Charting new territory: The Plasmodium falciparum tRNA modification landscape.

Ribonucleoside modifications comprising the epitranscriptome are present in all organisms and all forms of RNA, including mRNA, rRNA and tRNA, the three major RNA components of the translational machinery. Of these, tRNA is the most heavily modified and the tRNA epitranscriptome has the greatest diversity of modifications. In addition to their roles in tRNA biogenesis, quality control, structure, cleavage, and codon recognition, tRNA modifications have been shown to regulate gene expression post-transcriptionally in prokaryotes and eukaryotes, including humans. However, studies investigating the impact of tRNA modifications on gene expression in the malaria parasite Plasmodium falciparum are currently scarce. Current evidence shows that the parasite has a limited capacity for transcriptional control, which points to a heavier reliance on strategies for posttranscriptional regulation such as tRNA epitranscriptome reprogramming. This review addresses the known functions of tRNA modifications in the biology of P. falciparum while highlighting the potential therapeutic opportunities and the value of using P. falciparum as a model organism for addressing several open questions related to the tRNA epitranscriptome.

tRNA modification reprogramming contributes to artemisinin resistance in Plasmodium falciparum.

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

Cohesin contributes to transcriptional repression of stage-specific genes in the human malaria parasite.

The complex life cycle of the human malaria parasite, Plasmodium falciparum, is driven by specific transcriptional programs, but it is unclear how most genes are activated or silenced at specific times. There is an association between transcription and spatial organization; however, the molecular mechanisms behind genome organization are unclear. While P. falciparum lacks key genome-organizing proteins found in metazoans, it has all core components of the cohesin complex. To investigate the role of cohesin in P. falciparum, we functionally characterize the cohesin subunit Structural Maintenance of Chromosomes protein 3 (SMC3). SMC3 knockdown during early stages of the intraerythrocytic developmental cycle (IDC) upregulates a subset of genes involved in erythrocyte egress and invasion, which are normally expressed at later stages. ChIP-seq analyses reveal that during the IDC, SMC3 enrichment at the promoter regions of these genes inversely correlates with gene expression and chromatin accessibility. These data suggest that SMC3 binding contributes to the repression of specific genes until their appropriate time of expression, revealing a new mode of stage-specific gene repression in P. falciparum.

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis.

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyze the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identify large decreases in N2-methyladenosine (m2A) in both 23 S ribosomal RNA and transfer RNA. This we determine to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gives rise to a proteome that mimics the oxidative stress response, with an increase in levels of superoxide dismutase and decrease in virulence proteins. While tRNA modifications were established to be dynamic for fine-tuning translation, here we report the discovery of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to a model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, adding a different paradigm in which RNA modifications can directly regulate the proteome.

Malaria Parasite Stress Tolerance Is Regulated by DNMT2-Mediated tRNA Cytosine Methylation.

Malaria parasites need to cope with changing environmental conditions that require strong countermeasures to ensure pathogen survival in the human and mosquito hosts. The molecular mechanisms that protect Plasmodium falciparum homeostasis during the complex life cycle remain unknown. Here, we identify cytosine methylation of tRNAAsp (GTC) as being critical to maintain stable protein synthesis. Using conditional knockout (KO) of a member of the DNA methyltransferase family, called Pf-DNMT2, RNA bisulfite sequencing demonstrated the selective cytosine methylation of this enzyme of tRNAAsp (GTC) at position C38. Although no growth defect on parasite proliferation was observed, Pf-DNMT2KO parasites showed a selective downregulation of proteins with a GAC codon bias. This resulted in a significant shift in parasite metabolism, priming KO parasites for being more sensitive to various types of stress. Importantly, nutritional stress made tRNAAsp (GTC) sensitive to cleavage by an unknown nuclease and increased gametocyte production (>6-fold). Our study uncovers an epitranscriptomic mechanism that safeguards protein translation and homeostasis of sexual commitment in malaria parasites. IMPORTANCE P. falciparum is the most virulent malaria parasite species, accounting for the majority of the disease mortality and morbidity. Understanding how this pathogen is able to adapt to different cellular and environmental stressors during its complex life cycle is crucial in order to develop new strategies to tackle the disease. In this study, we identified the writer of a specific tRNA cytosine methylation site as a new layer of epitranscriptomic regulation in malaria parasites that regulates the translation of a subset of parasite proteins (>400) involved in different metabolic pathways. Our findings give insight into a novel molecular mechanism that regulates P. falciparum response to drug treatment and sexual commitment.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

Functional Characterization of the m6A-Dependent Translational Modulator PfYTH.2 in the Human Malaria Parasite.

Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen.IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that "reads" this modification and "translates" it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease.

Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite.

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

Hydrogen Sulfide Sensitizes Acinetobacter baumannii to Killing by Antibiotics.

