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.

Identification and structural validation of purine nucleoside phosphorylase from Plasmodium falciparum as a target of MMV000848.

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites. A global effort to identify lead compounds led to several initiatives such as the Medicine for Malaria Ventures (MMV), a repository of compounds showing promising efficacy in killing the parasite in cell-based assays. Here, we used mass spectrometry coupled with cellular thermal shift assay to identify putative protein targets of MMV000848, a compound with an in vitro EC50 of 0.5 µM against the parasite. Thermal shift assays showed a strong increase of P. falciparum purine nucleoside phosphorylase (PfPNP) melting temperature by up to 15 °C upon incubation with MMV000848. Binding and enzymatic assays returned a KD of 1.52 ± 0.495 µM and an IC50 value of 21.5 ± 2.36 µM. The inhibition is competitive with respect to the substrate, as confirmed by a cocrystal structure of PfPNP bound with MMV000848 at the active site, determined at 1.85 Å resolution. In contrast to transition states inhibitors, MMV000848 specifically inhibits the parasite enzyme but not the human ortholog. An isobologram analysis shows subadditivity with immucillin H and with quinine respectively, suggesting overlapping modes of action between these compounds. These results point to PfPNP as a promising antimalarial target and suggest avenues to improve inhibitor potency.

Employment of a high throughput functional assay to define the critical factors that influence vaccine induced cross-variant neutralizing antibodies for SARS-CoV-2.

The scale and duration of neutralizing antibody responses targeting SARS-CoV-2 viral variants represents a critically important serological parameter that predicts protective immunity for COVID-19. In this study, we describe the development and employment of a new functional assay that measures neutralizing antibodies for SARS-CoV-2 and present longitudinal data illustrating the impact of age, sex and comorbidities on the kinetics and strength of vaccine-induced antibody responses for key variants in an Asian volunteer cohort. We also present an accurate quantitation of serological responses for SARS-CoV-2 that exploits a unique set of in-house, recombinant human monoclonal antibodies targeting the viral Spike and nucleocapsid proteins and demonstrate a reduction in neutralizing antibody titres across all groups 6 months post-vaccination. We also observe a marked reduction in the serological binding activity and neutralizing responses targeting recently newly emerged Omicron variants including XBB 1.5 and highlight a significant increase in cross-protective neutralizing antibody responses following a third dose (boost) of vaccine. These data illustrate how key virological factors such as immune escape mutations combined with host demographic factors such as age and sex of the vaccinated individual influence the strength and duration of cross-protective serological immunity for COVID-19.

Comparative spatial proteomics of Plasmodium-infected erythrocytes.

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

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.

Rapid Evaluation of Vaccine Booster Effectiveness against SARS-CoV-2 Variants.

As the COVID-19 pandemic continues, countries around the world are switching toward vaccinations and boosters to combat the pandemic. However, waning immunity against SARS-CoV-2 wild-type (WT) and variants have been widely reported. Booster vaccinations have shown to be able to increase immunological protection against new variants; however, the protection observed appears to decrease quickly over time suggesting a second booster shot may be appropriate. Moreover, heterogeneity and waning of the immune response at the individual level was observed suggesting a more personalized vaccination approach should be considered. To evaluate such a personalized strategy, it is important to have the ability to rapidly evaluate the level of neutralizing antibody (nAbs) response against variants at the individual level and ideally at a point of care setting. Here, we applied the recently developed cellulose pulled-down virus neutralization test (cpVNT) to rapidly assess individual nAb levels to WT and variants of concerns in response to booster vaccination. Our findings confirmed significant heterogeneity of nAb responses against a panel of SARS-CoV-2 variants, and indicated a strong increase in nAb response against variants of concern (VOCs) upon booster vaccination. For instance, the nAb response against current predominant omicron variant was observed with medians of 88.1% (n = 6, 95% CI = 73.2% to 96.2%) within 1-month postbooster and 70.7% (n = 22, 95% CI = 66.4% to 81.8%) 3 months postbooster. Our data show a point of care (POC) test focusing on nAb response levels against VOCs can guide decisions on the potential need for booster vaccinations at individual level. Importantly, it also suggests the current booster vaccines only give a transient protective response against some VOC and new more targeted formulations of a booster vaccine against specific VOC may need to be developed in the future. IMPORTANCE Vaccination against SARS-CoV-2 induces protection through production of neutralization antibodies (nAb). The level of nAb is a major indicator of immunity against SARS-CoV-2 infection. We developed a rapid point-of-care test that can monitor the nAb level from a drop of finger stick blood. Here, we have implemented the test to monitor individual nAb level against wild-type and variants of SARS-CoV-2 at various time points of vaccination, including post-second-dose vaccination and postbooster vaccination. Huge diversity of nAb levels were observed among individuals as well as increment in nAb levels especially against Omicron variant after booster vaccination. This study evaluated the performance of this point-of-care test for personalized nAb response tracking. It verifies the potential of using a rapid nAb test to guide future vaccination regimens at both the individual and population level.

