in the β-propeller domain of the K13 protein. In light of the abovementioned facts, we performed Co-immunoprecipitation (Co-IP) studies where an anti-K13 antibody was crosslinked to AminoLink plus Coupling Resin to pull down the interacting partners from parasite lysate. The eluted fractions were subjected to mass spectrometry analysis. The hits obtained are provided in Fig. S1 and were used to generate protein–protein interaction network using STRING with special emphasis given to chaperones and chaperonin containing T complex. These data clearly demonstrate the possibility of interaction between PfPFDs with PfK13 (Fig. 1A).

Figure 1.

Interaction studies of Prefoldin subunits with PfK13.A, protein–protein interaction network generated using STRING showing the interaction of PfK13 with Prefoldin subunits. B, (i) Overexpression and purification of PfK13. Coomassie-stained gel showing the purified recombinant protein PfK13 (∼43 kDa). (ii) Evaluation of the interaction strength between PfK13 and PfPFD6. SPR-based interaction analysis upon immobilizing PfK13 and titrating PfPFD6 displayed a good binding strength with a K_D value of 174 nM. Data were fit to the two-state conformational change model using AutoLab SPR Kinetic Evaluation software. K_D value was calculated using the Integrated Rate Law (IRL) equation as described in experimental procedures. C, co-immunoprecipitation assays validating the binding of PfPFD6 with PfK-13. Anti-PfPFD6 antisera was cross-linked to amino coupling plus resin followed by incubation with P. falciparum 3D7 (i) and Pf3D7k13^R539T lysate (iv). Eluted fractions were resolved on 12% SDS-PAGE and subjected to Western blotting using anti-PfK13 (1:1000) antibodies and their respective HRP-conjugated anti-rat antibodies (1:5000). Immunoprecipitation assays were performed from two independent samples prepared from two independent experiments, and a representative blot is shown. (ii, v) Intensity plots of bands obtained for PfK13 in co-immunoprecipitation assays depicted in i and iv using ImageJ. AU depicts arbitory units. Error bars represent mean ± standard deviation among two independent biological replicates. (iii, vi) Eluted fractions of co-immunoprecipitation assays illustrated in i, iv were subjected to Western blotting using anti-GAPDH antibodies.

To confirm the direct interaction between prefoldins and PfK13, we performed an SPR-based interaction analysis. PfK13 was purified using Nickel NTA based affinity chromatography (Fig. 1Bi) and recombinant PfPFD6 and PfK13 were used to quantify the interaction strength by SPR. Here immobilized PfPFD6 was titrated with increasing concentrations of PfK13. A K_D value of 174 nM was observed for PfPFD6 - PfK13 interaction (Fig. 1B ii). The interaction of PfPFD6 with PfK13 was further validated using co-immunoprecipitation assays in Pf3D7 and artemisinin-resistant strain Pf3D7k13^R539T. Here, PfPFD6 antiserum was cross-linked to the AminoLink plus Coupling Resin, followed by incubation with the parasite lysate prepared from the mix-stage parasite population. Bound protein fractions were eluted and subjected to western blotting by probing with anti-PfK13 and GAPDH antibodies. The desired protein band of PfK13 was observed in the eluted fractions post-probing with anti-PfK13 antibodies (Fig. 1C i). These results suggest the interaction of PfPFD6 with PfK13 in Pf3D7. Interestingly, the desired protein band of PfK13 was observed in Pf3D7k13^R539T, further suggesting the interaction of both proteins in artemisinin-resistant strain (Fig. 1C iv). Intensity plots of PfK13 bands obtained are represented in Figure 1C ii, v. Blot probed with anti-GAPDH antibodies showed no band (Fig. 1C iii, vi) which suggest specific interaction pair of PfPFD6 are being pulled down in the assay. Additionally, Figure 2B reveals that anti-PfPFD6 antibody pulls the PFD complex as a band of each PfPFD protein was detected when probed with their specific antisera. However, no band was observed with pre-immune sera, further suggesting the specificity of PfPFD6 antibody towards pulling the specific interaction pairs of PfPFD6.

Figure 2.

Assembly of PfPFDs to form a complex.A, SDS-PAGE of purified recombinant prefoldin subunits. PfPFD (20 kDa), PfPFD (19 kDa), PfPFD3 (24 kDa), PfPFD4 (17 kDa), PfPFD5 (30 kDa), PfPFD6 (16 kDa). B, co-immunoprecipitation assay showing PfPFD complex formation. Anti-PfPFD6 antiserum was cross-linked to amino coupling plus resin followed by incubation with parasite lysate. Eluted fractions were resolved on 12% SDS-PAGE and subjected to Western blotting using in-house generated PfPFD1-6 antisera (1:1000 of each) and their respective HRP-conjugated anti-mice antibodies (1:5000). PIS represents samples crosslinked with preimmune sera. C, microscale thermophoresis for complex formation analysis. PfPFD3 (i, ii, iii, iv) and PfPFD5 (v, vi, vii, viii) (20 μM of each) were labelled using RED-NHS dye and titrated with increasing concentrations of other PfPFD subunits. Binding affinity (K_D) as determined by steady-state analysis, are as follows: (i) 81.6 nM (PfPFD3-PfPFD1), (ii) 682 nM (PfPFD3-PfPFD2), (iii) 11.3 μM (PfPFD3-PfPFD4), (iv) 1.88 μM (PfPFD3-PfPFD6) indicating higher binding affinity of PfPFD3 with PfPFD1 and PfPFD2. Similarly, K_D values of (v) 851 nM (PfPFD5-PfPFD1), (vi) 42.4 nM (PfPFD5-PfPFD2), (vii) 104 nM (PfPFD5-PfPFD4), and (viii) 324 nM (PfPFD5-PfPFD6) and (ix) 1.34 μM (PfPFD5-PfPFD3), were observed indicating higher binding affinity of PfPFD5 with PfPFD4, PfPFD4 and PfPFD6. D, overall architecture of the PfPFD_hexamer. Structural models of PfPFD1-6 were generated using I-TASSER, and PfPFD_hexamer structure was generated using human TRiC-PFD complex (PDB ID: 6NR8) as a template. Upon rigid-body superimposition of both the complexes, the overall RMSD value of the C-alpha atomic co-ordinates was found to be 1.45 Å, suggesting a reliable 3D structure to be taken further for in silico and in vitro interaction analysis, and inhibition studies. PfPFD_hexamer structural model revealed potential of PfPFDs to form a hetero-hexamer with a jellyfish-like architecture.

PfPFD1-6 expressed during asexual blood stages in Pf3D7 and artemisinin-resistant line Pf3D7k13^R539T

Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR) stands as a precise and convenient technique for quantifying target gene expression levels. RT-qPCR revealed that the transcripts encoding for PfPFD1-6 are expressed throughout the intra-erythrocytic life cycle of P. falciparum (Fig. 3A). As a positive control, transcripts encoding for 18S rRNA were amplified using Pf18s-specific primers.

Figure 3.

Expression and localization of PfPFDs in asexual blood stages of P. falciparum.A, RT-qPCR analysis. Bar graph plot representing Ct-values of PfPFD1-6 in different asexual stages of the parasite. Error bars represent values of mean ± standard deviation among independent measurements from three biological repeats done in duplicates. B, stage-specific expression of PfPFD1-6 at protein levels. (i) Parasite lysates were prepared for different asexual stages of the parasite and subjected to western blotting. Immunoblotting with PfPFD1-6 antisera (1:1000 of each) and HRP- conjugated anti-mice antibodies (1:5000) showed differential expression of PfPFD1-6 across different asexual stages of the parasite. (ii) Heat map shows differential expression of PfPFD at different intraerythrocytic stage. Shading of blue and red depict lower and higher expression levels, respectively. C, cellular localization of PfPFD1-6. Smears of methanol-fixed Pf3D7-infected erythrocytes were stained with anti-PfPFD1-6 antibodies (1:200) followed by incubation with Alexa Fluor-conjugated secondary antibodies (Alexa Fluor 488, green). DIC: differential interference contrast image, DAPI: nuclear staining 40, 6-diamidino-2-phenylindole (blue); PfPFD1-6: mouse anti-PfPFD1-6 (green) (Scale bar: 2 μm). IFA depicted that PfPFD1-6 are expressed at the trophozoite and schizont stage of the parasite. D, PfPFD1-6 are mostly confined to the cytoplasm of schizonts, as confirmed by its co-localization with a cytosolic protein marker, PfNapL. DIC: differential interference contrast image, DAPI: nuclear staining 40, 6-diamidino-2-phenylindole (blue); PfPFD1-6: mouse anti- PfPFD1-6 (green); PfNapL: anti-PfNapL antibody (red); merge 1: overlay of PfPFD1-6 with PfNapL; merge 2: overlay of DAPI, PfPFD1-6 and PfNapL (Scale bar: 2 μm).