The production of endogenous hydrogen sulfide (H2S) has been shown to confer antibiotic tolerance in all bacteria studied to date. Therefore, this mediator has been speculated to be a universal defense mechanism against antibiotics in bacteria. This is assuming that all bacteria produce endogenous H2S. In this study, we established that the pathogenic bacteria Acinetobacter baumannii does not produce endogenous H2S, giving us the opportunity to test the effect of exogenous H2S on antibiotic tolerance in a bacterium that does not produce it. By using a H2S-releasing compound to modulate the sulfide content in A. baumannii, we demonstrated that instead of conferring antibiotic tolerance, exogenous H2S sensitized A. baumannii to multiple antibiotic classes, and was able to revert acquired resistance to gentamicin. Exogenous H2S triggered a perturbation of redox and energy homeostasis that translated into hypersensitivity to antibiotic killing. We propose that H2S could be used as an antibiotic-potentiator and resistance-reversion agent in bacteria that do not produce it.

Discovery of a new predominant cytosine DNA modification that is linked to gene expression in malaria parasites.

DNA cytosine modifications are key epigenetic regulators of cellular processes in mammalian cells, with their misregulation leading to varied disease states. In the human malaria parasite Plasmodium falciparum, a unicellular eukaryotic pathogen, little is known about the predominant cytosine modifications, cytosine methylation (5mC) and hydroxymethylation (5hmC). Here, we report the first identification of a hydroxymethylcytosine-like (5hmC-like) modification in P. falciparum asexual blood stages using a suite of biochemical methods. In contrast to mammalian cells, we report 5hmC-like levels in the P. falciparum genome of 0.2-0.4%, which are significantly higher than the methylated cytosine (mC) levels of 0.01-0.05%. Immunoprecipitation of hydroxymethylated DNA followed by next generation sequencing (hmeDIP-seq) revealed that 5hmC-like modifications are enriched in gene bodies with minimal dynamic changes during asexual development. Moreover, levels of the 5hmC-like base in gene bodies positively correlated to transcript levels, with more than 2000 genes stably marked with this modification throughout asexual development. Our work highlights the existence of a new predominant cytosine DNA modification pathway in P. falciparum and opens up exciting avenues for gene regulation research and the development of antimalarials.

Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development.

Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N6-methyladenosine (m6A) over the course of blood-stage development. Using mass spectrometry and m6A RNA sequencing, we demonstrate that m6A is highly developmentally regulated, exceeding m6A levels known in any other eukaryote. We characterize a distinct m6A writer complex and show that knockdown of the putative m6A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m6A. In accordance, we find an inverse correlation between m6A methylation and mRNA stability or translational efficiency. We further identify two putative m6A-binding YTH proteins that are likely to be involved in the regulation of these processes across the parasite's life cycle. Our data demonstrate unique features of an extensive m6A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.

tRNA epitranscriptomics and biased codon are linked to proteome expression in Plasmodium falciparum.

Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites.

Targeted Phenotypic Screening in Plasmodium falciparum and Toxoplasma gondii Reveals Novel Modes of Action of Medicines for Malaria Venture Malaria Box Molecules.

The Malaria Box collection includes 400 chemically diverse small molecules with documented potency against malaria parasite growth, but the underlying modes of action are largely unknown. Using complementary phenotypic screens against Plasmodium falciparum and Toxoplasma gondii, we report phenotype-specific hits based on inhibition of overall parasite growth, apicoplast segregation, and egress or host invasion, providing hitherto unavailable insights into the possible mechanisms affected. First, the Malaria Box library was screened against tachyzoite stage T. gondii and the half-maximal effective concentrations (EC50s) of molecules showing ≥80% growth inhibition at 10 µM were determined. Comparison of the EC50s for T. gondii and P. falciparum identified a subset of 24 molecules with nanomolar potency against both parasites. Thirty molecules that failed to induce acute growth inhibition in T. gondii tachyzoites in a 2-day assay caused delayed parasite death upon extended exposure, with at least three molecules interfering with apicoplast segregation during daughter cell formation. Using flow cytometry and microscopy-based examinations, we prioritized 26 molecules with the potential to inhibit host cell egress/invasion during asexual developmental stages of P. falciparum. None of the inhibitors affected digestive vacuole integrity, ruling out a mechanism mediated by broadly specific protease inhibitor activity. Interestingly, five of the plasmodial egress inhibitors inhibited ionophore-induced egress of T. gondii tachyzoites. These findings highlight the advantage of comparative and targeted phenotypic screens in related species as a means to identify lead molecules with a conserved mode of action. Further work on target identification and mechanism analysis will facilitate the development of antiparasitic compounds with cross-species efficacy. IMPORTANCE The phylum Apicomplexa includes many human and animal pathogens, such as Plasmodium falciparum (human malaria) and Toxoplasma gondii (human and animal toxoplasmosis). Widespread resistance to current antimalarials and the lack of a commercial vaccine necessitate novel pharmacological interventions with distinct modes of action against malaria. For toxoplasmosis, new drugs to effectively eliminate tissue-dwelling latent cysts of the parasite are needed. The Malaria Box antimalarial collection, managed and distributed by the Medicines for Malaria Venture, includes molecules of novel chemical classes with proven antimalarial efficacy. Using targeted phenotypic assays of P. falciparum and T. gondii, we have identified a subset of the Malaria Box molecules as potent inhibitors of plastid segregation and parasite invasion and egress, thereby providing early insights into their probable mode of action. Five molecules that inhibit the egress of both parasites have been identified for further mechanistic studies. Thus, the approach we have used to identify novel molecules with defined modes of action in multiple parasites can expedite the development of pan-active antiparasitic agents.

Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

Modulation of the Vault Protein-Protein Interaction for Tuning of Molecular Release.

Vaults are naturally occurring ovoid nanoparticles constructed from a protein shell that is composed of multiple copies of major vault protein (MVP). The vault-interacting domain of vault poly(ADP-ribose)-polymerase (INT) has been used as a shuttle to pack biomolecular cargo in the vault lumen. However, the interaction between INT and MVP is poorly understood. It is hypothesized that the release rate of biomolecular cargo from the vault lumen is related to the interaction between MVP and INT. To tune the release of molecular cargos from the vault nanoparticles, we determined the interactions between the isolated INT-interacting MVP domains (iMVP) and wild-type INT and compared them to two structurally modified INT: 15-amino acid deletion at the C terminus (INTΔC15) and histidine substituted at the interaction surface (INT/DSA/3 H) to impart a pH-sensitive response. The apparent affinity constants determined using surface plasmon resonance (SPR) biosensor technology are 262 ± 4 nM for iMVP/INT, 1800 ± 160 nM for iMVP/INTΔC15 at pH 7.4. The INT/DSA/3 H exhibits stronger affinity to iMVP (K Dapp = 24 nM) and dissociates at a slower rate than wild-type INT at pH 6.0.

P. falciparum RH5-Basigin interaction induces changes in the cytoskeleton of the host RBC.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.

Synthesis and in vitro evaluation of hydrazinyl phthalazines against malaria parasite, Plasmodium falciparum.

In this report, we describe the synthesis of 1-(Phthalazin-4-yl)-hydrazine using bronsted acidic ionic liquids and demonstrate their ability to inhibit asexual stage development of human malaria parasite, Plasmodium falciparum. Through computational studies, we short-listed chemical scaffolds with potential binding affinity to an essential parasite protein, dihydroorotate dehydrogenase (DHODH). Further, these compounds were synthesized in the lab and tested against P. falciparum. Several compounds from our library showed inhibitory activity at low micro-molar concentrations with minimal cytotoxic effects. These results indicate the potential of hydralazine derivatives as reference scaffolds to develop novel antimalarials.

Synthesis, characterization and in vitro evaluation of novel enantiomerically-pure sulphonamide antimalarials.

Malaria parasites are currently gaining drug-resistance rapidly, across countries and continents. Hence, the discovery and development of novel chemical scaffolds, with superior antimalarial activity remain an important priority, for the developing world. Our report describes the development, characterization and evaluation of novel bepotastine-based sulphonamide antimalarials inhibiting asexual stage development of Plasmodium falciparum parasites in vitro. The screening results showed potent inhibitory activity of a number of novel sulphonamides against P. falciparum at low micromolar concentrations, in particular in late-stage parasite development. Based on computational studies we hypothesize N-myristoyltransferase as the target of the compounds developed here. Our results demonstrate the value of novel bepotastine-based sulphonamide compounds for targeting the asexual developmental stages of P. falciparum.

Single-cell evaluation of red blood cell bio-mechanical and nano-structural alterations upon chemically induced oxidative stress.

Erythroid cells, specifically red blood cells (RBCs), are constantly exposed to highly reactive radicals during cellular gaseous exchange. Such exposure often exceeds the cells' innate anti-oxidant defense systems, leading to progressive damage and eventual senescence. One of the contributing factors to this process are alterations to hemoglobin conformation and globin binding to red cell cytoskeleton. However, in addition to the aforementioned changes, it is possible that oxidative damage induces critical changes to the erythrocyte cytoskeleton and corresponding bio-mechanical and nano-structural properties of the red cell membrane. To quantitatively characterize how oxidative damage accounts for such changes, we employed single-cell manipulation techniques such as micropipette aspiration and atomic force microscopy (AFM) on RBCs. These investigations demonstrated visible morphological changes upon chemically induced oxidative damage (using hydrogen peroxide, diamide, primaquine bisphosphate and cumene hydroperoxide). Our results provide previously unavailable observations on remarkable changes in red cell cytoskeletal architecture and membrane stiffness due to oxidative damage. Furthermore, we also demonstrate that a pathogen that infects human blood cells, Plasmodium falciparum was unable to penetrate through the oxidant-exposed RBCs that have damaged cytoskeleton and stiffer membranes. This indicates the importance of bio-physical factors pertinent to aged RBCs and it's relevance to malaria infectivity.