Histone acetyltransferase PfGCN5 regulates stress responsive and artemisinin resistance related genes in Plasmodium falciparum

Abstract

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinin, which threatens malaria control and elimination strategies. Oxidative stress and protein damage responses have emerged as key players in the generation of artemisinin resistance. In this study, we show that PfGCN5, a histone acetyltransferase, binds to the stress-responsive genes in a poised state and regulates their expression under stress conditions. Furthermore, we show that upon artemisinin exposure, genome-wide binding sites for PfGCN5 are increased and it is directly associated with the genes implicated in artemisinin resistance generation like BiP and TRiC chaperone. Interestingly, expression of genes bound by PfGCN5 was found to be upregulated during stress conditions. Moreover, inhibition of PfGCN5 in artemisinin-resistant parasites increases the sensitivity of the parasites to artemisinin treatment indicating its role in drug resistance generation. Together, these findings elucidate the role of PfGCN5 as a global chromatin regulator of stress-responses with a potential role in modulating artemisinin drug resistance and identify PfGCN5 as an important target against artemisinin-resistant parasites.

Figure 1

PfGCN5 is associated with stress responsive genes. (A) An anti-PfGCN5 antibody was generated using the N-terminal peptide of the PfGCN5. Specificity of the antibody was tested using parasite protein lysate from asynchronous culture and uninfected RBCs. Western blotting results indicated presence of more than one form of PfGCN5 along with the full-length protein. The lower size bands are products of proteolytic cleavage as reported earlier and explained in Supplementary Figure S1B. (B) Heat map showing the ChIP-seq tag counts at 5712 P. falciparum genes for PfGCN5. PfGCN5 was found to be enriched mostly at the 3′ end of the genes and towards the centre of the genes. (C) ChIP-qPCR of selected genes confirms PfGCN5 binding to ChIP-seq targets. The results are shown as fold enrichment of ChIP performed with PfGCN5 α-peptide antibody versus non-immune IgG. Significance was determined using a paired t-test. *p < 0.05; ***p < 0.005. (D) RT-qPCR of the gene bound by PfGCN5 in the presence and absence of garcinol (10 µM). Significance was determined using a paired t-test. *p < 0.05; ***p < 0.005. (E) Gene ontology analysis of the PfGCN5 bound gene identified using ChIP sequencing. Stress responsive genes were overrepresented in gene ontology analysis.

Figure 2

PfGCN5 is not a general transcription coactivator; it is specifically associated with stimuli associated genes. (A) Box and whisker plots representing the correlation of genome-wide H3K9ac prevalence and PfGCN5 occupancy with the global gene expression. Absence of global correlation was found for the recruitment of PfGCN5 and gene expression. This indicates that PfGCN5 is not a general transcription coactivator. (B) The expression level of the genes bound by PfGCN5 in comparison to all the genes in P. falciparum is represented by the box plots. PfGCN5 is associated with highly as well as least expressed genes.

Figure 3

PfGCN5 is a specific stress regulator. (A) Schematic representation of the pipeline used for the stress experiment. (B) Change in the expression level of PfGCN5 during various stress conditions (N = 3). PfGCN5 is found to be upregulated during heat stress and artemisinin treatment conditions. Significance was determined using a paired t-test. *p < 0.05; ***p < 0.005. Data shows the mean ± SEM for three independent experiments. (C) MA plot showing the deregulation in the expression of protein coding genes during artemisinin treatment with 30 nM concentration for 6 h and during temperature stress at 40 °C for 6 h. (D) Venn diagram showing the genes upregulated during temperature stress and artemisinin treatment. Table representing the pathways enriched for the genes upregulated during temperature stress and artemisinin treatment. Gene ontology was performed using PlasmoDB. (E) Expression profiles of the genes bound to PfGCN5 during stress conditions. PfGCN5 bound genes are upregulated upon stress induction as compared to the control condition.

Figure 4

PfGCN5 shows prolific genomic binding during artemisinin treatment and in artemisinin resistant parasites. (A) Gene ontology analysis of the genes which are exclusively bound by PfGCN5 during artemisinin treatment. PfGCN5 is found to be enriched on the genes which are known to be deregulated in artemisinin resistant parasites indicating the possible role of PfGCN5 during resistance generation. (B) RT-qPCR results show upregulation of BiP and TCP1β during stress conditions. Garcinol treatment (10 µM) leads to decreased expression of BiP and TCP1β during stress conditions. Data shows the mean ± SEM for three independent experiments. Significance was determined using a paired t-test. *p < 0.05; ***p < 0.005. (C) Transcript level of expression of PfGCN5 in artemisinin resistant strains, K13-I543T (MRA-1241) and K13-C580Y (MRA-1236) in comparison to their sensitive counterparts, K13-I543wt (MRA-1253) and K13-C580wt (MRA-1254), respectively. (D,E) Change in the percentage parasite survival estimated through Ring Survival Assay (RSA) in presence of PfGCN5 inhibitor garcinol. (D) K13-I543T (MRA-1241) parasites were treated with 5 µM garcinol and (E) K13-C580Y (MRA-1236) parasites were treated with 250 µM garcinol. Presence of garcinol decreases the artemisinin resistance in K13-I543T (MRA-1241) at a concentration which has otherwise no effect on parasite growth. Higher concentration of garcinol was used with a significant decrease in resistance level in K13-C580Y (MRA-1236) parasites. (F) Gene ontology analysis of the genes which are bound by PfGCN5 in K13-I543T (MRA-1241), K13-C580Y (MRA-1236), K13-I543wt (MRA-1253), K13-C580wt (MRA-1254).

Figure 5

Mechanisms proposed for artemisinin resistance in P. falciparum. Model showing the role PfGCN5 in artemisinin resistance generation. Artemisinin treatment leads to random alkylation of proteins, which in turn are ubiquitinated by the protein ubiquitination and subjected to degradation by proteasome degradation. Massive alkylation by artemisinin exposure and/or oxidative stress activates PfGCN5 in the nucleus, which in turn upregulates the stress-responsive and unfolded protein response pathways that help in artemisinin resistance generation.