Western blot analysis of the native PfPFD1-6 in the parasite lysate prepared from all three intra-erythrocytic stages corroborated the expression of PfPFD1-6 in the parasite (Fig. 3B). At the trophozoite and schizont stages, distinct bands of PfPFD1-6 were detected with their anticipated molecular weights (18.4 kDa: PfPFD1, 16.6 kDa: PfPFD2, 22.6 kDa: PfPFD3, 15.3 kDa: PfPFD4, 29.1 kDa: PfPFD5, and 13.6 kDa PfPFD6). However, at the ring stage, the expression of PfPFD1-6 was not detected. PfαTubulin-I served as a loading control (Fig. 3B). Full uncropped blots of these results are represented in Fig. S2. Furthermore, expression of PFDs was not detected in the E. coli lysate or uninfected RBCs (both insoluble and soluble fractions) which served as negative controls (Fig. S3). We also tested the expression of PfPFD1-6 in artemisinin-resistant line Pf3D7k13^R539T and found that Prefoldins expression is upregulated in Pf3D7k13^R539T as compared to artemisinin-sensitive Pf3D7 (Fig. S4). RT-PCR and Western-based expression analysis were validated by Immunofluorescence Assays (IFAs), which depicted that PfPFD1-6 are expressed at the trophozoite and schizont stages of the parasite (Fig. 3C). Altogether, these findings suggest that PfPFD1-6 is expressed at both the transcript and protein levels in the intra-erythrocytic stages of the parasite. Furthermore, PfPFD1-6 is mostly confined to the cytoplasm of schizonts, as confirmed by its co-localization with a cytosolic protein marker, PfNapL (Fig. 3D).

PfPFD_s form a hetero-hexameric complex

CDS for pfpfd1, pfpfd2, pfpfd3, pfpfd4, pfpfd5, and pfpfd6 were cloned in pET-28a(+), over-expressed, and recombinant PfPFD subunits (6x-His-PfPFD1-6; or, rPfPFD1-6) were purified. rPfPFD1-6 resolved on SDS-PAGE as a protein species of 20, 19, 24, 17, 30, and 16 kDa, respectively (Fig. 2A). Polyclonal antibodies were raised against rPfPFD1-6 in male BALB/c mice. PfPFD complexation was investigated using Co-IP, in which PfPFD6 antiserum was cross-linked to AminoLink plus Coupling Resin followed by incubation with the parasite lysate. Eluted fractions were resolved on SDS-PAGE, followed by western blotting with the respective PfPFD1-6 antisera. Each blot showed a respective band of the PfPFD subunits (Fig. 3B), suggesting that PfPFD1-6 forms a complex similar to their counterparts in other species. Full uncropped blots are represented in Fig. S2.

In archaea and eukaryotes, PFD2 and PFD6 interact with PFD3 and PFD5, respectively, to generate sub-complexes. This is followed by the recruitment of PFD1 and PFD4, which are then assembled by these sub-complexes to form a complex (8). In light of the above facts, we attempted to identify how PFD subunits in P. falciparum assemble to form a complex. We performed an MST analysis to investigate the interaction of PfPFD3 and PfPFD5 with other subunits. Here labelled PfPFD3 and PfPFD5 were titrated with decreasing concentrations of other PfPFD subunits. Figure 2C shows the dose–response curve and MST signal for PfPFD3 and PfPFD5 binding with other subunits. Binding affinity (K_D) values obtained are as follows: 81.6 nM (PfPFD3-PfPFD1), 682 nM (PfPFD3-PfPFD2), 11.3 μM (PfPFD3-PfPFD4), and 1.88 μM (PfPFD3-PfPFD6), 851 nM (PfPFD5-PfPFD1), 42.4 nM (PfPFD5-PfPFD2), 1.34 μM (PfPFD5-PfPFD3), 104 nM (PfPFD5-PfPFD4), and 324 nM (PfPFD5-PfPFD6). Based on binding affinity values, PfPFD3 was found to have higher binding affinity for PfPFD1 and PfPFD2 as compared to PfPFD4 and PfPFD6. Also, PfPFD5 shows more binding affinity for PfPFD2 and PfPFD4 and PfPFD6 as compared to PfPFD1 and PfPFD3. These data suggest that PfPFDs can interact with each other to form a complex.

We next attempted to generate a reliable structural model of the PfPFD complex using in silico approach. The top threading templates used by I-TASSER to generate individual structural models of PfPFD subunits 1 to 6 are as follows: PFD from Pyrococcus horikoshii OT3, chain C (PDB ID: 2ZDI; for PfPFD1, PfPFD3, and PfPFD5); and, PFD-beta subunit from Thermococcus strain KS-1, chain A (PDB ID: 2ZQM; for PfPFD2, PfPFD4, and PfPFD6) (9, 10), and set to submit to generate PfPFD_hexamer complex structure by using X-Ray diffraction-based structural model of human TRiC-PFD complex as a suitable template (11). After optimal rigid-body superimposition of the generated structural model of PfPFD_hexamer with HsPFD, the overall Root-Mean-Square Deviation value of the C-alpha atomic co-ordinates was found to be 1.45 Å, suggesting a reliable 3D structure. Assessment of the stereochemical quality and accuracy of the generated structural model of PfPFD_hexamer displayed 91.8% of amino acid residues lying in the most favored (core) regions, with 6.8%, 0.9%, and 0.5% residues in additional allowed, generously allowed, and disallowed regions of Ramachandran plot, respectively.

PfPFD_hexamer structural model revealed a strong resemblance with its counterparts from other eukaryotes, forming a hetero-hexamer with a jellyfish-like architecture composed of two canonical classes of subunits: α (PFD subunits: 3 and 5) and β (subunits: 1, 2, 4, and 6), arranged in the following manner -PFD3-PFD2-PFD1-PFD5-PFD6-PFD4- (11, 12) (Fig. 2D). The core body of PfPFD_hexamer was found to consist of a double beta-barrel assembly, with six long tentacle-like coiled coils protruding from it in a regularly structured fashion. The comparable RMSD value and Ramachandran plot characteristics confirmed the reliability of the PfPFD_hexamer complex to be taken further for in silico and in vitro interaction analysis and inhibition studies.

Identification of BPD as a potential binder of PfPFD_hexamer

The in silico therapeutic repositioning approach which we adopted to screen the LOPAC¹²⁸⁰ library based on similarities with one of the potent Protein folding activity of the ribosome (PFAR) inhibitors, Metixene, generated BPD, with an overlay structural similarity index of 0.75 (Fig. 4A i). Similar to Metixene, BPD (IUPAC name: 1-{bicyclo[2.2.1]hept-5-en-2-yl}-1-phenyl-3-(piperidin-1-yl)propan-1-ol) is a member of piperidines and has a role as an antiparkinson drug and a muscarinic antagonist (Fig. 4A ii). In a recent investigation, BPD along with 16 other FDA-approved drugs, were identified to harbor anti-prion activity (13). In the study, seven out of the 17 compounds with lower IC₅₀ values were further examined for their ability to inhibit PFAR. However, because of its high IC₅₀ value, the PFAR activity of BPD was not investigated further. Given the fact that the structural features of BPD are similar to those of the PFAR-active compound, Metixene, we hypothesized that BPD would interact with additional genes whose activity is associated with protein folding and show an inhibitory effect, which prompted us to investigate the direct interaction of BPD with PfPFD.

Figure 4.

Identification of a drug ‘Biperiden’ targeting prefoldins and its effect on artemisinin-sensitive (Pf3D7) and -resistant parasite (PfKelch^R539T).A (i, ii), in silico therapeutic repositioning approach. LOPAC¹²⁸⁰ library was screened based on structural similarities with the Protein Folding Activity of Ribosomes (PFAR) inhibitor, Metixene. BPD was identified with an overlay structural similarity index of 0.75. B, schematic diagram of the BPD molecule. C, possible architecture of the PfPFD-BPD. The complex was generated using the modeled structure of the PfPFD complex. BPD was found to interact with PfPFD complex via two different conformations. (i) In conformation 1, BPD engaged at the interface of PfPFD subunits 2 and 6, with free binding energy (ΔG_bind) of −8.4 kCal/mol. (ii) In conformation 2, BPD was found to interact with the double beta-barrel assembly of the PfPFD complex, with ΔG_bind of −8.0 kCal/mol. D, BPD competes with PfPFD1 for binding to PfPFD3. Competing the PfPFD3-PfPFD1 interaction with BPD resulted in a decrease in the MST signal starting at 893 to 888.1 units (for PfPFD3-PfPFD1), thus enhancing the K_D values to 1.99 μM. E, in vitro anti-plasmodial activity of BPD in artemisinin sensitive (Pf3D7) (i) and resistant parasite (PfKelch^R539T) (iv). Ring stage Pf3D7 was treated with varying concentrations (250 nM to 5 μM) of BPD for 72 h. BPD inhibited the growth of the malaria parasite and displayed a potent anti-plasmodial effect with an IC₅₀ value of ∼1 μM in artemisinin sensitive and resistant parasite. Error bars in line graph define measurements (mean ± SD values) calculated for each data point from three independent experiments performed in triplicates each time. (ii, v) Bar graph plots depicting the percentage growth inhibition of parasite after treatment with different concentrations of artesunate in Pf3D7 (ii) and resistant PfKelch^R539T (v). Error bars in bar graph represent measurements (mean ± SD values) calculated for each concentration of artesunate treatment in three independent experiments performed in triplicates. The p values were calculated by Student’s t test (p-values ≤ 0.05 ∗, p-value ≤ 0.01∗∗, p-value ≤ 0.001∗∗∗, p < 0.0001∗∗∗∗). (iii, vi) Light microscopy-based Giemsa-stained images of artemisinin sensitive (Pf3D7, iii) and resistant parasites (PfKelch^R539T, vi) pre- and post-72 h, showing the formation of pyknotic bodies in BPD treated parasite (Scale bar = 2 μm).

