Biology subjects

Wirth, D. F.

Publications and source records attributed to Wirth, D. F..

15 recordsLinked to original sources

bioRxiv2026

De novo minibinders targeting RH5 achieve nanomolar inhibition of blood-stage malaria replication

Malaria kills {approx}600,000 people annually despite recently deployed vaccines. The parasite's blood-stage invasion of erythrocytes, driven by the essential, non-redundant interaction between Plasmodium falciparum RH5 and erythrocyte basigin, remains a leading therapeutic target. Here, we designed de novo mini-proteins (minibinders) to occlude this interface. From {approx}1,500 computationally generated designs, we synthesized 18 candidates ranging from 9.3 to 16.9 kDa which were easily expressed in E. coli as soluble, well-folded proteins. Of these, 7 minibinders blocked blood-stage replication of P. falciparum asexual stage parasites with greater potency than {approx}300nM IC50. Three potent candidate minibinders, mb-5, mb-7, and mb-21, were confirmed to bind RH5 via SPR and BLI and block the merozoite-to-ring transition in stage-synchronized parasite progression assays, with CryoEM of minibinder-RH5 complex confirming the predicted binding at RH5-basigin interface. Although pharmacological challenges remain, our data demonstrate that potent inhibitors of parasite growth, rivaling existing antimalarials, can be designed rather than discovered via extensive screening. Our data highlight the potential for artificial intelligence to change the development landscape of new antimalarial therapies.

microbiology↗

bioRxiv2026

Genetic background determines allele-specific Pfkelch13-mediated artemisinin tolerance and persistence in Plasmodium falciparum

Artemisinin-based combination therapies are the frontline treatment for Plasmodium falciparum malaria, but their efficacy is threatened by Artemisinin partial resistance (ARTR). ARTR in Plasmodium falciparum is mediated by Pfkelch13 (k13) mutations but remains rare in West Africa. A critical question is whether short-term drug survival intrinsically predicts long-term evolutionary persistence. Here, using CRISPR-Cas9 editing, and nanopore-assisted allele tracking in a contemporary Senegalese isolate, we evaluated ARTR receptivity using the ring-stage survival assay (RSA), a proxy for clinical resistance. We demonstrate that ARTR survival and drug-free persistence are biologically separable traits. While the WHO-validated Asian I543T mutation conferred the RSA phenotype, it was rapidly counter-selected during drug-free growth. In contrast, emerging African alleles (R561H, M579I) and a regional novel variant (C473S) establish stable resistance without a competitive penalty. These findings reveal that a parasite's genetic background acts as a strict evolutionary filter, uncoupling short-term tolerance from long-term persistence and establishing that genomic context determines allele-specific resistance trajectories.

microbiology↗

bioRxiv2026

Severe malarial anaemia is associated with parasite enrichment and host alterations in the bone marrow

BackgroundMalaria poses a significant global health challenge, with over 200 million infections and around 500,000 deaths annually, predominantly affecting children under 5 in sub-Saharan Africa. Severe malarial anaemia (SMA), a major contributor to morbidity and mortality in this demographic, results from various factors including red blood cell destruction and immune-mediated clearance. The role of these mechanisms in SMA differs based on age and infection history. Recent studies indicate increased accumulation of P. falciparum in the bone marrow and spleen, highlighting the need for understanding localized host-parasite interactions. MethodsThis study examines the bone marrow response to malarial infection in young children with SMA or mild malarial malaria in Mozambique, contrasting it with responses observed in peripheral blood in the two cohorts. Results and conclusionsThe study demonstrated that SMA is associated with increased red blood cell production, iron metabolism and tissue injury, as well as higher total parasite biomass including peripheral and bone marrow parasitaemia. We also demonstrate that direct analysis of bone marrow aspirates provides far more resolution in stratifying host signatures and drivers of malarial anaemia across a spectrum of severity than systemic measures from blood samples.

microbiology↗

bioRxiv2025

Shared Binding Site but Divergent Resistance Profiles Uncover Novel Resistance Mechanisms in Plasmodium HSP90 Inhibitors

