Biology subjects

Delves, M. J.

Publications and source records attributed to Delves, M. J..

11 recordsLinked to original sources

bioRxiv2026

DCX enables branching of subpellicular microtubules in Plasmodium falciparum gametocytes and is required for mosquito colonisation

Plasmodium falciparum, the causative agent of malaria, relies on specialised tubulin-based cytoskeletal structures to support its parasitic lifestyle. These include the conoid required for parasite motility and host-cell invasion, as well as subpellicular microtubules (SPMTs) that support parasite shape and rigidity. Here, we investigate the function of the doublecortin-domain protein DCX, a microtubule-binding protein previously associated with the Plasmodium conoid. We first show that, in P. falciparum, DCX is not expressed in the merozoite stage and is not required for the invasion of human erythrocytes. By contrast, DCX is expressed in ookinetes, the motile stage responsible for infecting the mosquito vector, where it associates with conoid tubulin fibres, consistent with a role in stabilising the conoid architecture. Unexpectedly, we find that DCX is required for P. falciparum transmission to the mosquito independently of conoid function. We further link this requirement to the distinctive organisation of SPMTs in P. falciparum gametocytes, which display an unusual branching architecture comprising multiple microtubules of 15 to 18 protofilaments. Deletion of DCX leads to a reduction in SPMT branching and is associated with higher protofilament numbers, revealing a previously unrecognised role for DCX in shaping the ultrastructure of SPMTs in P. falciparum gametocytes. Altogether, our findings uncover the repurposing of DCX across distinct microtubule systems in transmission stages and identify DCX as a key factor mediating microtubule branching and stabilisation in SPMTs required for efficient mosquito transmission.

microbiology↗

bioRxiv2026

The translatome of quiescent Plasmodium falciparum gametocytes reveals parasite pyridoxal 5'-phosphate (PLP) biosynthesis is essential for efficient mosquito stage development

The ability of Plasmodium falciparum gametocytes to remain quiescent within the vertebrate host but poised for rapid onward development in the mosquito is an adaptation essential to maximise the onward spread of malaria. In this dormant state, mature infectious stage V gametocytes are largely unaffected by most antimalarial drugs and our limited understanding of how gametocytes prepare for mosquito transmission has hindered the identification of new molecular targets for transmission-blocking therapeutics. In this study, we move beyond the total proteome of gametocytes and define the translatome of mature stage V gametocytes using L-azidohomoalanine incorporation into nascent proteins, click chemistry purification and proteomic analysis. We identify the proteins and pathways that gametocytes sustain in preparation for transmission during this dormant period and through genetic disruption, we validate this approach by demonstrating the importance of parasite pyridoxal 5-phosphate biosynthesis for mosquito transmission.

microbiology↗

bioRxiv2026

Comparing the transmission blocking efficacy of Primaquine and Tafenoquine with in vivo pre-clinical models

The 8-aminoquinoline family are a well-established class of antimalarial drugs containing two clinically relevant analogues, primaquine and tafenoquine. These compounds have two therapeutically significant activities across the plasmodial lifecycle; elimination of Plasmodium vivax hypnozoites as part of a radical cure (relapse prevention), and as prophylactic transmission blocking compounds. Primaquine is currently recommended as a single low (0.25 mg/kg) dose administered with artemisinin-based combination therapy to reduce malarial transmission in areas at high risk of artemisinin partial resistance. Tafenoquine was approved in 2018 for radical cure when co-administered with chloroquine however, its transmission blocking efficacy in humans has not yet been fully evaluated, and direct head-to-head comparisons of transmission-blocking efficacy of primaquine and tafenoquine have not been performed. Given that primaquine and tafenoquine are presumed to have similar mechanisms of action, tafenoquine may have potential use as a transmission blocking intervention. Furthermore, tafenoquine has a substantially longer half-life than primaquine, which could provide a pharmacokinetic advantage if such efficacy is confirmed. However, assessment of 8-aminoquinoline efficacy against the sexual stages of Plasmodium is complicated by the requirement for metabolic activation to generate parasite-reactive species, limiting the utility of in vitro assays. Here, we directly compare the transmission blocking effects of primaquine and tafenoquine using two in vivo preclinical models. We titrate the transmission blocking efficacy of each compound, and evaluate the pharmacokinetics of each compound over time, linking drug exposure to efficacy. The evidence presented here suggests that beyond 24 hours, a single dose of tafenoquine is likely to have more clinically desirable pharmacokinetics, resulting in higher transmission blocking efficacy than primaquine. These findings are observed across multiple models, both in the absence or presence of a partner schizonticide and thus demonstrate a potential advantage of the utilisation of tafenoquine when compared to primaquine.