A plausible architecture of the PfPFD-BPD complex was constructed using the generated structural model of the PfPFD_hexamer. Structural representation of BPD is depicted in Figure 4B and a schematic representation of complex formation between PfPFD_hexamer and BPD is shown in Figure 4C i. BPD was found to interact with PfPFD_hexamer via two different conformations. In conformation 1, BPD was found to engage at the interface of PfPFD subunits 2 and 6, with free binding energy (ΔG_bind) of −8.4 kCal/mol, via polar contact (H-bonds) with Glu⁶⁸ PfPFD6 with a bond length of 3.5 Å (Fig. 4C ii). Contrastingly, in conformation 2, BPD was found to interact with the core body of the PfPFD complex consisting of a double beta-barrel assembly, although with a lower ΔG_bind of −8.0 kCal/mol (Fig. 4C iii). It was, therefore, hypothesized that PfPFD complexation with BPD in either of the conformations would result in a diminished ability to bind and stabilize newly synthesized proteins, thereby impairing the correct folding of the nascent polypeptides.

To further evaluate whether BPD can inhibit the interaction of prefoldin subunits, we performed an MST-based competition experiment, in which PfPFD3 was used as a labeled protein; and, BPD and PfPFD1 as the unlabelled competing ligands. The interaction of PfPFD3 with PfPFD1 exhibited increasing MST signals (F_norm [%ₒ]) starting at 886.1 to 892.1 units (for PfPFD1), resulting in the K_D value of 81.6 nM (Fig. 2C i). Competing the interactions with BPD resulted in a decrease in the MST signal starting at 893 to 888.1 units, thus enhancing the K_D value to 1.99 μM (Fig. 4D). The difference in the MST signal in the absence and presence of BPD indicated that it competes with PfPFD1 for binding to PfPFD3.

BPD treatment inhibits in vitro growth of P. falciparum

BPD was evaluated for its growth-inhibitory effect on Pf3D7 and Pf3D7k13^R539T using an in vitro intra-erythrocytic Growth Inhibition Assay (GIA), wherein, parasitemia levels were determined at 72 h post-treatment. BPD inhibited the growth of the malaria parasite and displayed a potent anti-plasmodial effect with an IC₅₀ value of ∼1 μM in Pf3D7 and Pf3D7k13^R539T (Fig. 4E i, iv). Artesunate served as a control, which is a well-known anti-malarial drug, and killed ∼50% of the parasites at a concentration of 8 nM and 20 nM in Pf3D7 and Pf3D7k13^R539T respectively. (Fig. 4E ii, v). Giemsa-stained images of BPD-treated and untreated parasites are shown in Figure 4F iii, vi.

PfPFD2 interacts with Pfα-tubulin-I and their interaction is inhibited by BPD

Previous reports in eukaryotes indicate that PFD interacts with cytoskeletal proteins (14, 15, 16). Protein-Protein Interaction data available in the PlasmoDB database also indicate the interaction of PfPFD2 with Pfα-tubulin-I. To validate the interaction, preliminary screening was carried out with semi-quantitative ELISA, wherein, PfPFD2 upon titration with Pfα-tubulin-I depicted the interaction in a concentration-dependent manner (Fig. 5A). The interaction of PfPFD2 with Pfα-tubulin-I was confirmed with co-IP, in which PfPFD2 antiserum was cross-linked to the AminoLink plus Coupling Resin, followed by incubation with the parasite lysate prepared from the mix-stage parasite population. The desired protein band of Pfα-tubulin-I was observed in the eluted fraction (Fig. 5B i). Similarly, reverse co-IP, in which Pfα-tubulin-I antisera was cross-linked to the resin, followed by incubation with the parasite lysate, confirmed the interaction between the two proteins (Fig. 5B ii). Collectively, these findings point to a possible interaction between PfPFD2 and Pfα-tubulin-I. Further, Western blot analysis demonstrated that the Pfα-tubulin-I levels get markedly reduced upon the treatment of the parasites with BPD (Fig. 5C i), suggesting the inhibitory effect of BPD in blocking PfPFD2 interaction with its substrate protein Pfα-tubulin-I. The full uncropped blot is represented in Fig. S2. Band intensity of Pfα-tubulin-I before and after BPD treatment is given in Figure 5C ii.

Figure 5.

Interaction studies of PfPFD2 with Pfα-tubulin-I and effect of BPD on native Pfα-tubulin-I and PfMSP1 expression.A, PfPFD2 interacts with Pfα-tubulin-I. Semi-quantitative ELISA was done in which PfPFD2 upon titration with Pfα-tubulin-I depicted the interaction in a concentration-dependent manner. The experiments were performed in three independent biological replicates done in triplicate each time, and in line graph shown with error bars define mean ± SD values calculated for each data point. B, Co-IP-based interaction analysis of PfPFD2 and Pfα-tubulin-I. (i) PfPFD2 antiserum was cross-linked to the AminoLink plus Coupling Resin, followed by incubation with the parasite lysate prepared from the mix-stage parasite population. The desired protein band of Pfα-tubulin-I was observed in the eluted fraction. (ii) Similarly, reverse co-IP confirmed the interaction between the two proteins. C, BPD destabilizes Pfα-tubulin-I. (i) Western blot analysis demonstrated that the Pfα-tubulin-I levels get markedly reduced upon the treatment of the parasites with BPD for 6 h (1 μM and 5 μM). (ii) Bar graphs representing the intensity plots of Pfα-tubulin-I in treated and untreated samples. AU depicts arbitory units. (p-value ≤ 0.01∗∗; ns-non significant). D, BPD destabilizes PfMSP-1. (i) Western blot analysis demonstrating reduction in PfMSP-1 expression level in BPD-treated parasites at different time points of BPD treatment (1 μM; 6 h, 12 h). (ii) Intensity of PfMSP1 under each condition is shown as an intensity graph (p-values ≤ 0.05 ∗). AU depicts arbitory units. (iii) Western blot analysis demonstrating reduction in PfMSP-1 expression level in BPD-treated parasites at 48 h of BPD treatment. (iv) Western blot showing GAPDH as loading control in the assay. (v) Intensity plot of PfMSP-1 levels in the parasite treated with BPD for 48 h. (p-value ≤ 0.01∗∗). AU depicts arbitory units. Data represented in C (i) and D (i, iii) were obtained from two independent biological replicates and a representative blot is shown in each case. Error bars in intensity plots (C ii, D ii, v) define measurements (mean ± SD) taken from two independent experiments. E, confocal microscopy-based analysis demonstrated reduced expression of PfMSP-1 in the BPD-treated schizonts (Scale bar =2 μm). F, effect of BPD on parasite viability. Trophozoite-parasitized RBCs were treated with BPD (1 and 5 μM) and DHA (700 nM) for 6 h and post-treatment, parasites were subjected to PI/SYTO9 (Red/Green) co-staining. (i) Microscopic imaging data revealed that the BPD-treated parasites were SYTO9-positive and PI-negative, suggesting that the viability of the parasites was not compromised upon BPD-treatment. DHA treated parasites served as positive control while untreated parasites were taken as negative control. Viability experiments using SYTO9/PI were performed in three independent biological replicates and representative images of a biological repeat are shown. (Scale bar =2 μm). (ii) Mean fluorescence intensity of SYTO9 and PI in BPD, DHA treated and untreated parasites (n = 10) are plotted and shown as bar graphs. AU depicts arbitory units. Error bars define standard deviation among no. of cells taken for intensity measurements (n = 10). The p values were calculated by Student’s t test (p-value ≤ 0.01∗∗, p-value ≤ 0.001∗∗∗, ns-non-significant). G, BPD inhibits egress and invasion of the parasite. (i) Bar graph plot representing percent egress inhibition at different concentrations of BPD treatment. BPD significantly inhibited the egress of the parasite by ∼60% at 1.25 μM, and ∼75% at 2.5 μM and 5 μM concentrations. The p values were analysed by Student’s t test (p-values ≤ 0.05 ∗, p-value ≤ 0.01∗∗). (ii) Bar graph plot showing no. of rings per egress of schizont at different concentrations of BPD treatment. The number of rings formed per egress of schizont was also significantly reduced in the presence of BPD, as compared to the control. The p values were calculated by Student’s t test (p-values ≤ 0.05 ∗, p-value ≤ 0.01∗∗). Egress and invasion experiments were performed in three independent biological replicates and error bars in bar graph plots depict measurements (mean ± SD) calculated from three independent replicates.

BPD destabilizes PfMSP-1, a substrate of PfPFD

We previously reported that Merozoite surface protein-1 (PfMSP-1), a critical protein involved in the egress and invasion of the parasite, is a substrate of PfPFD6 (17). To evaluate how BPD affects the function of PfPFD subunits, the expression, and localization of PfMSP-1 were assessed in the BPD-treated parasites. Expression of PfMSP-1 was tested at two different time points (6 h and 12 h) of drug treatment (1 μM) using western blotting. Our results indicate reduced PfMSP-1 expression with prolonged drug treatment (Fig. 5D i; Full uncropped blots are represented in Fig. S2). Band intensity of PfMSP-1 obtained in untreated and BPD treated parasite are plotted in Figure 5D ii. Furthermore, PfMSP-1 expression gets reduced when parasites were treated with BPD (1 μM) for 48 h, (Fig. 5D iii), which is also shown by plots of band intensity of treated and untreated samples (Fig. 5D v). GAPDH served as a loading control (Fig. 5D iv). Confocal microscopy validated the western-based analysis, wherein, reduced expression of PfMSP-1 was observed in the BPD-treated schizonts (Fig. 5E); NapL served as a control (Fig. S5). These findings imply that BPD destabilizes PfMSP-1 expression by interacting with PfPFD.

