PfHDAC1 is an essential regulator of parasite asexual growth with its altered genomic occupancy and activity associated with artemisinin drug resistance in Plasmodium falciparum

  1. Abhishek Kanyal
  2. Heledd Davies
  3. Bhagyashree Deshmukh
  4. Dilsha Farheen
  5. Moritz Treeck
  6. Krishanpal Karmodiya

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Abstract

Plasmodium falciparum is a deadly protozoan parasite and the causative agent of malaria, which accounts for close to 200 million cases and 400,000 deaths every year. It has been identified to possess a tightly regulated gene expression profile that is integrally linked to its timely development during the intraerythrocytic stage. Epigenetic modifiers of the histone acetylation code have been identified as key regulators of the parasite’s transcriptome. In this study, we characterize the solitary class I histone deacetylase PfHDAC1 and demonstrate that phosphorylation is required for its catalytic activity. PfHDAC1 binds to and regulates parasite genes responsible for housekeeping and stress-responsive functions. We show that PfHDAC1 activity in parasites is crucial for normal intraerythrocytic development and that its cellular abundance is correlated with parasitemia progression. We further show that PfHDAC1 has differential abundance and genomic occupancy in artemisinin drug-resistant vs sensitive parasites and that inhibition of its deacetylase activity can modulate the sensitivity of parasites to the drug. We also identify that artemisinin exposure can interfere with PfHDAC1 phosphorylation and its genomic occupancy. Collectively, our results demonstrate PfHDAC1 to be an important regulator of basic biological functions in parasites while also deterministic of responses to environmental stresses such as antimalarial drugs.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    This manuscript attempts to link different aspects of HDAC1 function to Plasmodium falciparum biology. HDAC1 is essential so is likely to have important functions in parasite development.

    The emphasis is upon the potential gene regulation aspects of HDAC1 function, but it is well known that acetylation of other proteins is regulated by HDAC1 orthologues. While they examine the genome occupancy of HDAC1, it's not clear whether the phenotypic effects described can be ascribed to effects upon histone modifications. For RNA-seq analysis and ChIPseq, they generally use one time point so they have not controlled for potential differences in cell cycle to explain differences in gene expression or genome occupancy. These weaknesses in the experimental design make it difficult to evaluate the significance of their data with artemesinin and drug resistant lines.

    The authors suggest that CKII is important for regulating the function of HDAC1. This is biologically plausible, but the link could be more convincing. In addition, the evidence that the potential gene regulation effects are critical for the phenotype observed could be stronger.

    Major comments:

    Figure 1: They perform phosphorylation studies with recombinant CKII and HDAC1, but they do not demonstrate whether the phosphorylated residues correspond to the predicted residues S391, S397 and S440 or if mutation of the predicted residues affects activity.

    The inhibitor data are consistent with the predicted effects, but kinase inhibitors do not always have the same target in vivo or in cells as they do in protein assays. Concentrations of inhibitors used should be provided in the materials and methods.

    They also claim that CK2 and HDAC1 interact in parasites (p5). They do not provide data to support this statement, nor do they provide any data about other proteins that might be interacting with HDAC1. If they were able to purify enough HDAC1 for mass spec identification, they should provide further documentation about interacting proteins and potential post-translational modifications.

    In addition, they should provide more detailed characterization with Western/IFA of when HDAC1 is expressed and whether CKII is always expressed at the same time.

    Overall the importance and significance of CKII in regulation of HDAC1 activity is not clear and would be much strengthened if experiments performed with recombinant protein could be replicated in IP parasite lysates with appropriate controls and a time series.

    Figure 2: Using an HDAC1 GFP line they perform ChIP-seq. The ChIP-seq experiments seem to be well performed with high correlation between replicates but were performed at a single time point in the life cycle of erythrocytic stages. It's not clear if the distribution or abundance of HDAC1 changes during the cell cycle, though they suggest it does, and given changes noted in genome occupancy, one cannot determine if the differences seen could be completely explained by parasites being in different stages of the cell cycle with different levels of HDAC1. They show enrichment of different pathways, but do not comment on whether these are just pathways that are enriched in trophozoites.

    Figure 3 They characterize the growth rate of parasites treated with sublethal concentrations of HDAC1 inhibitor and see effects. The images presented in panel A are not good quality and parasite morphology is difficult to evaluate. They perform RNA-seq at a single time point and the choice of time point and drug concentration used is not justified. Changes are reported but again with a single time point, it's difficult to interpret the significance of the changes-are these dying parasites or parasites slowly progressing through the life cycle? To really understand the effects of these drugs a better characterization of dose response and time point series is needed.

    Figure 4 Upon overexpression of PfHDAC1-GFPglmS there appear to be more parasites. It is unclear if this due to more merozoites per schizont, better invasion with more rings. Again, better characterization of time points would be helpful to understand how overexpression of HDAC1 affects proliferation.