Drug resistance is a widespread problem across therapeutic areas including malaria, but what accounts for resistance propensity remains poorly understood. Here, we reveal that two HSP90 inhibitors targeting the identical ATP-binding site exhibit dramatically different resistance profiles in P. falciparum. Geldanamycin readily selected 10 distinct resistance mutations conferring up to 22-fold resistance, while AUY-922 required 44 weeks to yield a single A41S mutation with only 2-fold resistance to AUY-922 but not to geldanamycin. Resistance mapping in parasites and yeast revealed geldanamycin resistance mutations distributed throughout the binding pocket whereas AUY-922 resistance mutations localized close to the ATP-binding site. Unexpectedly, the A41S mutation enhanced AUY-922 binding affinity without changing geldanamycin binding. In silico analysis suggested this enhancement occurs through additional hydrogen bonding, yet stronger binding correlated with resistance. In yeast, A41S had opposite effects, hypersensitizing cells to all HSP90 inhibitors tested. Additionally, conditional HSP90 knockdown increased geldanamycin sensitivity but left AUY-922 activity unaffected, indicating different target dependencies despite shared binding sites. Based on these data, we propose a multi-target hypothesis where AUY-922s lower resistance risk stems from engaging multiple HSP90 family members. Our findings reveal how enhanced drug-target binding can paradoxically correlate with resistance and demonstrate that resistance risk cannot be predicted from binding site identity alone, providing insights for developing more durable drugs across therapeutic areas.

microbiology↗

bioRxiv2025

Cellular and molecular basis of PfCoronin function in artemisinin resistance in Plasmodium falciparum

Artemisinin-based therapies are central to malaria treatment, but their efficacy is threatened by the emergence of resistant Plasmodium falciparum strains. While the role of Pfkelch13 mutations in clinical resistance is well established, recent reports from Africa implicate Pfcoronin mutations in treatment failure, adding to the complexity of resistance mechanisms. Here, we show that PfCoronin, a non-essential actin regulator active during the ring-stage, facilitates efficient hemoglobin uptake. We demonstrate that resistance-associated Pfcoronin mutations disrupt PfCoronins interaction with PfActin and its ring-stage localization, leading to impaired endocytosis and reduced hemoglobin acquisition. PfCoronin and PfKelch13 function in distinct cellular regions; mutations in both converge to limit heme availability and artemisinin activation. Although PfCoronin is dispensable for parasite viability, our findings demonstrate that Pfcoronin mutations reduce endocytosis and modulate artemisinin susceptibility during the clinically relevant ring stage--highlighting how non-essential, temporally restricted proteins can shape antimalarial drug response and resistance.

cell biology↗

bioRxiv2024

The Plasmepsin-Piperaquine Paradox Persists

Malaria is still a major health issue in many parts of the world, particularly in tropical and subtropical regions of Africa, Asia, and Latin America. Despite significant efforts to control and eliminate the disease, malaria remains a leading cause of illness and death, mainly due to the occurrence of drug-resistant parasites to the frontline antimalarials such as dihydroartemisinin-piperaquine (DHA-PPQ). Artemisinin resistance has been linked to kelch13 mutations, while decreased PPQ sensitivity has been associated with higher plasmepsin II and III gene copies and mutations in the chloroquine resistance transporter. In this study, we demonstrate the effective use of CRISPR/Cas9 technology to generate single knockouts (KO) of plasmepsin II and plasmepsin III, as well as a double KOs of both genes, in two isogenic lines of Cambodian parasites with varying numbers of plasmepsin gene copies. The deletion of plasmepsin II and/or III increased the parasites sensitivity to PPQ, evaluated by the area under the curve. We explored several hypotheses to understand how an increased plasmepsin gene copy number might influence parasite survival under high PPQ pressure. Our findings indicate that protease inhibitors have a minimal impact on parasite susceptibility to PPQ. Additionally, parasites with higher plasmepsin gene copy numbers did not exhibit significantly increased hemoglobin digestion, nor did they produce different amounts of free heme following PPQ treatment compared to wildtype parasites. Interestingly, hemoglobin digestion was slowed in parasites with plasmepsin II deletions. By treating parasites with digestive vacuole (DV) function modulators, we found that changes in DV pH potentially affect their response to PPQ. Our research highlights the crucial role of increased plasmepsin II and III gene copy numbers in modulating response to PPQ and begins to uncover the molecular and physiological mechanisms underlying PPQ resistance in Cambodian parasites. Author SummaryGlobal malaria control has plateaued, with drug-resistant Plasmodium falciparum posing a significant challenge. Artemisinin-based combination therapies (ACTs) are becoming less effective, especially in South-East Asia, where resistance to dihydroartemisinin-piperaquine (DHA-PPQ) is leading to treatment failures, notably in Cambodia. Genome-wide studies link artemisinin resistance to kelch13 mutations, while decreased PPQ sensitivity is tied to higher plasmepsin II and III gene copies and mutations in chloroquine resistance transporter. We previously showed a connection between increased plasmepsin gene copies and reduced PPQ sensitivity. In this study we try to understand the biological role of the plasmepsins in PPQ sensitivity. Therefore, we knocked out plasmepsin II and III genes in Cambodian strains using CRISPR/Cas9, and found increased PPQ sensitivity, confirming these genes roles in resistance. Plasmepsins are proteases that participate in the hemoglobin degradation cascade in the digestive vacuole of the parasites. Protease inhibitor experiments and hemoglobin digestion studies indicate that digestive vacuole pH fluctuations affect PPQ response, highlighting the need for further research into PPQ resistance mechanisms.