To further confirm the specific effect of BPD on MSP-1 expression, the viability of the BPD-treated parasites was further assessed by co-staining the parasites with Propidium Iodide (PI) and SYTO9. PI is a nuclear counterstain that fluoresces red and is used to identify dead cells in a cell population; and, SYTO9 is a green fluorescent nucleic acid that stains both live and dead cells. Our data suggest that BPD exposure for 6 h didn’t have any cytotoxic effect (Fig. 5F i and ii), and further suggest the specific inhibitory potency of BPD on MSP1 expression.

We next evaluated the effect of BPD on the egress and invasion of the parasite. BPD significantly inhibited the egress of the parasite by ∼60% at 1.25 μM, and ∼75% at 2.5 μM and 5 μM concentrations (Fig. 5G i). Similarly, the number of rings formed per egress of schizont was significantly reduced in the presence of BPD, as compared to the control (Fig. 5G ii). This indicates that disturbing the PfPFD-mediated proteostasis by BPD inhibits the egress and invasion processes of the parasite.

Complementation of PfPFD-6 in yeast mutants restores growth and reverts to artemisinin resistance

To ascertain the identity and function of P. falciparum Prefoldins, we exploited S. cerevisiae orthologous model. Functional characterization was done in a yeast orthologous model by expressing PfPFD6 in cells harboring mutants. We cloned PfPFD6 in p416-GPD expression vector, followed by transformation of the resulting plasmid (p416-GPD-PfPFD6) in Scpfd6-deleted strain of S. cerevisiae (YTM1304). Following transformation, spot assay analysis was performed that revealed restoration of cell growth in YTM1304 complemented with PfPFD6 (YTM1304-Pfpfd6), as depicted in (Fig. 6A). WT (BY4742) was used as a positive control in the assay.

Figure 6.

Complementation of Pf Prefoldin-6 in yeast orthologous system and effect of BPD and artemisinin in mutant yeast strains.A, complementation with Pfpfd6 rescues YTM 1304 cell growth as depicted using Spot assays. Cultures of wild-type (BY4742), Scpfd6Δ (YTM 1304), and Scpfd6Δ complemented with PfPFD6 were harvested and adjusted to an optical density, A600 of 0.1 using sterile YPD media. Serial 10-fold dilutions of each culture were spotted onto YPD agar plates. Upon complementing YTM 1304 with Pfpfd6, partial restoration of cell growth was observed. B, PfPFD6-targeting effect of BPD. The growth pattern of YTM1304 and YTM1304 complemented with PfPFD6 was analyzed in the presence and absence of BPD. Cultures were harvested and adjusted to A600 of 0.1 using sterile YPD media. Serial 10-times dilutions of each culture were spotted onto solid YPD-agar plates and incubated for 2 days at 30 °C. Interestingly, PfPFD6-complemented yeast exhibited reduced growth in the presence of BPD as compared to the control (untreated). C, effect of artemisinin on the growth pattern of YTM1304 complemented with PfPFD6. The yeast cells YTM1304-p416GPD, and YTM1304-p416GPD-PfPFD6 were grown overnight and cultures were diluted to initial A₆₀₀ = 0.1. Cultures were allowed to grow for 12 h in the presence and absence of artemisinin (8 μM). Each culture was harvested and adjusted to an A600 value of 0.1. Subsequently, it was 10-fold serially diluted and was spotted onto a YPD agar plate followed by incubation at 30 °C for 2 days. PfPFD6 renders the mutant cells less sensitive to artemisinin. D, immunofluorescence images showing the expression of PfPFD6 in mutant yeast strains (YTM1304) transformed with PfPFD6-p416GPD. Expression was not observed in mutants transformed with only p416GPD. (Scale bar = 2 μm). E, (i) Schematic representation showing the methodology followed for ring survival assay, wherein ART-resistant ring staged Pf3D7k13^R539T parasites were treated with DHA followed by treatment with BPD. The surviving parasites were scored and the percentage survival was calculated for each condition. (Scale bar = 2 μm). (ii) Bar graph plot depicting the percent survival of DHA and BPD treated rings and its comparison with DHA alone treated Pf3D7k13^R539T parasites. BPD potently decreases the survival of DHA pretreated resistant rings when compared to DHA alone. Ring survival assays were carried out in two independent biological replicates. Values in error bars represent mean ± standard deviation among two independent experiments. (p-values ≤ 0.05 ∗).

Having demonstrated the restored growth of YTM 1304 upon complementation with PfPFD6, we next investigated BPD specificity for PfPFD6. Two sets of yeast cultures (Wild type and YTM 1304-PfPFD6) were used in the assay and were treated with 20 μM BPD to assess their growth patterns through spot assay analysis. Interestingly, YTM 1304-PfPFD6 exhibited reduced growth in the presence of BPD as compared to the control (untreated) (Fig. 6B). However, no significant difference was observed in BPD-treated and untreated wild-type S. cerevisiae (Fig. 6B). These results suggest that the decline in growth is primarily attributed to the targeted effect of BPD on PfPFDs. Further, to elucidate the effect of artemisinin, two batches of yeast cultures were cultivated: one comprising YTM 1304-p416-GPD and the other consisting of YTM 1304-PfPFD6. These cultures were grown in the presence and absence of ART. Our Spot assay analysis showed the reduced growth of YTM1304-p416-GPD in the presence of ART, while, complemented strain YTM 1304-PfPFD6 exhibited enhanced growth (Fig. 6C). These data point out that PfPFD6 has a significant role in the development of resistance against artemisinin.

We also checked the expression of PfPFD6 in YTM 1304 mutants transformed with PfPFD6-p416GPD through immunofluorescence assays. Our data suggests the expression of PfPFD6 in transformed yeast cells (Fig. 6D). We could not observe any expression in YTM 1304 mutants transformed with empty vector p416GPD, suggesting the specificity of our functional complementation assays.

BPD inhibits the growth of ART-resistant parasites recovered after DHA treatment

Since our previous results suggest the gain of artemisinin resistance on complementing Pf prefoldin in yeast, we tested whether BPD has any inhibitory potential on ART-resistant parasites recovered after DHA treatment. In vitro studies involve the use of ring-stage survival assay (RSA) that are considered golden standard for in vitro measurement of artemisinin resistance. In RSA, the parasites are tightly synchronized in order to assay during the short window (0–3 h rings) that can differentiate ART-resistant parasites from ART-sensitive ones (Fig. 6E i). We employed this assay in ART-resistant line Pf3D7k13^R539T to investigate the effect of BPD on artemisinin survivors. The percentage survival of DHA pretreated rings decreased in the presence of BPD (Fig. 6E ii), suggesting that BPD is potent in eliminating resistant parasites surviving post-DHA treatment. These data further shed light on the role of prefoldins in artemisinin-resistant mechanism.

BPD inhibits parasite growth in vivo

The anti-plasmodial activity of BPD was also evaluated in vivo, in mice infected with P. berghei ANKA. A schematic representation of the methodology followed for the experiment is shown in Figure 7A. On the ninth day post-infection, parasitemia in the untreated mice reached over 23%, and all mice died (Fig. 7B i). However, the parasitemia was around 7% in infected mice treated with BPD or artesunate (Fig. 7Bi). On the 23rd day post-infection, survival of the infected mice treated with BPD was found to be 50% (Fig. 7B (ii)). These findings suggest that BPD-treated mice possess reduced parasitemia and a higher survival rate than the untreated ones.

Figure 7.

BPD inhibits the growth of the rodent malaria parasite and increases the survival rate.A, schematic representation of the methodology used for dose administration of BPD in mice. B, in vivo anti-malarial activity of BPD. (i) Graph showing percent parasitemia in five experimental groups. On day 9, parasite load in the untreated group was ∼23%, whereas, in BPD- and artesunate-treated group, ∼7% parasitemia was observed. (ii) Graph showing percent survival of P. berghei-infected mice. On day 9, all mice of the untreated group died while more than 50% of mice survived for 20 days in the treated group. Statistical significance was calculated for treated groups and compared with untreated group of mice, and is shown with p-value ≤ 0.01∗∗.

Discussion

Artemisinin resistance has become a major threat to the efficacy of artemisinin-based combination therapies. Although artemisinin resistance has become prevalent, there is less knowledge about the molecular mechanisms that make P. falciparum insensitive to artemisinins. A study by Mok et al. carried out transcriptome analyses of 1043 clinical P. falciparum isolates and identified the underlined mechanisms on the transcriptional level that mediate artemisinin resistance (5). The same study reported that artemisinin resistance is linked with the coordinated transcription of several chaperone partners including the unexplored Prefoldins of P. falciparum. In light of these facts, the present study attempted to elucidate the functions of Pf Prefoldins in malaria parasites and their relevance to artemisinin resistance. This piece of knowledge can fill the key gaps to malaria biology and can help to combat artemisinin resistance.

Since PfK13 protein is reported to be responsible for mediating ART resistance, we performed Co-IP studies coupled with mass spectrometry analysis to identify the possibility of interaction between PfK13 and Pf Prefoldin. The protein-protein interaction network generated using STRING suggests the interaction of PfK13 with Prefoldins. This interaction was further confirmed by SPR and co-IP studies. These data prompted us to explore the role of Prefoldin in Pf and their contribution towards artemisinin resistance.