    Figure 5. They state that there is less HDAC1 in art resistant lines, but given that they have not provided any information about cell cycle expression of HDAC1 and growth of these lines in comparison to wild-type, it is unclear if there are differences in biology or if the cells differ where they are in the cell cycle.

    This is particularly important because of the known differences of artemisinin effects depending upon cell cycle stage.

    Figure 6 Genome occupancy data are difficult to interpret given possible differences in cell cycle.

    Minor comments:

    The general quality of images and gels should be improved.

    More information should be provided about the validation and specificity of the in house HDAC1 antibodies.

    Concentrations of inhibitors used should be provided.

    Referees cross-commenting

    There is consensus amongst all reviewers that the experiments as presented cannot be readily interpreted and are lacking adequate controls. The amount of experimental work and further analysis is considerable.

    Significance

    Understanding gene expression and the role of HDAC1 is potentially significant, particularly if these can be linked to important biological processes such as artemisinin resistance. Potentially the audience would be broad. The link between these processes is not well supported by the data as currently presented.

    Expertise: epigenetics, parasite gene expression.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Kanyal et al present a functional analysis of the Plasmodium falciparum Histone deacetylase 1 (PfHDAC1). PfHDAC1 is the only predicted class I HDAC in P. falciparum and has been shown to be the target of several established and novel compounds with anti-malarial activity across parasite stages. In this work, the authors showed that recombinant HDAC1 is phosphorylated in the presence of CKII in vitro and can de-acetylate P. falciparum histones (although no loading control was presented for this latter assay). ChIPseq of GFP tagged HDAC1 identifies target sites relating to diverse cellular processes, and sublethal treatment with the proposed HDAC1 inhibitor rhomidepsin has an impact on cell cycle progression, suggesting that HDAC1 may act in cell cycle control. However, overexpression of Histone deacetylase seems to enhance parasite multiplication by increasing invasion gene expression, which seems counterintuitive as overexpression is expected to cause decreased histone acetylation and thereby gene repression, and hence this pattern may be due to indirect effects, which the authors acknowledge but the relevance of which they do not discuss further. Interestingly, HDAC1 expression is reduced in Art resistant parasites and inhibition of HDAC1 (at what concentrations?) increases ART resitance both in wt and in K13 mutant parasites, suggesting regulation of HDAC1 may be involved in adapting to artemisinin treatment. ChIPseq of artemisinin resistant versus sensitive parasites suggests that HDAC1 is relocated to various different loci, although replicates for this experiment seem to be missing and hence the validity of these results would need further support, particularly because ChIP results conducted with anti-HDAC1 antibodies and anti-GFP antibodies seem to diverge considerably. Lastly, the authors propose that artemisinin treatment results in mistrageting of HDAC1 but find no correlation with gene expression. Generally, the study raises some interesting aspects related to a function of HDAC1 in artemisinin resistance but would benefit from more rigorous analyses and comparisons of the NGS data presented in correlation to each other (e.g. ChIP anti-GFP vs ChIP anti-HDAC1) as well as to published data sets (e.g. Huang et al). Sometimes it is difficult to assess which data set the authors refer to and whether transcriptional data were derived from RNA harvested in parallel to ChIPseq (matched) or whether they were performed independently or by others. Also, many assays seem to lack replicates and controls as outlined below.

    Major comments:

    • Transcriptomic data of parasites treated with Romidepsin are presented as a proxy for HDAC1 function and indicate deregulation of invasion pathways, however what is the evidence that romidepsin targets (exlusively) HDAC1? This could for example be addressed by comparing the Romidepsin IC50 in HDAC1 overexpressing parasites versus parasites with WT levels of HDAC1.
    • How do the rhomidepsin treatment data correlate with JX21108 RNAseq results, a validated HDAC1 targeting compound? The authors need to thoroughly cross evaluate their data with the RNAseq data set from HDAC1 knockdown parasites and JX21108 treated parasites presented in Huang et al, 2020.
    • What is the overlap between genes deregulated after rhomidepsin treatment and ChIPseq targets?
    • What are the target genes that show strong enrichment in the gene body in Fig. 2E? How are the data sorted? It is expected that HDAC1 may affect gene expression differently when it is present in the gene body to when it is present in the promoter region, therefore it would be useful to stratify the target genes by peak position relative to genetic elements.
    • How do the anti-GFP ChIPseq data in K13WT strains (496 target genes, Fig 5) correlate with the anti-HDAC1 ChIPseq data (1409 target genes Fig 2 and 6)? There seems to be limited overlap in the target sites in number and quality, but it is difficult to assess just from looking at gene numbers and GO analyses. The data sets need to be more thoroughly cross-validated. What proportion of peaks overlap and where?
    • How many replicates were performed for each experiment? Many of the Figures showing recombinant assays and ChIPseq assays seem to represent only a single biological replicate (e.g. Fig 1E histone deacetylation assay, Fig. 5A, Fig 6C: ChIP under Artemisinin treatment).
    • Several critical controls are missing, for example Figure 1E/ Suppl. Figure 2D loading control (anti-H3). How was densitometry normalized?
    • What was the parasite age in RNAseq of HDAC1-GFP-GlmS parasites? Were the two data sets from different parasite lines adjusted for parasite age? How many replicates for RNAseq?
    • The data in Figure 6A Lanes 1-5 are evidently the same as shown in Fig 1D. The presentation of Art treated data as a single lane 6 without direct reference is not convincing as this does not allow a direct comparison of loading and between conditions.
    • How does histone acetylation change in response to Art treatment?