molecular biology↗

bioRxiv2023

Artemisinin resistance mutations in Pfcoronin impede hemoglobin uptake

Artemisinin (ART) combination therapies have been critical in reducing malaria morbidity and mortality, but these important drugs are threatened by growing resistance associated with mutations in Pfcoronin and Pfkelch13. Here, we describe the mechanism of Pfcoronin-mediated ART resistance. PfCoronin interacts with PfActin and localizes to the parasite plasma membrane (PPM), the digestive vacuole (DV) membrane, and membrane of a newly identified preDV compartment--all structures involved in the trafficking of hemoglobin from the RBC for degradation in the DV. Pfcoronin mutations alter PfActin homeostasis and impair the development and morphology of the preDV. Ultimately, these changes are associated with decreased uptake of red blood cell cytosolic contents by ring-stage Plasmodium falciparum. Previous work has identified decreased hemoglobin uptake as the mechanism of Pfkelch13-mediated ART resistance. This work demonstrates that PfCoronin appears to act via a parallel pathway. For both Pfkelch13-mediated and Pfcoronin-mediated ART resistance, we hypothesize that the decreased hemoglobin uptake in ring stage parasites results in less heme-based activation of the artemisinin endoperoxide ring and reduced cytocidal activity. This study deepens our understanding of ART resistance, as well as hemoglobin uptake and development of the DV in early-stage parasites.

cell biology↗

bioRxiv2022

Collateral sensitivity as a strategy to suppress resistance emergence: the challenge of diverse evolutionary pathways

Drug resistance remains a major obstacle to malaria control and eradication efforts, necessitating the development of novel therapeutic strategies to treat this disease. Drug combinations based on collateral sensitivity, wherein resistance to one drug causes increased sensitivity to the partner drug, have been proposed as an evolutionary strategy to suppress the emergence of resistance in pathogen populations. In this study, we explore collateral sensitivity between compounds targeting the Plasmodium dihydroorotate dehydrogenase (DHODH). We profiled the cross-resistance and collateral sensitivity phenotypes of several DHODH mutant lines to a diverse panel of DHODH inhibitors. We focus on one compound, TCMDC-125334, which was active against all mutant lines tested, including the DHODH C276Y line, which arose in selections with the clinical candidate DSM265. We found that selection of the DHODH C276Y mutant with TCMDC-125334 yielded additional genetic changes in the dhodh locus. These double mutant parasites exhibited decreased sensitivity to TCMDC-125334 and were highly resistant to DSM265. Finally, we tested whether collateral sensitivity could be exploited to suppress the emergence of resistance in the context of combination treatment by exposing wildtype parasites to both DSM265 and TCMDC-125334 simultaneously. This selected for parasites with a DHODH V532A mutation which were cross-resistant to both compounds and were as fit as the wildtype parent in vitro. The emergence of these cross-resistant, evolutionarily fit parasites highlights the mutational flexibility of the DHODH enzyme.