Abnormalities in protein synthesis, folding, or clearance disrupt cellular processes, leading to pathological consequences. To maintain a functional proteome, cells rely on a complex network of surveillance mechanisms directed by molecular chaperones, which fold newly synthesized polypeptides, refold misfolded proteins, and guide protein degradation (18). Genomic cataloging reveals that approximately 2% of the P. falciparum genes encode for an extensive array of molecular chaperones that are believed to play a crucial role in assisting the parasite to adapt to infection-induced stress (19). Investigating chaperones in the parasitic system, therefore, may not only provide potential therapeutic targets but also offer insights into novel principles of chaperone function in parasite biology. Among the chaperones, Prefoldin, a highly acclaimed co-chaperone protein, aids in the proper folding of essential proteins. Prefoldin has garnered considerable attention for its significance in emerging domains such as nanoparticles, biomaterials, and tumor biology. Furthermore, each subunit of Prefoldin possesses distinct and independent roles apart from its involvement in the Prefoldin complex (8). Although archaea and eukaryotes have been well-characterized in terms of Prefoldin function, Plasmodium species lack sufficient studies investigating the potential importance of Prefoldin subunits in managing the proteostasis of critical plasmodial proteins. The transcriptome data obtained from PlasmoDB indicates the presence of all six Prefoldin subunits in Plasmodium (PF3D7_1107500, PF3D7_1416900, PF3D7_0718500, PF3D7_0904500, PF3D7_1128100, and PF3D7_0512000). CDS for the full-length Prefoldin subunits were cloned into a bacterial expression system and subsequently overexpressed and purified. Mice were immunized to generate polyclonal antibodies against each Prefoldin subunit. These antibodies exhibited specific binding to the corresponding subunits, enabling their detection and analysis. qRT-PCR and Western blot analysis confirmed the expression of Prefoldin subunits at both transcript and protein levels during the intra-erythrocytic stages of the malaria parasite. Also, expression of Prefoldin subunits was observed to be upregulated in artemisinin-resistant strains as compared to sensitive parasites, underlining their importance during resistant mechanism. Furthermore, our immunofluorescence-based experiments illustrate the localization of Prefoldin subunits within the cytosol of the parasite. Notably, the co-localization of Prefoldin subunits with the Plasmodium cytosolic marker protein 'PfNapL' reveals a substantial overlap of localization signals, indicating their coexistence within the parasite cytosol. These findings align with previous reports in archaea and eukaryotes, suggesting the presence of the Prefoldin complex in the cytoplasm (20). Moreover, this suggests that the role and localization of Prefoldin subunits may be conserved across different organisms.

The Prefoldin complex, which functions as a cytoplasmic chaperone protein, is assembled through the interaction of its six distinct subunits, resulting in the formation of a hybrid oligomer. Based upon previous research (8), we conducted Nano temper-based interaction analysis, revealing the high affinity of PFD3 for PFD1 and PFD2, while PFD5 exhibited greater affinity towards PFD2, PFD4, and PFD6. These data suggest the ability of PfPFDs to interact with each other and form a complex.

To gain insights into the role of the PfPFD complex and explore potential antimalarial small molecules, we conducted an analysis of various chemotypes approved by the FDA and identified biperiden as a probable PfPFD-binding molecule. In silico analysis suggested that BPD binds to the PFD complex, demonstrating a significant one-to-one interaction of PfPFD subunits with BPD. Our competition experiments using MST revealed disruption of protein-protein interactions involving the Prefoldin subunits by BPD. Further, through parasite growth inhibition assay, we evaluated the effect of BPD treatment on the in-vitro growth of P. falciparum in artimisinin-sensitive Pf3D7 and resistant line Pf3D7k13^R539T, and observed an IC₅₀ of 1 μM, indicating its efficacy in inhibiting parasite growth in both strains.

Actin and tubulins are highly abundant cytoskeletal proteins that play crucial roles in numerous cellular functions, including cellular mobility, morphogenesis, polarity establishment, cell division, and intracellular transport. Considering the importance of these functions, we observed that Plasmodium Prefoldin also interacts with α-tubulin-I, thus supporting the crucial role of Prefoldin abundance in microtubule function. This is consistent with the previous findings in Arabidopsis, wherein, Pfd1-6 mutant showed considerable microtubule defects, including oryzalin hypersensitivity, impaired cell division, cortical array disorganization, and reduced microtubule dynamics (21).

Previously, we reported the interaction of PfPFD6 with MSP-1 (17). Moreover, treatment with BPD efficiently impeded the interaction between the PFD complex and its substrate, resulting in the degradation of α-tubulin-I and MSP-1. These proteins are known to be essential for the growth and proliferation of the parasite, prompting us to investigate the impact of Prefoldin inhibition on parasite growth and proliferation. Our results demonstrated that treatment with BPD led to the inhibition of parasite egress and reduced the formation of rings per schizont egress. Understanding the processes involved in Plasmodium egress inhibition could pave the way for the development of new pharmacological targets and more effective antimalarial therapies.

We next used a yeast orthologous system to show that PfPFD-6 complementation in yeast mutants restores the growth of the mutant strain. The yeast model system was also used to show the selectivity of the compound BPD for Pf Prefoldins. Interestingly, we observed that Prefoldin decreases the sensitivity of artemisinin in yeast, providing evidence for the role of PFDs in providing resistance to Pf.

Clinical ART resistance is defined as a parasite clearance half-life ≥ 5 h from a patient’s blood post-treatment. To measure ART resistance in vivo, assays that measure the linear decline of parasitemia in patients after drug treatment are used (22). In vitro studies involve the use of ring-stage survival assay (RSA) that is considered the golden standard for in vitro measurement of artemisinin resistance. Here, the parasites are tightly synchronized in order to assay during the short window (0–3 h rings) that can differentiate ART-resistant parasites from ART-sensitive ones. We employed this assay in ART-resistant line Pf3D7k13^R539T to investigate the effect of BPD on artemisinin survivors. Our data clearly demonstrated that artemisinin-resistant parasites can be inhibited by treatment with Prefoldin targeting molecule BPD. These observations further shed light on the unexplored role of Pf Prefoldins in the artemisinin resistance mechanism.

Assessing the in vivo efficacy of a drug is crucial for identifying promising leads in drug development. Therefore, we also evaluated the in vivo efficacy of BPD in a rodent malaria model. The results showed that the drug efficiently inhibited parasite development and increased the survival rate of mice at a dosage of 12.5 mg/kg.

Based on our results, we have proposed a model depicting the role of Prefoldins in the artemisinin resistance mechanism (Fig. 8). The model demonstrates the contribution of Prefoldin in providing artemisinin resistance to parasites, and probable action of BPD targeting this process to overcome resistance. Altogether, this study is the first to shed light on the unexplored Prefoldin subunits during the asexual stage of P. falciparum with special relevance to understanding their role in providing artemisinin resistance and identifying them as potential pharmacological targets. Complementation of prefoldin in yeast mutants provides evidence that PfPFD6 expression in yeast leads to the gain of artemisinin resistance. Additionally, we identified the interaction between the drug molecule, BPD, and the Prefoldin subunit complex, and demonstrated its impact on the proteostasis of key interacting proteins, namely α-tubulin-I and MSP-1. Furthermore, our in vitro and in vivo results establish BPD as an anti-plasmodial inhibitor in both artemisinin-sensitive Pf3D7 and resistant line Pf3D7k13^R539T. This research contributes to ongoing efforts in combating resistance management and reducing the burden of this deadly disease worldwide.

Figure 8.

Model depicting the role of Prefoldin in providing artemisinin resistance to parasites, and probable action of BPD targeting this process to overcome resistance. Prefoldin upregulation in resistant parasites enhances their interaction with α-tubulin-I and PfMSP1, and is responsible for the transfer of nascent α-tubulin-I polypeptide and PfMSP1 to the TRiC/CCT complex, ensuring the accurate folding of proteins. As a consequence, there is no proteotoxic effect, and fitness cost is managed. On the contrary, targeting Prefoldins with BPD disrupts its interaction with substrate proteins and inhibits their folding process. Accumulation of protein aggregates leads to a more proteotoxic effect. Subsequently, fitness cost of resistant parasites is compromised.

Experimental procedures

In vitro culture of P. falciparum

P. falciparum laboratory-adapted strain 3D7 was cultured in vitro using the standard protocols, as described previously (23). Briefly, the parasites were cultured in RPMI 1640 (GibcoTM) medium supplemented with 5.9 gm/L HEPES (Sigma-Aldrich), 50 mg/L hypoxanthine (Sigma-Aldrich), 2 gm/L sodium bicarbonate (Sigma-Aldrich), 5 gm/L AlbuMax I (for 3D7, R539T; GibcoTM) and 10 mg/L Gentamicin (Sigma-Aldrich). The culture was maintained in 75 cm2 culture flasks (Corning) using fresh O-positive (O+) human erythrocytes, under an ambient mixed gas environment (5% O2, 5% CO2, and 90% N2) at 37 °C. Before every experiment, the parasite culture was tightly synchronized with 5% D-sorbitol for two successive intra-erythrocytic proliferative cycles, followed by enrichment of trophozoites or schizonts parasitized erythrocytes with 65% percoll. Percoll synchronization was performed post 24 to 28 h of sorbitol treatment.

Co-IP assays and mass spectrometry analysis

The co-IP assays were carried out using AminoLink Plus Coupling Resin (Pierce Biotechnology) following the manufacturer’s protocol. Briefly, the resin was washed with coupling buffer (0.1 M Na₃PO₄, 0.15 M NaCl; pH = 7.2) followed by incubation with purified anti-PfK13 antibodies and preimmune sera for 2 h. Post this, the resin was washed and incubated with 50 mM sodium cyanoborohydride (NaCNBH₃) at 4°C for overnight. The parasite lysate was then added with the resin and incubated overnight at 4 °C followed by washing with wash buffer. Bound proteins were eluted using elution buffer (0.1–0.2 M glycine-HCl; pH = 2.5–3.0) and neutralized with neutralizing buffer (1M Tris; pH = 9.0) and run on 10% SDS-PAGE. The eluted proteins were digested by using trypsin (20 μg/ml). Extracted peptides were acidified to 0.1% formic acid and assessed by Orbitrap VelosPro mass spectrometer coupled with nano-LC 1000 (Thermo Fisher Scientific Inc). Using STRING online tool, interactome of proteins were generated that showed chaperone functions.

