Investigation of stress responses as determinants of artemisinin resistance and cellular heterogeneity in Plasmodium falciparum

Abstract

Plasmodium falciparum has evolved resistance to almost all front-line drugs including artemisinins, 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 and multi-variant family genes in poised state and regulates their expression under stress conditions. We have also provided biochemical and cellular evidences that PfGCN5 regulates stress responsive genes by acetylation of PfAlba3. 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. Moreover, inhibition of PfGCN5 in artemisinin resistant parasites reverses 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 potential role in modulating artemisinin drug resistance. Moreover, stress responses and drug resistance can show significant variation in cellular adaptation due to phenotypic cell-to-cell variability. To investigate this, we performed single cell RNA-sequencing (scRNA-seq) to quantify the cellular heterogeneity under temperature stress condition. High-resolution clustering of scRNA-seq datasets and a combination of gene signatures allow identification of cellular heterogeneity and stage transition during stress adaptation. Interestingly, we identified a rare population of cells, which is only emerged during the stress condition, showing the reactive state of the pathogen against the temperature stress condition. Thus, in this study, we have identified PfGCN5 as a global modulator of artemisinin resistance and revealed magnitude of gene expression heterogeneity under temperature stress condition within populations of P. falciparum.