Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A₂ family protease that hydrolyzes ubiquitinated proteasome substrates. Treatment of P. falciparum parasites with ARTs leads to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of ubiquitinated proteins by inhibiting the PfDdi1 protease activity. Besides, whereas the PfDdi1 is predominantly localized in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, we show that Ddi1 knock-out Saccharomycescerevisiae cells are more susceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act in multiple ways; by inducing DNA and protein damage and might be impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.

Despite significant progress in apicomplexan genome sequencing and genomics, the current list of experimentally validated transcription factors (TFs) in these genomes is incomplete and mainly consists of AP2 family of proteins, with only a limited number of non-AP2 family TFs and transcription-associated co-factors (TcoFs). We have performed a systematic bioinformatics-aided prediction of TFs and TcoFs in apicomplexan genomes and developed the ApicoTFdb database which consists of experimentally validated as well as computationally predicted TFs and TcoFs in 14 apicomplexan species. The predicted TFs are manually curated to complement the existing annotations. The current version of the database includes 1292 TFs which includes experimentally validated and computationally predicted TFs, representing 20 distinct families across 14 apicomplexan species. The predictions include TFs of TUB, NAC, BSD, HTH, Cupin/Jumonji, winged helix and FHA family proteins, not reported earlier as TFs in the genomes. Apart from TFs, ApicoTFdb also classifies TcoFs into three main subclasses: TRs, CRRs and RNARs, representing 2491 TcoFs in 14 apicomplexan species, are analyzed in this study. The database is designed to integrate different tools for comparative analysis. All entries in the database are dynamically linked with other databases, literature reference, protein–protein interactions, pathways and annotations associated with each protein. ApicoTFdb will be useful to the researchers interested in less-studied gene regulatory mechanisms mediating the complex life cycle of the apicomplexan parasites. The database will aid in the discovery of novel drug targets to much needed combat the growing drug resistance in the parasites.

The meiotic recombination 11 protein (MRE11) plays a key role in DNA damage response and maintenance of genome stability. However, little is known about its function during development of the malaria parasite Plasmodium. Here, we present a functional, ultrastructural and transcriptomic analysis of Plasmodium parasites lacking MRE11 during its life cycle in both mammalian and mosquito vector hosts. Genetic disruption of Plasmodium berghei mre11 (PbMRE11) results in significant retardation of oocyst development in the mosquito midgut associated with cytoplasmic and nuclear degeneration, along with concomitant ablation of sporogony and subsequent parasite transmission. Further, absence of PbMRE11 results in significant transcriptional downregulation of genes involved in key interconnected biological processes that are fundamental to all eukaryotic life including ribonucleoprotein biogenesis, spliceosome function and iron–sulfur cluster assembly. Overall, our study provides a comprehensive functional analysis of MRE11′s role in Plasmodium development during the mosquito stages and offers a potential target for therapeutic intervention during malaria parasite transmission.

Cells use fatty acids (FAs) for membrane biosynthesis, energy storage and the generation of signaling molecules. 3-hydroxyacyl-CoA dehydratase – DEH – is a key component of very long chain FA (VLCFA) synthesis. Here, we further characterized in-depth the location and function of DEH, applying in silico analysis, live cell imaging, reverse genetics and ultrastructure analysis using the mouse malaria model Plasmodium berghei . DEH is evolutionarily conserved across eukaryotes, with a single DEH in Plasmodium spp. and up to three orthologs in the other eukaryotes studied. DEH-GFP live-cell imaging showed strong GFP fluorescence throughout the life-cycle, with areas of localized expression in the cytoplasm and a circular ring pattern around the nucleus that colocalized with ER markers. Δ deh mutants showed a small but significant reduction in oocyst size compared to WT controls from day 10 post-infection onwards and endomitotic cell division and sporogony were completely ablated, blocking parasite transmission from mosquito to vertebrate host. Ultrastructure analysis confirmed degeneration of Δ deh oocysts, and a complete lack of sporozoite budding. Overall, DEH is evolutionarily conserved, localizes to the ER and plays a crucial role in sporogony.

Despite significant progress in apicomplexans genome sequencing and genomics, the current list of experimentally validated TFs in these genomes is incomplete and mainly consists of AP2 family of proteins, with only a limited number of non-AP2 family TFs and TAFs. We have performed systematic bioinformatics aided prediction of TFs and TAFs in apicomplexan genomes, and developed ApicoTFdb database which consists of experimentally validated as well as computationally predicted TFs and TAFs in 14 apicomplexan species. The predicted TFs are manually curated to complement the existing annotations. The current version of the database includes 1310 TFs, out of which 833 are novel and computationally predicted TFs, representing 22 distinct families across 14 apicomplexan species. The predictions include TFs of TUB, NAC, BSD, CCAAT, HTH, Cupin/Jumonji, winged-helix, and FHA family proteins, not reported earlier in the genomes.Apart from TFs, ApicoTFdb also classifies TAFs into three main subclasses-TRs, CRRs and RNARs, representing 3047 TAFs in 14 apicomplexan species are analyzed in this study. The database is equipped with a set of useful tools for comparative analysis of a user-defined list of the proteins. ApicoTFdb will be useful to the researchers interested in less-studied gene regulatory mechanisms mediating the complex life cycle of the apicomplexan parasites. The database will aid the discovery of novel drug targets to much needed combat the growing drug resistance in the parasites.