Transcriptome-module phenotype association study implicates extracellular vesicles biogenesis in Plasmodium falciparum artemisinin resistance

Abstract

Plasmodium falciparum malaria is still an important disease in sub-Saharan Africa (sSA). Great strides have been made in its control spear-headed by artemisinin (ART)-based combination therapies (ACTs). However, concerns about the imminent spread of ART-resistant (ARTr) malaria parasites to sSA threaten gains already made. Attempts to mitigate this risk have highlighted the need to discover novel P. falciparum drug targets. Therefore, studies to deepen our understanding of the biology of P. falciparum are needed. The role of extracellular vesicles (EVs) in the biology of malaria parasites is not fully understood. Recently, the ART resistance-associated transcriptional profile has been reported to involve several biological processes connected to vesicular trafficking, proteotoxic stress, erythrocyte remodelling, and mitochondrial metabolism. We explored a role for EVs in developing the P. falciparum ARTr phenotype using bulk RNA sequencing of unsynchronized parasite cultures under untreated, 0.1% dimethyl sulfoxide and 700nM dihydroartemisinin treated conditions for six hours. As pathway and gene ontology analysis is limited in its curated knowledge repertoire on EVs biogenesis in P. falciparum, we used a modular (gene set) analysis approach to explore whether an EVs biogenesis module is associated with the ARTr phenotype in P. falciparum. We first generated well-defined EVs modules of interest and used statistical tools to determine differences in their expression among the parasite and treatment conditions. Then we used gene set enrichment analysis to determine the strength of the association between each EVs module of interest and the ARTr phenotype. This transcriptome-module phenotype association study (TMPAS) represents a well-powered approach to making meaningful discoveries out of bulk gene expression data. We identified four EVs module of interest and report that one module representing gene sets with correlated expression to PF3D7_1441800 – involved with EVs biogenesis in P. falciparum - is associated with the ARTr phenotype (R539T_DHA_treated versus R539T_untreated: normalized enrichment score (NES) = 1.1830174, FDR q-value < 0.25; C580R_DHA_treated versus C580R_untreated: NES = 1.2457103, FDR q-value < 0.25). PF3D7_1441800 has been reported to reduce EVs production when knocked out in P. falciparum. Altogether, our findings suggest a role for EVs in developing ART resistance and warrant further studies interrogating this association.

Exosome‐derived long noncoding RNAs: Mediators of host–Plasmodium parasite communication

“…Second, Tandoh and his colleagues revealed the involvement of EVs in the dissemination of artemisinin-resistant malaria parasites, representing a potential novel drug target for P. falciparum (Tandoh et al, 2022). In addition, EVs derived from P. vivax liver hypnozoite were found to carry inflammation markers associated with active schizont replication, demonstrating the diagnostic biomarker potential of EVs in malaria through mass spectrometry identification (Gualdron-Lopez et al, 2022).…”

“…Second, Tandoh and his colleagues revealed the involvement of EVs in the dissemination of artemisinin‐resistant malaria parasites, representing a potential novel drug target for P . falciparum (Tandoh et al, 2022). In addition, EVs derived from P .…”

Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites

“…This preliminary implication of EVs in the expression of ART resistance in malaria parasites requires further validation. However, this finding has implicated EV biogenesis as a potential drug discovery space for mitigating the risk of ART resistance in malaria parasites [12,18,19].…”

“…The findings of this study provided the first evidence of the putative genes involved in EV biogenesis in P. falciparum. [18] This study leveraged on the findings of [16] to show that a module (set of genes with similar expression profiles) containing the PfVps60 gene was associated with the ART-resistant phenotype. It provided nascent evidence to support the predictions of the EV export hypothesis.…”

“…Finally, we used these modules as the exposure/independent variable to determine their association with the ART‐resistant phenotype and opine that the advantage of this transcriptome module–phenotype association study was that it was not limited by the lack of representation on EV biogenesis genes in gene ontology databases and it did not assume that only quantitatively differentially expressed genes were drivers of the biological phenotype of interest. In conclusion, we used a transcriptome module–phenotype association study to show that two modules of EV interest were associated with the ART‐resistant phenotype in P. falciparum [18]. This preliminary implication of EVs in the expression of ART resistance in malaria parasites requires further validation.…”