microbiology↗

bioRxiv2025

Targeting Aurora kinases as essential cell cycle regulators to deliver multi-stage antimalarials against Plasmodium falciparum

Kinases that play critical roles in the development and adaptation of Plasmodium falciparum present novel opportunities for chemotherapeutic intervention. Of particular interest are mitotic kinases that regulate the proliferation of the parasites by controlling nuclear division, segregation and cytokinesis. We evaluated the potential of human Aurora kinase (Aur) inhibitors to inhibit P. falciparum development by targeting members of the Aurora-related kinase (Ark) family in this parasite. Several human AurB inhibitors exhibited multistage potency (<250 nM) against all proliferative stages of parasite development, including asexual blood stages, liver schizonts and male gametes. Among the most potent compounds, hesperadin and AT83 exhibit >1000x selectivity towards the parasite without concerns about mammalian cell toxicity. Importantly, we identified PfArk1 as the principal vulnerable Ark family member, with specific inhibition of PfArk1 as the primary target for hesperadin and the human anaplastic lymphoma kinase (ALK) inhibitor TAE684. Hesperadins whole-cell and protein activity validates it as a unique PfArk1 tool compound. Inhibition of PfArk1 results in the parasites inability to complete mitotic processes, presenting with unsegregated, multi-lobed nuclei caused by aberrant microtubule organization. This suggests that PfArk1 is the main Aur mitotic kinase in proliferative stages of Plasmodium, characterized by bifunctional AurA and B activity. This paves the way for drug discovery campaigns based on hesperadin targeting PfArk1.

molecular biology↗

bioRxiv2024

A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo

The Plasmodium falciparum cytoplasmic tyrosine tRNA synthetase (PfTyrRS) is an attractive drug target that is susceptible to reaction-hijacking by AMP-mimicking nucleoside sulfamates. We previously identified an exemplar pyrazolopyrimidine ribose sulfamate, ML901, as a potent pro-inhibitor of PfTyrRS. Here we examined the stage specificity of action of ML901, showing very good activity against the schizont stage, but lower trophozoite stage activity. We explored a series of ML901 analogues and identified ML471, which exhibits improved potency against trophozoites and enhanced selectivity against a human cell line. Additionally, it has no inhibitory activity against human ubiquitin-activating enzyme (UAE) in vitro. ML471 exhibits low nanomolar activity against asexual blood stage P. falciparum and potent activity against liver stage parasites, gametocytes and transmissible gametes. It is fast-acting and exhibits a long in vivo half-life. ML471 is well-tolerated and shows single dose oral efficacy in the SCID mouse model of P. falciparum malaria. We confirm that ML471 is a pro-inhibitor that is converted into a tight binding Tyr-ML471 conjugate by the PfTyrRS enzyme. A crystal structure of the PfTyrRS/ Tyr-ML471 complex offers insights into improved potency, while molecular docking into UAE provides a rationale for improved selectivity.

biochemistry↗

bioRxiv2024

Mitochondrial ATP synthesis is essential for efficient gametogenesis in Plasmodium falciparum

Interrupting parasite transmission from humans to mosquitoes is vital for malaria elimination and eradication. Plasmodium male and female gametocytes are the gatekeepers of human to mosquito transmission. Whilst dormant in the human host, their divergent roles during transmission become visually apparent soon after ingestion by the mosquito after rapid transformation into gametes - the males forming eight motile sperm-like cells that each aim to fertilise a single female gamete. Here we report that antibodies raised against PfLDH2 allow accurate identification of male gametocytes. Using this novel tool and functional mitochondrial labelling, we show that the male gametocyte mitochondrion is less active than that of female gametocytes. Rather than being a vestigial organelle discarded during male gametogenesis, we demonstrate that mitochondrial ATP synthesis is essential for male gametocytes to complete gametogenesis and inhibition leads to early arrest. Additionally, using a genetically encoded ratiometric sensor of ATP, we show that gametocytes can maintain cytoplasmic ATP homeostasis in the absence of mitochondrial respiration, indicating the essentiality of the gametocyte mitochondrion for transmission alone. Together, this reveals how gametocytes balance the conflicting energy demands of a dormant and active lifestyle and highlights the mitochondria as a rich source of transmission-blocking targets for future drug development.