Expression and purification of PfK13

Purification of recombinant PfK13 was performed as described previously (24).

Surface plasmon resonance (SPR)-based interaction analysis

The interaction of PfK13 with PfPFD6 was evaluated by the Auto LAB ESPRIT SPR instrument (Kinetic Evaluation Instruments BV). Briefly, recombinant PfK13 (20 μM) was immobilized on a gold sensor chip previously activated through amine coupling. PBS was used as an immobilization and binding buffer. PfPFD6 was injected in increasing concentrations (25 nM, 100 nM, 1 μM, 2.5 μM, and 5 μM) over the PfPFD6-immobilized chip surface. 50 mM NaOH was used to regenerate the chip surface. Data were analyzed using Auto Lab ESPRIT kinetic evaluation software. K_D value was estimated using the Integrated Rate Law (IRL) equation:

$$R\left(t\right)=E.\left(\mathit{1}-e^{Ks.t}\right)+R\left(\mathit{0}\right)$$

Where E is the maximal extent of change in response at a certain concentration and is equal to k_a.C.R_max/(k_a.C + k_d); K_s is equal to (k_a.C+ k_d); and R(0) is the response unit at t=0. E and K_s were assessed at each concentration by minimizing the residual sum of squares between observed data and the model equation using Solver in MS Excel. Calculation of K_s at different concentrations of the ligand was performed to estimate K_D by using following equation:

$$K_s=k_a.C+kd$$

Where K_s is a concentration-dependent factor that relies on k_a and k_d. The ratio of intercept (k_d) and slope (k_a) of the above line was determined to be the dissociation constant or K_D.

Cloning, expression, and purification of PfPFD1-6

PfPFD1-6 were PCR amplified from the cDNA of Pf3D7 using gene-specific primers. CDS (coding sequence) encoding for PfPFD1-6 (PF3D7_1107500: PfPFD1, PF3D7_1416900: PfPFD2, PF3D7_0718500: PfPFD3, PF3D7_0904500: PfPFD4, PF3D7_1128100: PfPFD5, and PF3D7_0512000: PfPFD6) were cloned in pET-28a(+) vector (Novagen, Merck KGaA) at BamHI and XhoI restriction sites, and over-expressed in E. coli BL21 (ʎDE3). Ni-NTA (Qiagen) affinity purification of PfPFD1-6 was done in lysis buffer (50 mM Tris/HCl, 300 mM NaCl, and 0.02% Na-azide, pH 8.0).

Generation of antisera against PfPFDs

To raise antibodies against PfPFD1-6, three male BalB/c mice (6–8 weeks old) were each administered (i.p.) with 50 μg of the recombinant PfPFDs (in 0.9% saline), in a prime and boost regimen. A formulation for the priming dose (day 0) was prepared by thoroughly combining equal parts of Freund's complete adjuvant and saline containing the protein. Freund's incomplete adjuvant was used to make formulations for the following booster doses (days 21 and 42). After primary immunization, blood samples were collected from the retro-orbital sinus of the mice on days 31 and 52 (terminal bleed). Extracted blood samples were incubated at 37 °C for 30 min before being centrifuged at 1,200g for 15 min at 4 °C, and serum samples were collected and stored at −80 °C until further analysis. The raised antisera were checked for specificity by performing western blotting.

Co-immunoprecipitation assay to confirm the interaction of PfK13 with PfPFD6, and PfPFD with other subunits and their substrates

Co-immunoprecipitation assay was performed using a PierceTM Co-Immunoprecipitation (Co-IP) kit to confirm the interaction of PfK13 with PfPFD6. Briefly, the anti-PfPFD6 antibody was cross-linked to AminoLink plus coupling beads. After extensive washing, beads were incubated with mixed-stage Pf3D7 and PfKelch13^R539T parasite lysate. Parasite lysate was generated by subjecting the mixed-stage culture (∼8% parasitemia) to 0.15% saponin lysis, followed by RIPA lysis of the purified parasites. Bound protein was eluted in the elution buffer, separated on 12% SDS-PAGE, and subjected to western blotting. The blot was probed with a polyclonal anti-K13 antibody (1:1000) followed by a secondary anti-rat (1:5000) antibody. The blot was developed by using the ECL substrate.

A similar protocol was followed to confirm the assembly of the PfPFD complex and its interaction with specific protein substrate α-tubulin-I. To evaluate PfPFD complex formation, PfPFD6 antisera was cross-linked to AminoLink plus coupling beads followed by extensive washing with wash buffer. Mixed-stage culture (∼8% parasitemia) was subjected to saponin lysis, followed by RIPA lysis of the purified parasites. Beads cross-linked with the antisera were incubated with the parasite lysate overnight at 4 °C. Elutes were collected, divided into six groups, and resolved (along with appropriate control, rPfPFDs) on 12% SDS-PAGE, and transferred to nitrocellulose membrane. Blots were individually probed with PfPFD1-6 antisera (1:1000 of each) and secondary HRP conjugated anti-mice antibodies (1:5000; Sigma Aldrich), and developed using diaminobenzidine/H₂O₂ substrate (Sigma-Aldrich). To test PfPFD2-α-tubulin-I interaction, anti PfPFD2 antibodies were crosslinked and incubated with parasite lystae. Western blotting was performed by probing with anti-α-tubulin-I antibodies (1:1000). Reverse Co-IP was carried out by crosslinking α-tubulin-I antibody and eluted fractions were probed with anti-PfPFD2 antibodies (1:1000).

Real-time PCR analysis of PfPFD1-6

Expression of PfPFD1-6 at transcript levels was evaluated in intra-erythrocytic stages of Pf3D7 using Real-Time PCR (StepOnePlusTM Real-Time PCR system, Applied Biosystems). 18S rRNA served as a positive control. The primer sequences for the real-time PCR analysis of PfPFD1-6 and 18S rRNA are mentioned in Table S1. The reaction mixture (10 μl) consists of cDNA, SYBR Green PCR Master Mix (5 μl), and PfPFD1-6 specific forward and reverse primers (5 μM). Initial denaturation in PCR was set at 95 °C for 5 min, followed by amplification for 40 cycles of 15 s each at 95 °C, 5 s at 55 °C, and 1 min at 72 °C. Post this, amplification was performed by a melt program comprising of 15 s at 95 °C, 1 min at 60 °C, and a stepwise temperature increment of 0.3 °C/s until 95 °C. Fluorescence acquisition was carried out at each temperature transition. All samples were analyzed in duplicates.

Expression analysis of PfPFD1-6 by immunoblotting

Mixed-stage asexual cultures of Pf3D7 (5–10% parasitemia) were subjected to saponin lysis, followed by RIPA lysis of the purified parasites. The parasite lysate (10 μg of total protein), recombinant proteins (positive control), the hemoglobin from the cytosolic fraction of parasitized RBCs, unparasitized RBCs, and crude extract of E. coli (negative control) were resolved on 12% SDS-PAGE and transferred to nitrocellulose membrane. The transferred blots were blocked with 5% BSA in PBS overnight at 4 °C, and probed with mice PfPFD1-6 antisera (1:5000 of each) followed by incubation with Horseradish Peroxidase (HRP)-conjugated goat anti-mice IgG (1:2000). Blots were developed by using diaminobenzidine/H₂O₂ substrate (Sigma-Aldrich).

To check for the stage-specific expression of PfPFD1-6, synchronized ring, trophozoite, and schizont stages of the parasite were harvested separately and subjected to saponin lysis. Parasite pellets, thus obtained, were lysed with RIPA buffer for 30 min at 4 °C to rupture the parasite’s membrane and release its cytosolic content. Supernatants of the parasite lysate (10 μg of the total protein) prepared for all three stages were resolved on 12% SDS-PAGE and transferred to the nitrocellulose membrane. The blot was blocked with 5% BSA in PBS overnight at 4 °C, and probed with PfPFD1-6 antisera (1:5000 of each) followed by incubation with HRP-conjugated goat anti-mice IgG (1:2000). Blots were developed using diaminobenzidine/H₂O₂ substrate (Sigma-Aldrich).

A similar protocol was followed to check the expression of PfPFDs in P. falciparum 3D7 sensitive and ART-resistant PfKelch13^R539T parasites. Synchronized ring, trophozoite, and schizont stage parasite lysate were prepared in RIPA buffer, and an equal amount of protein was loaded on 12% SDS-PAGE and transferred to the nitrocellulose membrane. The membrane was blocked in 5% skimmed milk, followed by probing with primary PFDs anti-sera (1:1000) and secondary anti-mice (1:1000) antibodies.

Immunofluorescence assays

Thin smears of mixed-stage parasite culture were fixed in ice-cold methanol for 30 min at −20ºC. Fixed smears were permeabilized with PBS/Tween-20, and blocked with 5% BSA (w/v) in PBS for 2 h at RT. For localization studies, PfPFD1-6 antisera (1:200 of each) were added followed by incubation at RT for 2 h. Alexa Fluor 488 conjugated anti-mice (1:250; green; Molecular Probes, Invitrogen) was used as a secondary antibody. For co-localization studies, anti-mice PfPFD1-6 (1:200 of each), anti-rabbit PfNapL (1:250), and anti-rabbit PfMSP1 (1:250) were used. Alexa Fluor 488 conjugated anti-mice and Alexa Fluor 546 conjugated anti-rabbit (1:250; red; Molecular Probes) were used as secondary antibodies. DAPI-antifade (Invitrogen, Life Technologies corporation) was used to counterstain parasite nuclei followed by mounting the slides with coverslips. The slides were viewed under a confocal microscope at 100× magnification (Olympus Corporation).