Finger stick blood test to assess postvaccination SARS-CoV-2 neutralizing antibody response against variants.

There is clinical need for a quantifiable point-of-care (PoC) SARS-CoV-2 neutralizing antibody (nAb) test that is adaptable with the pandemic's changing landscape. Here, we present a rapid and semi-quantitative nAb test that uses finger stick or venous blood to assess the nAb response of vaccinated population against wild-type (WT), alpha, beta, gamma, and delta variant RBDs. It captures a clinically relevant range of nAb levels, and effectively differentiates prevaccination, post first dose, and post second dose vaccination samples within 10 min. The data observed against alpha, beta, gamma, and delta variants agrees with published results evaluated in established serology tests. Finally, our test revealed a substantial reduction in nAb level for beta, gamma, and delta variants between early BNT162b2 vaccination group (within 3 months) and later vaccination group (post 3 months). This test is highly suited for PoC settings and provides an insightful nAb response in a postvaccinated population.

Development and translation of a paper-based top readout vertical flow assay for SARS-CoV-2 surveillance.

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

MalariaCometChip for high-throughput quantification of DNA damage in Plasmodium falciparum.

Comet assay is a standard approach for studying DNA damage in malaria, but high-throughput options are not available. The CometChip was previously developed using mammalian cells as a high-throughput version of the comet assay. It is based on the same principle as the comet assay but provides greater efficacy, automated data processing, and improved consistency between experiments. In this protocol, we present MalariaCometChip to quantitatively assess drug-induced DNA damage in Plasmodium falciparum. For complete details on the use and execution of this protocol, please refer to Xiong et al. (2020).

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.

Vertical Flow Cellulose-Based Assays for SARS-CoV-2 Antibody Detection in Human Serum.

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies.

Background: Neutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive. Method: We develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor-RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper. Results: Here we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma. Conclusions: A rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.

Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture.

In vitro erythroid cultures from human hematopoietic stem cells produce immature red blood cells (RBCs) called reticulocytes, which are important for RBCs production, and are widely used in scientific studies of malaria pathology, hematological diseases and protein translation. However, in vitro reticulocyte cultures contain expelled cell nuclei and erythroblasts as undesirable by-products and current purification methods such as density gradient centrifugation and fluorescence-activated cell sorting (FACS) are not optimal for integrated bioprocessing and downstream therapeutic applications. Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) microfluidic sorting methods are ideal alternatives due to label-free size sorting, throughput scalability and low manufacturing cost. DFF sorting of reticulocytes from whole erythroid culture showed a 2.4-fold increase in cell recovery compared to FACS albeit with a lower purity; DLD sorting showed comparable cell recovery and purity with FACS using an inverse-L pillar structure to emphasize size and deformability sorting of reticulocytes. The viability and functional assurance of purified reticulocytes showed conserved cell deformability and supported the propagation of malaria parasites. Collectively, our study on label-free RBCs isolation represents a significant technical advancement towards developing in vitro generated viable human RBCs, opening opportunities for close-loop cell manufacturing, downstream therapeutic and research purposes.

