The ER chaperone PfGRP170 is essential for asexual development and is linked to stress response in malaria parasites

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the Red Blood Cell (RBC) is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In other eukaryotes, ER chaperones assist with protein folding and unfolding, the crossing of biological membranes, ER stress, lipid metabolism, and protein trafficking. In this study we studied the role of an uncharacterized ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localizes to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that knockdown of PfGRP170 leads to the activation of the Plasmodium eIF2□ kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

oligomerizes and becomes active, phosphorylating the cytoplasmic translation initiation  Since PfGRP170 knockdown resulted in EIF2-alpha phosphorylation, which has been 2 4 8 shown to be required for resistance to artemisinin exposure, we tested if PfGRP170 2 4 9 plays a role in drug resistance. For this purpose, we utilized PfGRP170-BirA parasites,  Several Plasmodium kinases have been shown to phosphorylate EIF2-alpha in late 2 5 5 developmental stages or in response to other cellular stress or artemisinin 2 5 6 treatment 16,50-52 . We were therefore interested in identifying the specific kinase, which is 2 5 7 responsible to the phosphorylation of EIF2-alpha during PfGRP170 knockdown. We the presence or absence of the PK4 inhibitor GSK2606414. Parasite lysates were used 2 6 0 to determine the phosphorylation state of EIF2-alpha. We observed that in the presence 2 6 1 of the PK4 inhibitor, EIF2-alpha phosphorylation was blocked, demonstrating that 2 6 2 PfGRP170 knockdown specifically results in PK4 activation, that leads to 2 6 3 phosphorylation of EF2-alpha ( Figure 6B). We present in this work the first characterization of PfGRP170 in the asexual life cycle 2 6 6 of P. falciparum. We have generated conditional mutants that allow us to probe the role 2 6 7 of this protein using the DDD conditional system 29-34 . Additionally, taking advantage of 2 6 8 the GFP fused to PfGRP170, we were able to isolate an exceptionally rare clonal further implemented not only for rare events but also to significantly cut down the time 2 7 2 from transfection to a clonal cell population. We demonstrate here that PfGRP170 is an ER resident protein that is essential for chaperone BiP, serving as the nucleotide exchange factor to regulate BiP activity 27,53 .

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Unlike Plasmodium falciparum, Yeast null for GRP170 are viable due to the 2 7 9 upregulation of Sil1, another nucleotide exchange factor, that usually plays a role in the 2 8 0 IRE1 stress response pathway 54 . Plasmodium genome does not encode Sil1 and IRE1, 2 8 1 which goes in line with the observed essentiality of PfGRP170 during the blood stages. Additionally, research in mammalian systems suggests that GRP170 also has BiP-  Previously it was shown that apicoplast transit peptides are predicted to bind the ER 2 8 8 chaperone BiP, and when these predicted binding sites were mutated, targeting to the 2 8 9 apicoplast was disrupted 55 . Moreover, an Hsp70 inhibitor with an antimalarial activity 2 9 0 was shown to inhibit apicoplast targeting 56,57 . These data, combined with the predicted 2 9 1 transit peptide of PfGRP170, led us to investigate the role of this chaperone in 2 9 2 apicoplast trafficking ( Figure 3A). Interestingly, when the putative transit peptide was 2 9 3 tagged with a GFP reporter and without an ER retention signal, the fusion protein was 2 9 4 retained in the ER. Typically, proteins with a signal peptide that lack a trafficking signal 2 9 5 or ER retention signal, are secreted to the parasitophorous vacuole 58-61 . One possibility 2 9 6 is that this GFP fusion was unfolded and therefore retained in the ER 59 . Regardless, this 2 9 7 reporter was not sent to the apicoplast indicating that it is not a functional apicoplast 2 9 8 transit peptide ( Figure 3B). In addition, PfGRP170 knockdown did not lead to any 2 9 9 defects in trafficking to the apicoplast, nor could be rescued with the essential  Protein trafficking to the host RBC originates in the parasite ER and is essential for 3 0 2 parasite viability, and therefore could potentially account for the observed death with PfGRP170 in our study (Table 1 and Figure 4). However, our data show that there 3 0 7 is no significant difference in the trafficking of some exported proteins upon knockdown 3 0 8 of PfGRP170, suggesting that protein export is not broadly affected (Supplemental 3 0 9 Figure 5). The possibility that PfGRP170 activity is required for the trafficking of a 3 1 0 subset of specific exported proteins remains to be tested. ER chaperones are known in other eukaryotes to be vital to managing cellular 3 1 2 stress 17,21 . Our data demonstrate that PfGRP170 is important for coping with a specific 3 1 3 form of cellular stress, namely heat shock ( Figure 2F). This finding highlights a potential encode many of the UPR orthologues, but a single ER stress pathway (PK4 signaling) 3 2 0 has been previously described and was shown to be activated following artemisinin  Finally, our data suggest that unlike its homologs in other eukaryotes, the essential 3 2 5 function of PfGRP170 is not linked to its role in regulating BiP, since PfGRP170 binds to 3 2 6 BiP upon removal of TMP and knockdown leads to upregulation of the stress response and future work will determine which of the key substrates are solely dependent on PfGRP170 activity for their function. These data also suggest that given the divergence PfGRP170 could be a viable antimalarial drug target.  unfolded DDD and is inhibited from interacting with client proteins.    were taken as a Z-stack using super resolution microscopy and SIM processing was 4 2 1 performed on the Z-stacks. Images are displayed as a maximum intensity projection.