MicroScale thermophoresis assays

Binding affinities among the PfPFD subunits were evaluated by MST analyses, using Monolith NT.115 instrument (NanoTemper Technologies). MST detects binding-induced changes in the thermophoretic mobility of a macromolecule, influenced by factors like particle charge, size, conformation, hydration state, and solvation entropy. Under the same buffer conditions, unbound proteins displayed different thermophoresis compared to proteins bound to their interacting partners. In this experiment, 20 μM of PfPFD3 and PfPFD5 were labeled using NanoTemper’s Protein Labelling Kit RED-NHS (L001, NanoTemper technologies) (25). These labeled proteins were titrated with decreasing concentrations of other PfPFD subunits in 1× PBS (pH 7.5) containing 0.01% tween-20. Samples were pre-mixed and incubated for 10 min at room temperature in the dark, then loaded into standard treated capillaries (K002 Monolith NT.115). Interaction analysis measured changes in thermophoresis which is reflected in fluorescence change in the MST signal and is defined as Fhot/Fcold (Fhot being the hot region after IR laser heating and Fcold the cold region at 0 s). Titration of the non-labeled ligand resulted in a gradual change in thermophoresis and is plotted as ΔFnorm to produce a dose-response curve. This dose–response curve is fitted to estimate binding constants. Data analysis was performed using Monolith software (Nano Temper).

Competitive MST analysis was done to check whether BPD hinders the interaction among the PFD subunits. Towards this, the labelled PfPFD3 was mixed with PfPFD1 (7.5 uM), and incubated for 10 to 15 min. The PfPFD3-PfPFD1 complexes, thus formed, were titrated with serial dilutions of BPD in PBS (with 0.01% tween-20) starting from 100 μM, and the interaction analysis was done under the same conditions as described above.

Generating the 3D-structure model of the PfPFD complex

Amino acid sequences of the PFD subunits 1 to 6 from P. falciparum strain 3D7 (1: PF3D7_1107500; 2: PF3D7_1416900, 3: PF3D7_0718500, 4: PF3D7_0904500, 5: PF3D7_1128100, and 6: PF3D7_0512000) were retrieved from the PlasmoDB database (https://plasmodb.org/plasmo/app) (26). A multiple threading approach, which is one of the most common structure prediction methods in structural genomics and proteomics, was employed to generate 3D-structural coordinates of the PfPFD subunits. To accomplish this feat, individual structural models of each subunit were generated using I-TASSER (Iterative Threading ASSEmbly Refinement), a web server that uses a hierarchical approach to protein structure prediction and structure-based function annotation (https://zhanggroup.org/I-TASSER/) (27), as described previously (28). Structural models of PfPFD subunits 1 to 6, thus generated, with higher values of Confidence-score (C-score) were selected and subjected to structural refinement by using ModRefiner (https://zhanglab. ccmb.med.umich.edu/ModRefiner/) which is an algorithm-based approach for atomic-level, high-resolution protein structure refinement (29). The refined structural models of PfPFD subunits were rendered with PyMOL Molecular Graphics System, v2.1 by Schrödinger, LLC (http://pymol.org/2/) (30), and set to submit to generate PfPFDhexamer structure.

The X-Ray diffraction-based structural model of the human TRiC (T-complex protein Ring Complex, also known as Chaperonin Containing TCP-1 (CCT))-PFD complex (PDB ID: 6NR8; resolution: 7.80 Å) (11) was used as a suitable template to generate a 3D structural model of the PfPFDhexamer, as described previously (31). The reliability of the PfPFD structural model was assessed by examining backbone dihedral (torsion) angles: phi (Ø) and psi (Ψ) of the amino acid residues lying in the energetically favourable regions of Ramachandran space (32). This was done by using PROCHECK v.3.5 which checks the stereochemical quality of a protein structure, producing several PostScript plots analyzing its overall and residue-by-residue geometry (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK/) (33). Percent quality measurement of the protein structures was evaluated by using four sorts of occupancies called “core,” “additional allowed,” “generously allowed,” and “disallowed” regions. The 3D structural model of PfPFD, thus generated, was subsequently used for in silico and in vitro interaction analysis, and inhibition studies.

LOPAC1280 library screening for a possible inhibitor of PfPFD-mediated protein folding

In a recent investigation by Aline Bamia et al., novel small molecules with Prion (PrPSc) propagation-inhibitory activities were identified, which interfered with the Protein Folding Activity of the Ribosome (PFAR), and significantly prolonged the survival of prion-infected mice (13). Using an in silico therapeutic repositioning approach, we screened LOPAC1280 Library of 1280 Pharmacologically Active Compounds (Sigma-Aldrich) based on similarities with one of the potent PFAR inhibitors identified in the study, Metixene (https://www.sigmaaldrich.com/IN/en/product/sigma/lo1280). Metixene is a member of piperidines and has a role as an antiparkinson drug and a muscarinic antagonist. Structural Data Format (SDF) files of Metixene and LOPAC1280 library were retrieved from PubChem, a database of freely accessible chemical information of chemical molecules and their activities against biological assays (https://pubchem.ncbi.nlm.nih.gov/), and Sigma-Aldrich, respectively. Structural superimposition of LOPAC1280 ligands with Metixene was done by using Discovery Studio Visualizer v20.1.0.19295, developed by Dassault Systèms Biovia Corp. (https://www.3ds.com/products-services/biovia/), and overlay structural similarity for each of the 1280 compounds were evaluated, taking Metixene as a reference compound. Structural superimposition analysis was done by using ChemDraw Ultra v12.0.2.1076, one of the CambridgeSoft products for producing a nearly unlimited variety of biological and chemical drawings (https://perkinelmerinformatics.com/products/research/chemdraw).

In silico interaction analysis of BPD with PfPFD

Structural Data Format (SDF) file of BPD.HCl was retrieved from PubChem, and converted to standard PDB format, followed by the generation of its energy-minimized 3D-structural model by using Chem3D Pro 12.0, as described previously (28, 34). Molecular docking studies were performed by using Autodock Vina Tools 1.5.6 to rationalize the inhibitory activity of BPD against PfPFD (28, 34, 35, 36, 37). We ensured that the entire PfPFD complex was covered while constructing a virtual 3D grid for the in silico interaction analysis. A grid of 80 × 100 × 80 with x, y, and z coordinates of the center of energy, 209.514, 147.278, and 209.119, respectively was constructed through the Autogrid module of AutoDock Tools, with default spacing. Top scoring docked conformations of BPD were selected based on their most negative free binding energies and visualized for polar contacts (H-bonds; if any) with the amino acid residues of PfPFD complex using PyMOL Molecular Graphics System (30).

Parasite growth inhibition assay

To evaluate the effect of BPD on the intra-erythrocytic proliferation of the parasite, a growth inhibition assay was performed. Briefly, Pf3D7 culture synchronized at ring stage (0.8% parasitemia and 2% hematocrit) was treated with BPD (250 nM, 500 nM, 750 nM, 1 μM, 2.5 μM, and 5 μM). Dilutions of BPD were prepared in iRPMI (Incomplete RPMI; (GibcoTM) medium supplemented with 5.9 gm/L HEPES (Sigma-Aldrich), 50 mg/L hypoxanthine (Sigma-Aldrich), 2 gm/L sodium bicarbonate (Sigma-Aldrich), and 10 mg/L Gentamicin (Sigma-Aldrich). The assay plate was maintained at 37 °C in a controlled atmosphere (5% O₂, 5% CO₂, and 90% N₂) for 72 h. Post-incubation, Giemsa-stained thin blood smears of P. falciparum were prepared and ∼3000 RBCs were counted in each smear. The experiment was performed in triplicates. The percentage of growth inhibition was calculated by using the formula: % Inhibition = [1 - % Parasitemia treatment/% Parasitemia control] ∗ 100. The graph was plotted using GraphPad PRISM software.

Enzyme-linked immune sorbent assay (ELISA) to evaluate the interaction of PfPFD1-6 with α-tubulin-I

To assess the interaction of PfPFD1-6 with α-tubulin-I, a 96-well ELISA plate was coated with purified PfPFD1-6 (bait; 100 ng of each) in PBS at RT for 5 h, and blocked overnight at 4°C with 5% BSA in PBS, followed by incubation at RT for 2 h with increasing concentrations (0–100 ng) of α-tubulin-I (prey). After washing with PBS, antisera (1:10,000) against the respective PfPFD subunits was added to each well, followed by incubation with secondary anti-mice HRP conjugated antibody at RT for 2 h. After washing with PBS, a detection reagent (TMB; HIMEDIA) was added, followed by the addition of 3M HCl to stop the HRP reaction. Absorbance was measured in a microplate reader at 450 nm. The graph was plotted using GraphPad PRISM software.