microbiology↗

bioRxiv2023

Aryl amino acetamides prevent the development of Plasmodium falciparum rings via inhibition of the lipid transfer protein PfSTART1

With resistance to most antimalarials increasing, it is imperative that new antimalarial drugs are developed to replace or complement front-line artemisinin therapies. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited ring development of newly invaded merozoites. Here, we selected parasites resistant to M-833 and identified independent mutations arising in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introduction of the identified PfSTART1 mutations into wildtype parasites reproduced resistance to both M-833 and highly potent analogues, confirming PfSTART1 mutations were sufficient to confer resistance. The analogues bound to recombinant PfSTART1 with nanomolar affinity. We also demonstrated selective PfSTART1 engagement by the analogues using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assay for the first time in Plasmodium. Imaging of newly invaded merozoites showed the inhibitors prevented the conversion into larger amoeboid ring-stage parasites potentially through the inhibition of phospholipid transfer from the parasite to the encasing parasitophorous vacuole membrane (PVM) and/or within the parasite. We show that these PfSTART1 inhibitors also block transmission. With multiple stages of the parasites lifecycle being targeted by PfSTART1 inhibitors, this protein therefore represents a novel drug target with a new mechanism of action.

cell biology↗

bioRxiv2023

Machine Learning-based Phenotypic Imaging to Characterise the Targetable Biology of Plasmodium falciparum Male Gametocytes for the Development of Transmission-Blocking Antimalarials

Preventing parasite transmission from humans to mosquitoes is recognised to be critical for achieving elimination and eradication of malaria. Consequently developing new antimalarial drugs with transmission-blocking properties is a priority. Large screening campaigns have identified many new transmission-blocking molecules, however little is known about how they target the mosquito-transmissible Plasmodium falciparum stage V gametocytes, or how they affect their underlying cell biology. To respond to this knowledge gap, we have developed a machine learning image analysis pipeline to characterise and compare the cellular phenotypes generated by transmission-blocking molecules during male gametogenesis. Using this approach, we studied 40 molecules, categorising their activity based upon timing of action and visual effects on the organisation of tubulin and DNA within the cell. Our data both proposes new modes of action and corroborates existing modes of action of identified transmission-blocking molecules. Furthermore, the characterised molecules provide a new armoury of tool compounds to probe gametocyte cell biology and the generated imaging dataset provides a new reference for researchers to correlate molecular target or gene deletion to specific cellular phenotype. Our analysis pipeline is not optimised for a specific organism and could be applied to any fluorescence microscopy dataset containing cells delineated by bounding boxes, and so is extendible to any disease model.

microbiology↗

bioRxiv2021

Plasmodium falciparum protein Pfs16 is a target for transmission-blocking antimalarial drug development

Phenotypic cell-based screens are critical to the discovery of new antimalarial lead compounds. However, identification and validation of cellular targets of lead compounds is required following discovery in a phenotypic screen. We recently discovered a Plasmodium transmission-blocking N-((4-hydroxychroman-4-yl)methyl)-sulfonamide (N-4HCS) compound, DDD01035881, in a phenotypic screen. DDD01035881 and its potent derivatives have been shown to block Plasmodium male gamete formation (microgametogenesis) with nanomolar activity. Here, we synthesised a photoactivatable N-4HCS derivative, probe 2, to identify the N-4HCS cellular target. Using probe 2 in photo-affinity labelling coupled with mass spectrometry, we identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as the likely cellular target of the N-4HCS series. Further validating Pfs16 as the cellular target of the N-4HCS series, the Cellular Thermal Shift Assay (CETSA) confirmed DDD01035881 stabilised Pfs16 in lysate from activated mature gametocytes. Additionally, photo-affinity labelling combined with in-gel fluorescence and immunoblot analysis confirmed the N-4HCS series interacted with Pfs16. High-resolution, widefield fluorescence and electron microscopy of N-4HCS-inhibited parasites was found to result in a cell morphology entirely consistent with targeted gene disruption of Pfs16. Taken together, these data strongly implicate Pfs16 as the target of DDD01035881 and establish the N-4HCS scaffold family as a powerful starting point from which future transmission-blocking antimalarials can be developed.