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The scale bar is 2µM. GFP and streptavidin IPs were cross-referenced to create a list of 28 common proteins.      The table includes the 28 proteins identified using two independent mass spectroscopy  University Integrated Proteomics Core for mass spectrometry. This work was supported All primer sequences used in this study can be found in Supplemental Table 2. per the manufacturer's protocol 31 .

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Genomic DNA was isolated using the QIAamp DNA blood kit (Qiagen). gDNA used in 4 9 4 this study was isolated from either 3D7 or Plasmepsin I knockout parasites (PM1KO) 31 .

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The pPfGRP170-DDD plasmid used to generate the PfGRP170-GFP-DDD mutants was The pGRP170-HA-BirA-KDEL plasmid was prepared by amplifying PfGRP170 (without sequence of an ER retention signal (KDEL). These PCR products were fused together 5 0 8 using SLIC as described previously and subsequently PCR amplified using primers P5 5 0 9 and P8 29 . The resulting product was then inserted into pCEN-DHFR 66 that was digested 5 1 0 with Nhe1 and BglII (New England Biolabs) using SLIC and transformed into bacteria as The pPfGRP170TP-GFP plasmid was prepared by amplifying the first 450 bp (includes using primers P5 and P9. The GFP sequence used was amplified from pGDB using 5 1 6 primers P10 and P11. The PfGRP170 transit peptide PCR was digested with Nhe1 and 5 1 7 AatII (New England Biolabs) and the GFP PCR was digested with AatII and BglII (New England Biolabs). The two fragments were then ligated together (via the AatII digest 5 1 9 site) using a T4 ligase (kit from New England Biolabs) and subsequently PCR amplified Biolabs) using a T4 ligase and transformed into bacteria as described previously 29,30 . Parasites were grown in RPMI 1640 media supplemented with Albumax 1 (Gibco) and 5 2 6 transfected as described previously 28-32 .

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Following drug cycling, GFP positive cells were enriched using an S3 Cell Sorter for clones 1B2 and 1B11, parasites were shifted into media containing 2.5μg/ml BSD 5 3 6 and 20μM TMP to facilitate optimal growth.

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Integration tests for PfGRP170-DDD mutants 5 4 2 Genomic DNA was isolated from parasites using the QIAamp DNA blood kit (Qiagen).

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Control primers to amplify the genome were P4 and P12 and primers used to amplify 5 4 4 integrated DNA were P12 and P13. parasites (1B2 and 1B11) as described previously 29,30 . The assay was also performed probe on the southern blot was detected using IRDye 800CW streptavidin-conjugated (LICOR Biosciences).

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Growth assays using flow cytometry factor. Parasitemia was normalized by using the highest parasitemia as one hundred 5 6 8 percent. Using Prism software (GraphPad Software Inc), the parasitemia data were fit to 5 6 9 an exponential growth curve equation.

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To determine the EC 50 of TMP for PfGRP170-DDD cell lines, parasites were washed as 5 7 1 described above and seeded into a 96 well plate with 2.5μg/ml Blasticidin and varying 5 7 2 TMP concentrations. Parasitemia was measured after 48 hours using flow cytometry as Prism.

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For the IPP rescue experiment, asynchronous PfGRP170-DDD parasites were washed 5 7 6 as described above and resuspended in media either with 2.5μg/ml Blasticidin or (Isoprenoids LC). Parasitemia were monitored using flow cytometry as described above and the data were fit to an exponential growth curve equation using Prism. without it and all parasites were shifted back to 37ºC. Parasitemia was monitored using 5 8 5 flow cytometry as described above and the data were fit to an exponential growth curve PfGRP170-DDD Parasites were synchronized as described previously by sorbitol 5 8 9 (VWR), followed by percoll (Genesee Scientific) the next day and then sorbitol four hours later to obtain 0-4 hour rings 30,31 . Parasites were washed as described above to 5 9 1 remove TMP from the media and incubated in media either with 2.5μg/ml Blasticidin or 5 9 2 2.5μg/ml Blasticidin and 20μM TMP. Thin blood smears using the Hema 3 Staining Kit 5 9 3 (PROTOCOL/Fisher) were prepared every few hours to monitor parasite growth and 5 9 4 morphology. Slides were imaged using a Nikon Eclipse E400 microscope with a Nikon 5 9 5 DS-L1-5M imaging camera.

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For testing PK4 activity, synchronized ring stage PfGRP170-DDD parasites were 6 1 1 incubated in media with either 2.5μg/ml Blasticidin or 2.5μg/ml Blasticidin and 20μM 6 1 2 TMP in the presence or absence of a PK4 inhibitor GSK2606414 (Millipore Sigma) at 6 1 3 2μM for 24 hours. After 24 hours, the parasites were lysed for western blot analysis 6 1 4 using 0.04% saponin in 1X PBS as described above. To visualize PfGRP170-DDD live parasites, 100μL of parasite culture was pelleted. The 37°C for 20 minutes. The parasites were then pelleted again and 90% of the medium 6 2 0 was removed. Parasites were resuspended in the remaining medium and 8μL of this culture was placed on a glass slide and covered with a coverslip. The edges were 6 2 2 sealed with nail polish and the cells were imaged using a DeltaVision II Microscope. 6 2 3