K13-Mediated Reduced Susceptibility to Artemisinin in Plasmodium falciparum Is Overlaid on a Trait of Enhanced DNA Damage Repair.

Southeast Asia has been the hotbed for the development of drug-resistant malaria parasites, including those with resistance to artemisinin combination therapy. While mutations in the kelch propeller domain (K13 mutations) are associated with artemisinin resistance, a range of evidence suggests that other factors are critical for the establishment and subsequent transmission of resistance in the field. Here, we perform a quantitative analysis of DNA damage and repair in the malaria parasite Plasmodium falciparum and find a strong link between enhanced DNA damage repair and artemisinin resistance. This experimental observation is further supported when variations in seven known DNA repair genes are found in resistant parasites, with six of these mutations being associated with K13 mutations. Our data provide important insights on confounding factors that are important for the establishment and spread of artemisinin resistance and may explain why resistance has not yet arisen in Africa.

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.

Rapid activation of distinct members of multigene families in Plasmodium spp.

The genomes of Plasmodium spp. encode a number of different multigene families that are thought to play a critical role for survival. However, with the exception of the P. falciparum var genes, very little is known about the biological roles of any of the other multigene families. Using the recently developed Selection Linked Integration method, we have been able to activate the expression of a single member of a multigene family of our choice in Plasmodium spp. from its endogenous promoter. We demonstrate the usefulness of this approach by activating the expression of a unique var, rifin and stevor in P. falciparum as well as yir in P. yoelii. Characterization of the selected parasites reveals differences between the different families in terms of mutual exclusive control, co-regulation, and host adaptation. Our results further support the application of the approach for the study of multigene families in Plasmodium and other organisms.

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation.

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand-receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in-situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.

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 profiling reveals functional variation in Plasmodium falciparum parasites from controlled human malaria infection studies.

BACKGROUND: The transcriptome of Plasmodium falciparum clinical isolates varies according to strain, mosquito bites, disease severity and clinical history. Therefore, it remains a challenge to directly interpret the parasite's transcriptomic information into a more general biological signature in a natural human malaria infection. These confounding variations can be potentially overcome with parasites derived from controlled-human malaria infection (CHMI) studies. METHODS: We performed CHMI studies in healthy and immunologically naïve volunteers receiving the same P. falciparum strain ((Sanaria® PfSPZ Challenge (NF54)), but with different sporozoite dosage and route of infection. Parasites isolated from these volunteers at the day of patency were subjected to in vitro culture for several generations and synchronized ring-stage parasites were subjected to transcriptome profiling. FINDINGS: We observed clear deviations between CHMI-derived parasites from volunteer groups receiving different PfSPZ dose and route. CHMI-derived parasites and the pre-mosquito strain used for PfSPZ generation showed significant transcriptional variability for gene clusters associated with malaria pathogenesis, immune evasion and transmission. These transcriptional variation signature clusters were also observed in the transcriptome of P. falciparum isolates from acute clinical infections. INTERPRETATION: Our work identifies a previously unrecognized transcriptional pattern in malaria infections in a non-immune background. Significant transcriptome heterogeneity exits between parasites derived from human infections and the pre-mosquito strain, implying that the malaria parasites undergo a change in functional state to adapt to its host environment. Our work also highlights the potential use of transcriptomics data from CHMI study advance our understanding of malaria parasite adaptation and transmission in humans. FUND: This work is supported by German Israeli Foundation, German ministry for education and research, MOE Tier 1 from the Singapore Ministry of Education Academic Research Fund, Singapore Ministry of Health's National Medical Research Council, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA and the German Centre for Infection Research (Deutsches Zentrum für Infektionsforschung-DZIF).

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.