Effect of BPD on the expression levels of PfPFD substrates

To evaluate the effect of BPD on the expression levels of α-tubulin-I, trophozoite-parasitized RBCs were treated with BPD (1 and 5 μM). After 6 h of treatment, parasitized erythrocytes were harvested and lysed with 0.05% saponin. The purified parasites, thus obtained, were lysed with RIPA buffer. The parasite lysate was used for western blotting with α-tubulin-I anti-serum to check for the expression levels. Similarly, to assess the effect of BPD on the expression levels of PfMSP-1, trophozoite-parasitized RBCs (at 0.8% parasitemia and 3% hematocrit) were treated with BPD at a concentration equivalent to its IC₅₀ (i.e., 1 μM) for 6 and 12 h; whereas, ring parasitized RBCs were treated with 1 μM BPD for 48 h. Thin smears of the BPD-treated cultures were prepared on glass slides for IFA. Similarly, BPD-treated parasites were harvested for western blotting with PfMSP-1 antiserum to check for the expression levels of PfMSP-1.

Cytotoxic evaluation of BPD on the parasite

To evaluate the cytotoxic effect of BPD on the parasite, BPD (1 and 5 μM) treated trophozoite-parasitized RBCs were co-stained with Propidium Iodide (PI) and SYTO9 fluorescent dyes. Untreated parasitized RBCs served as a negative control. 6 h post-treatment, cells were washed with iRPMI and stained with PI and SYTO9 (100 μM of each; InvitrogenTM Thermo Fisher Scientific) in a 1:1 ratio. Cells were incubated in the dark at RT for 15 min, washed with iRPMI and transferred onto a glass slide for visualization under an Olympus fluorescence microscope. Parasites were treated with DHA (700 nM) served as positive control.

Parasite egress and invasion assay in vitro

To determine the effect of BPD on the invasion and egress rate of the parasite, mature schizonts (45–47 h post-invasion, hpi) were diluted to ∼4% parasitemia and 2% hematocrit. Parasites were treated with varying concentrations of BPD (5, 2.5, 1.25, and 0.625 μM). Untreated parasites were taken as control. After 8 h of treatment, thin smears were prepared on glass slides and stained with Giemsa. Approximately 3000 RBCs were counted under a light microscope at 100× magnification. Percent egress was calculated by using the formula: (No. of schizonts at 0 h - No. of schizonts in treated sample)/(No. of schizonts at 0 h - No. of schizonts in untreated sample) ∗ 100. The initial number of schizonts was taken as 100%. Percent egress inhibition was calculated as 100 percent egress. The number of rings formed per schizont egress: No. of rings/(No. of schizonts pre-treatment - No. of schizonts post-treatment) (38, 39).

Generation of PfPFD6 complementation strain in yeast mutants

Full-length sequence of PfPFD6 (1–360 bp) was amplified using P. falciparum cDNA as a template, and gene-specific primers. The purified insert and p416-GPD vector, with a host range in bacteria and yeast, harbors GPD promotor and bears the Uracil-encoding gene (ura+) for selection, were digested with BamHI/SalI (New England Biolabs) and ligated overnight at 4 °C using T4 DNA ligase (New England Biolabs). The ligation mix was transformed into E. coli DH5-α competent cells and positive clones were screened by colony PCR.

The PfPFD6-p416-GPD and p416-GPD constructs were transformed in S. cerevisiae mutants YTM1304::pfd6Δ (gim1Δ), using Frozen-EZ Yeast Transformation IITM kit (Zymo Research) as per the manufacturer's protocol and plated on YNB (yeast nitrogen base) agar plate (supplemented with 2% glucose and 1× amino acid mix without uracil) as selective media. Cells were allowed to grow at 30 °C. The transformed colonies were confirmed by colony PCR using gene-specific primers. S. cerevisiae BY4742 (MATα; his3Δ; leu2Δ; lys2Δ; ura3Δ) was used as the wild-type control wherever applicable. YTM1304-p416-GPD was also used as a control in experiments.

Assessment of biperiden selectivity for PfPFD6

To confirm the selectivity of BPD towards PfPFDs, YTM 1304-p416-GPD and YTM 1304-Pfpfd6 were cultured in their respective media overnight until sufficient growth was achieved. Subsequently, the cultures were diluted to an optical density, A₆₀₀ of 0.1 using sterile fresh media and further incubated in the absence and presence of 20 μM BPD at 30 °C for 12 h. The resulting cell suspension was then serially diluted to 10-folds, and each dilution was spotted onto a YPD agar plate. The culture plate was incubated for 2 days at 30 °C.

Assessment of artemisinin effect on growth of complemented mutant yeast strains

To elucidate the impact of ART treatment on the yeast cell growth pattern, YTM1304-PfPFD6, and YTM1304-p416-GPD were grown in YPD media and used as a pre-culture. Once the culture reached appropriate growth, it was diluted to an initial A₆₀₀ value of 0.1 and subjected to further incubation at 30 °C for 12 h, with and without the addition of artemisinin (8 μM). Post-incubation cultures were harvested and adjusted to an A₆₀₀ value of 0.1. The cultures were then subjected to ten-fold serial dilution, spotted on the YPD agar plate, and incubated at 30 °C for the next 2 days.

Expression analysis of PfPFD6 in YTM 1304 mutants of S. cerevisiae

Immunofluorescence assays were performed to check the expression of PfPFD6 in YTM 1304 mutants of S. cerevisiae. 5 ml of secondary yeast culture with O.D 0.5 to 0.7 was fixed with 37% formaldehyde and incubated for 30 min at 30 °C with intermitted tapping. Cells were then pelleted and washed thrice with 1X PBS and resuspended in 25 μl of 1M DTT and again incubated for 20 min at 30 °C. The cell wall was digested using zymolase (10 mg/ml) and 5ul β-mercaptoethanol, further, the suspension was incubated at 30 °C for 20 min. The culture was pelleted and washed twice with 1XPBS. 0.1% Triton X-100 was used for cell permeabilization and incubated at RT for 20 min. Cells were then blocked using a blocking solution (5 mg/ml BSA+22.5 mg/ml glycine in 1X PBS) and incubated for 10 min at RT. Yeast cells were then pelleted and incubated for 1 h with anti-mice PfPFD6 antisera (1:300) followed by three PBST washes. Post-washing, the cells were incubated for 45 min with anti-mice Alexa fluor-488 antibody (1:300) followed by PBST washes to remove unbound antigens. Yeast pellets were resuspended in 100 μl of 1× PBS and 3 μl of yeast cells were then spotted onto the microscope slide along with DAP1 and a coverslip was applied. Imaging was done using an Olympus fluorescence microscope at 100× magnification.

Ring survival assays

To evaluate the effect of Biperiden in PfKelch13 ^R539T ART-resistance parasites, a ring survival assay was performed as previously described (22, 40). Briefly, Percoll purified early ring stage parasite (0–3-h ring post-invasion) was treated with DHA (700 nM) for 6 h. Post-washing with iRPMI, the parasite was treated with BPD (1 μM), and incubated at 37º C for 66 h. The parasitemia was calculated by counting the number of ∼3000 parasitized cells. Percent survival was calculated using the following formula:

$$\%\text{survival}=100-\%\text{Inhibition}$$

$$\%\text{Inhibition}=\left[\left(\mathrm{C}-\mathrm{T}/\mathrm{C}\right)\right]100$$

C: Parasitaemia of untreated PfKelch13 R539T, T: Parasitaemia of either DHA treated or DHA plus BPD treated parasites.

Parasite growth inhibition in vivo

BALB/c female mice (6 weeks old) were divided into five groups and each group consisted of four animals. At day 0, mice were infected intra-peritoneally with 1 × 10⁶ infected RBCs (100 μl, diluted in PBS), obtained from P. berghei ANKA-infected donor mice. Group 1 mice were treated with artesunate at a concentration of 6 mg/kg (positive control), Group 2 was treated with 12.5 mg/kg of BPD, while Group 3 was treated with a combination of artesunate (6 mg/kg) and BPD (12.5 mg/kg). Group 4 was left untreated (negative control), while Group 5 was not infected by P. berghei ANKA. Blood samples from the tail end of the infected mice were taken daily. Percent parasitemia was determined by observing the Giemsa-stained smears of the blood samples under a microscope at 100× magnification (Olympus Corporation).

Statistical analysis

All graphs were generated by the software GraphPad Prism (version 8) and statistical analysis was calculated by using unpaired two-tailed Student’s t-tests. p value ≤ 0.05 was considered significant.

Ethics approval

The mice-based studies were conducted at Animal House, Jawaharlal Nehru University and were approved by the Institutional Animal Ethics Committee (IAEC) of Jawaharlal Nehru University, New Delhi, India (code no. 35/2019); and, the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Mice-based in vivo experiments are also reported following the guidelines of “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines (https://arriveguidelines.org/).

Data availability

Data will be made available on request.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Biographies

Rumaisha Shoaib is a Ph.D student at Jamia Millia Islamia, New Delhi, India, and she is working under the supervision of Prof. Shailja Singh at Jawaharlal Nehru University and Dr. Mohammad Abid at JMI. She has expertise in molecular biology and infectious diseases. In this research, she unveils the role of co-chaperone Prefoldin in drug resistance development in human malaria parasites. She aims to demonstrate how understanding chaperones' involvement in drug resistance could transform therapeutic strategies.

Nidha Parveen is a Ph.D. scholar of Prof. Shailja Singh at Jawaharlal Nehru University, India. Her research aims to enhance our understanding of prefoldin as novel regulators, with a primary focus on correlating these molecular players with artemisinin resistance. This work seeks to identify the involvement of these co-chaperones in the development of artemisinin resistance, in addition to the well-known Kelch13 protein, potentially opening new avenues for the management of resistant parasites.

Reviewed by members of the JBC Editorial Board. Edited by Elizabeth J. Coulson