microbiology↗

bioRxiv2020

Artemisinin-resistant malaria parasites show enhanced transmission to mosquitoes under drug pressure

Resistance to artemisinin combination therapy (ACT) in the Plasmodium falciparum parasite is threatening to reverse recent gains in reducing global deaths from malaria. Whilst resistance manifests as delayed asexual parasite clearance in patients following ACT treatment, the phenotype can only spread geographically via the sexual cycle and subsequent transmission through the mosquito. Artemisinin and its derivatives (such as dihydroartemisinin, DHA) as well as killing the asexual parasite form are known to sterilize male, sexual-stage gametes from activation. Whether resistant parasites overcome this artemisinin-dependent sterilizing effect has not, however, been fully tested. Here, we analysed five P. falciparum clinical isolates from the Greater Mekong Subregion, each of which demonstrated delayed clinical clearance and carried known resistance-associated polymorphisms in the Kelch13 gene (PfK13var). As well as demonstrating reduced sensitivity to artemisinin-derivates in in vitro asexual growth assays, certain PfK13var isolates also demonstrated a marked reduction in sensitivity to these drugs in an in vitro male gamete activation assay compared to a sensitive control. Importantly, the same reduction in sensitivity to DHA was observed when the most resistant isolate was assayed by standard membrane feeding assays using Anopheles stephensi mosquitoes. These results indicate that ACT use can favour resistant over sensitive parasite transmission. A selective advantage for resistant parasite transmission could also favour acquisition of further polymorphisms, such as mosquito host-specificity or antimalarial partner-drug resistance in mixed infections. Favoured transmission of resistance under ACT coverage could have profound implications for the spread of multidrug resistant malaria beyond Southeast Asia. ONE SENTENCE SUMMARYArtemisinin-resistant clinical isolates can also demonstrate resistance to the transmission-blocking effects of artemisinin-based drugs, favouring resistance transmission to the mosquito.

microbiology↗

bioRxiv2017

Failure of in vitro differentiation of Plasmodium falciparum gametocytes into ookinetes arises because of poor gamete fertilisation

A critical step towards malaria elimination will be the interruption of Plasmodium transmission from the human host to the mosquito. At the core of the transmission cycle lies Plasmodium sexual reproduction leading to zygote formation and mosquito midgut colonisation by ookinetes. Whilst in vitro ookinete culture from the murine and avian malaria parasites, Plasmodium berghei and P. gallinaceum, has greatly increased our knowledge of transmission biology; efforts to mimic the process in the human parasite P. falciparum have, to date, had only limited success. Using fluorescence microscopy and flow cytometry with antibodies specific to the male gametocyte and developing ookinetes, we sought to evaluate P. falciparum ookinete production using previously published in vitro protocols. We then compared in vitro versus in vivo ookinete production in both P. falciparum and P. berghei parasites, exploring potential barriers to complete development. Finally, we sought to test a wide range of literature-led culture conditions towards further optimisation of in vitro P. falciparum ookinete production. Despite extensive testing, our efforts to replicate published methods did not produce appreciable quantities of fully formed P. falciparum ookinetes in vitro. In parallel, however, gametocyte cultures that failed to differentiate fully in vitro successfully developed into ookinetes in vivo with an efficiency approximating that of P. berghei. Flow cytometry analysis showed that this disparity likely lies with the poor fertilization of P. falciparum gametes in vitro. Attempts to improve gametocyte fertility or define conditions more permissive to fertilisation/ookinete survival in vitro were also unsuccessful. Current in vitro conditions for P. falciparum ookinete production are not optimal for gamete fertilisation either due to the lack of parasite-species-specific mosquito factors missing from in vitro culture, or non-permissive cues contaminating culture preparations.

microbiology↗