microbiology↗

bioRxiv2021

Defining the Yeast Resistome through in vitro Evolution and Whole Genome Sequencing

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in the model microbe, Saccharomyces cerevisiae. Analysis of 355 curated, laboratory-evolved clones, resistant to 80 different compounds, demonstrates differences in the types of mutations that are identified in selected versus neutral evolution and reveals numerous new, compound-target interactions. Through enrichment analysis we further identify a set of 137 genes strongly associated with or conferring drug resistance as indicated by CRISPR-Cas9 engineering. The set of 25 most frequently mutated genes was enriched for transcription factors and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function, single nucleotide variants found in 170-amino-acid domains in two Zn2C6 transcription factors, YRR1 and YRM1 (p < 1x 10 -100). This remarkable enrichment for transcription factors as drug resistance genes may explain why it is challenging to develop effective antifungal killing agents and highlights their important role in evolution.

genetics↗

bioRxiv2019

P. falciparum gametocyte density and infectivity in peripheral blood and skin tissue of naturally infected parasite carriers

Transmission of Plasmodium falciparum depends on the presence of mature gametocytes that can be ingested by mosquitoes taking a bloodmeal when feeding on human skin. It has long been hypothesised that skin sequestration contributes to efficient transmission. Although skin sequestration would have major implications for our understanding of transmission biology and the suitability of mosquito feeding methodologies to measure the human infectious reservoir, it has never been formally tested. In two populations of naturally infected gametocyte carriers from Burkina Faso, we assessed transmission potential to mosquitoes and directly quantified male and female gametocytes and asexual parasites in: i) finger prick blood, ii) venous blood, iii) skin biopsies, and in pools of mosquitoes that fed iv) on venous blood or, v) directly on the skin. Whilst more mosquitoes became infected when feeding directly on the skin compared to venous blood, concentrations of gametocytes in the subdermal skin vasculature were identical to that in other blood compartments. Asexual parasite densities, gametocyte densities and sex ratios were identical in the mosquito blood meals taken directly from the skin of parasite carriers and their venous blood.\n\nWe also observed sparse gametocytes in skin biopsies from legs and arms of gametocyte carriers by microscopy. Taken together, we provide conclusive evidence for the absence of significant skin sequestration of P. falciparum gametocytes. Gametocyte densities in peripheral blood are thus informative for predicting onward transmission potential to mosquitoes. Quantifying this human malaria transmission potential is of pivotal importance for the deployment and monitoring of malaria elimination initiatives.\n\nIMPORTANCEOur observations settle a long-standing question in the malaria field and close a major knowledge gap in the parasite cycle. By deploying mosquito feeding experiments and stage-specific molecular and immunofluorescence parasite detection methodologies in two populations of naturally infected parasite carriers, we conclusively reject the hypothesis of gametocyte skin sequestration. Our findings provide novel insights in parasite stage composition in human blood compartments, mosquito bloodmeals and their implications for transmission potential. We demonstrate that gametocyte levels in venous or finger prick blood can be used to predict onward transmission potential to mosquitoes. Our findings thus pave the way for methodologies to quantify the human infectious reservoir based on conventional blood sampling approaches to support the deployment and monitoring of malaria elimination efforts for maximum public health impact.

microbiology↗

bioRxiv2018

Amplicon deep sequencing of low-density Plasmodium falciparum infections: an evaluation of analysis approaches