    Minor comments:

    • Page 4 top paragraph: check whether Ref 16 and 17 are correctly cited here.
    • In all Figures: specify drug concentrations and number of replicates.
    • What concentrations of etinostat, DHA, Romidepsin were used to treat parasites? Please provide exact concentrations of treatments ( not just +, ++, for example for TBB Fig 1, Artemisinin 6A), what was the "continuous sublethal dosage of romidepsin" exactly, what is the IC50 of romidepsin?
    • What is referred to as control in Fig. 1E?
    • Fig 4F please specify in the figure legend what the control was
    • Fig 4G the labelling of the circus plot is unreadably small.
    • Figure 5G and H: what RNAseq data set is shown here? Are these matched RNAseq data from these ChIP assays or other?
    • The calculation of how the growth curves were corrected as "GFP-glmS corrected growth curves" is unclear, please provide exact formula. Generally, the multiplication rates even in untreated conditions appear rather low in all experimetns (for example Fig 4D, only less than 2-fold growth after 1 cycle, 4 fold growth after 2 cycles.... Do the parasites under the normal growth conditions really only duplicate in each cycle? This seems a very low multiplication rate even for static in vitro culture of P. falciparum.
    • What is the relevance of the 2xFKBP in the tagging construct?

    Referees cross-commenting

    All reviewers agree that the manuscript in its current form would benefit from the addition of controls and replicates as well as additional time points for RNAseq and ChIP experiements.

    Significance

    Generally, the study raises some interesting aspects related to a function of HDAC1 in artemisinin resistance but would benefit from more rigorous analyses and comparisons of the NGS data presented in correlation to each other (e.g. ChIP anti-GFP vs ChIP anti-HDAC1) as well as to published data sets (e.g. Huang et al). Sometimes it is difficult to assess which data set the authors refer to and whether transcriptional data were derived from RNA harvested in parallel to ChIPseq (matched) or whether they were performed independently or by others. Also, many assays seem to lack replicates and controls as outlined below.

    My personal field of research is chromatin biology and antigenic variation in malaria parasites.

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    Referee #1

    Evidence, reproducibility and clarity

    This study characterises a Plasmodium class I Histone deacetylase (PfHDAC1). The manuscript reports a wide range of experiments - some of them complex and involved, but not all of these experiments appear to be well controlled, and some are insufficiently described to know if they have been appropriately designed and interpreted. A link to HDAC1 regulation and artemisinin resistance is advanced, but the evidence here is very indirect and inconclusive.

    The study shows that HDAC1 interacts with PfCKII- a homologue of the mammalian casein kinase known to interact with mammalian HDAC1. They also demonstrate that, at least in vitro, HDAC1 can serve as a substrate for phosphorylation by PfCKII, and that this phosphorylation impacts HDAC1's deacetylation of histones. Such assays where a kinase is provided with a single, abundant substrate in vitro, are not always rigourous tests for kinase specificity, but do in this case at least indicate that HDAC1 associated with its activity.

    Major issues:

    1. The authors conduct CHiP seq experiments on a GFP tagged HDAC. It is unclear from the methods and results section what control is used in these experiments. The ENCODE consortium has established minimum standards (Landt et al 2012) for conducting and reporting CHiP seq experiments, and states that the "recommended control for epitope-tagged measurements is an immunoprecipitation using the same antibody against the epitope tag in otherwise identical cells that do not express the tagged factor.". These experiments appear to lack that control and the enrichments reported should be approached with caution in the absence of such a control.
    2. The genes with apparently altered ChiP seq were subjected to gene ontology enrichment analysis, and the authors report potential enrichments - which appear to impact a range of unconnected biological pathways throughout the parasite and throughout the lifecycle, despite the CHIP seq being conducted only at a single time stage. No mention is made of correction for multiple hypothesis testing, known to present a considerable problem for such analyses, and no correction is described for background GO distributions in the P. falciparum genome, so again it's unknown if or how that was performed. The reported enriched categories must be also treated with considerable caution given the absence of description of these crucial steps. The authors report from this section that HDAC1 is associated with stress responses, but really, by their criteria, HDAC1 is associated with 1/3 of the whole genome, so it's a bit selective to regard it as a stress regulator
    3. The authors preform a well-designed series of transfection experiments with modulation of HDAC1 to show that an overexpression of HDAC1 leads to increased growth rate, and that this increase reduces when the overexpression of HDAC1 is inducibly repressed. However, I found the presentation of results from these experiments difficult to understand and there is considerable transformation of the data prior to plotting - they would be easier to understand if no background subtraction to normalise for GFP were conducted, and if all strains were plotted on the same axes. A potential confounding factor in this experiment is that many lines overexpressing GFP grow more slowly, and that this growth defect can be localisation dependent, so that over-expression of GFP alone may cause a different growth penalty than GFP on a nuclear protein. I am uncertain that the conclusion of 50% faster growth is a safe one based on these graphs - at some time intervals the over-expressor appears to grow just as slow or even slower (as a percentage of the previous timepoint) than the control, and these appear to have been based on technical replicates of a single biological experiment. The authors contend that the growth rate is due to changed expression of invasion genes (among many other substrate gene categories) giving rise to enhanced invasion - such a phenomenon is readily testable, and the authors should dissect this if they wish to substantiate the frankly surprising claim that overexpression of HDAC leads to increased growth rate.
    4. The authors also report an apparent down regulation of HDAC abundance in artemisinin resistant parasites. This conflicts with previous global proteomic analyses of artemisinin resistant parasites which found no such change in HDAC1 regulation or abundance (eg Siddiqui et al 2017, Yang et al 2019). Stage matching is a particular challenge in such experiments given the differences in cycle progression between ARTR and ARTS parasites, and it isn't clear that this has been adequately controlled for to have confidence in these results, particularly given their contradiction of previous analyses. The abundance of PfHDAC1 changes considerably throughout the asexual intraerythrocytic cycle, (out of synch with the control used here actin), so potential stage-mismatch might contribute to apparent differences here. Again, explicit mention of replicates is lacking. The authors also mention genes regulated by HDAC1 as including genes related to processes related to artemisin resistance, but this is hard to sustain - indeed with so many genes apparently substrates of HDAC1 it would be highly surprising if there were no overlap with some genes in pathways related to artemisin resistance. An accompanying experiment demonstrating an increase in survival (of both ART resistant and ART sensitive lines) in an artemisinin ring stage survival assay is intriguing, after using a possible inhibitor of HDAC but these results are hard to reconcile with a dynamic transcriptional response. (Why was this done with an uncharacterised inhibitor, rather than the more specific HDAC1 overexpressor/knockdown system? An accompanying RNAseq analysis is described, but the analysis is piecemeal and selective, with the authors pointing out candidate genes representing categories plausibly linked to artemisinin resistance. I found this section unconvincing and indirect - lots of genes are changed in these experiments, and so they inevitably include some that are feasibly linked to artemisinin resistance, but the one gene convincingly known to modulate resistance, K13, isn't mentioned, and presumably wasn't specifically changed in this analysis.
    5. A previous study by the laboratory of Christian Doerig (Eukaryot Cell. 2010 Jun; 9(6): 952-959.) reported that HDAC1 activity (unclear which of the HDACs) is associated with Pfcrk-3). This activity may not correspond to the HDAC1 characterised here, but deserves some discussion.
    6. The Western blots are letterboxed and in some cases appear to crop out bands on the limit of the image (eg Fig 5, 6). Please provide fuller pictures of the blots and indicate the relevant bands if there are several background bands.

    Minor issues

    The text uses breaking spaces for the gap between genus abbreviation and species throughout. Replace with non-breaking spaces. Abstract: "is correlated with parasitemia progression" - Unclear meaning. Reword. Introduction "closes in on 400,000 deaths annually" Unclear meaning/vernacular usage. Reword. Very long paragraph on pages 3-4. Reorder logical flow and break into smaller paragraphs to make this more easily read. "Given the evidence of the role of HDAC inhibition in the emergence of chemotherapeutic resistance in mammalian system" - needs a reference - no mention of this phenomenon up until this point of the manuscript

    Referees cross-commenting

    I agree with the other reviewers comments. Although the manuscript contains a very large number of complex experiments, necessary controls, sufficient replicates, and appropriate analysis are missing from many of the experiments.

    I appreciate that the experiments referred to would require a very substantial time and resource commitment to complete, but in their current form, many of these experiments are not safely interpretable.

    Significance

    This manuscript makes major claims for HDAC1, in particular for its role in artemisinin resistance. Such a link would be significant, but I regard few of these claims as having been robustly substantiated in this manuscript. The CHIP-seq evidence is of interest as a useful dataset, particularly if accompanied by relevant controls

  5. Version published to 10.1101/2022.05.01.490232v1 on bioRxiv