BackgroundDeep sequencing of targeted genomic regions is becoming a common tool for understanding the dynamics and complexity of Plasmodium infections, but its lower limit of detection is currently unknown. Here, a new amplicon analysis tool, the Parallel Amplicon Sequencing Error Correction (PASEC) pipeline, is used to evaluate the performance of amplicon sequencing on low-density Plasmodium DNA samples. Illumina-based sequencing of two P. falciparum genomic regions (CSP and SERA2) was performed on two types of samples: in vitro DNA mixtures mimicking low-density infections (1-200 genomes/l) and extracted blood spots from a combination of symptomatic and asymptomatic individuals (44-653,080 parasites/l). Three additional analysis tools--DADA2, HaplotypR, and SeekDeep--were applied to both datasets and the precision and sensitivity of each tool were evaluated. ResultsAmplicon sequencing can contend with low-density samples, showing reasonable detection accuracy down to a concentration of 5 Plasmodium genomes/l. Due to increased stochasticity and background noise, however, all four tools showed reduced sensitivity and precision on samples with very low parasitemia (<5 copies/l) or low read count (<100 reads per amplicon). PASEC could distinguish major from minor haplotypes with an accuracy of 90% in samples with at least 30 Plasmodium genomes/l, but only 61% at low Plasmodium concentrations (<5 genomes/l) and 46% at very low read counts (<25 reads per amplicon). The four tools were additionally used on a panel of extracted parasite-positive blood spots from natural malaria infections. While all four identified concordant patterns of complexity of infection (COI) across four sub-Saharan African countries, the COI values obtained for individual samples differed in some cases. ConclusionsAmplicon deep sequencing can be used to determine the complexity and diversity of low-density Plasmodium infections. Despite differences in their approach, four state-of-the-art tools resolved known haplotype mixtures with similar sensitivity and precision. Researchers can therefore choose from multiple robust approaches for analyzing amplicon data, however, error filtration approaches should not be uniformly applied across samples of varying parasitemia. Samples with very low parasitemia and very low read count have higher false positive rates and call for read count thresholds that are higher than current recommendations.

microbiology↗

bioRxiv2017

De Novo Mutations Resolve Disease Transmission Pathways in Clonal Malaria

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a set of clinical samples and novel methods of sequencing, library preparation, and genotyping, we have genotyped low-complexity regions of the genome with a high degree of accuracy. Despite its slower evolutionary rate compared to bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2 years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

genomics↗

bioRxiv2017

Within-infection diversity of Plasmodium falciparum antigens reflects host-mediated selection

Host immunity exerts strong selection on pathogens, but it does not act in a uniform manner across individual hosts. By providing a direct approach for understanding host-specific selection pressures, patterns of intra-host pathogen diversity complement population genetic analyses performed on broad geographic scales. Here, we perform a combined analysis of inter- and intra-host diversity for the malaria parasite Plasmodium falciparum with haplotype sequences of three antigens sampled from over 4,500 natural infections in sub-Saharan Africa using targeted deep sequencing. We find that multi-strain infections in young children contain non-random combinations of parasite genotypes, and identify individual amino acid positions that may contribute to strain-specific blocking of infections. These results demonstrate for the first time that natural host defenses to Plasmodium detectably impact which infections proceed to the blood stage within a given host. This selection partially explains the extreme amino acid diversity observed at many parasite antigens and suggests that vaccines targeting such proteins should account for the impact of allele-specific immunity.

evolutionary biology↗

bioRxiv2017

hmmIBD: software to infer pairwise identity by descent between haploid genotypes

SummaryWe introduce hmmIBD, software to estimate pairwise identity by decent between haploid genomes, such as those of the malaria parasite, sampled from one or more populations. We verified hmmIBD using simulated data, benchmarked it against a previously published method for detecting IBD within populations, and demonstrated its utility using Plasmodium falciparum data from Cambodia and Ghana.\n\nSupplementary informationSupplementary data include Appendices S1, S2 and S3, and are available online.\n\nAvailability and ImplemetationSource code written in C99/C11-compliant C and requiring no external libraries, is freely available for download at https://github.com/glipsnort/hmmIBD/releases, alongside test datasets.\n\nContactsfs@broadinstitute.org

bioinformatics↗

bioRxiv2017

Mapping The Malaria Parasite Drug-Able Genome Using In Vitro Evolution And Chemogenomics

Chemogenetic characterization through in vitro evolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262 Plasmodium falciparum parasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of the P. falciparum resistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.\n\nOne Sentence SummaryWhole genome sequencing reveals how Plasmodium falciparum evolves resistance to diverse compounds and identifies new antimalarial drug targets.

genomics↗