Biology subjects

Wilson, D. W.

Publications and source records attributed to Wilson, D. W..

13 recordsLinked to original sources

bioRxiv2026

Plasmodium berghei is resistant to aryl amino acetamides that inhibit P. falciparum growth by targeting the phospholipid transfer protein PfSTART1.

In a previous screen for compounds that inhibit Plasmodium falciparum merozoite invasion of red blood cells, we identified the Medicines for Malaria Venture compound MMV006833. This compound inhibits PfSTART1, a protein implicated in the expansion of the nascent parasitophorous vacuole membrane following invasion, to accommodate the developing ring-stage parasite. Live-cell lattice light-sheet microscopy of invading merozoites revealed that mNeonGreen-tagged PfSTART1 is released from structures within the merozoite into the nascent parasitophorous vacuole approximately 109 seconds after invasion. Expansion microscopy of PfSTART1-HA merozoites further showed that these punctate PfSTART1-containing structures do not colocalise with known secretory organelles (rhoptries, micronemes and dense granules). Although analogues of MMV006833 are highly potent against P. falciparum, they were previously found to be ineffective against P. berghei parasites in the mouse malaria model. Here, we demonstrate that PbSTART1 is highly resistant to MMV006833 and its analogues when expressed in P. falciparum, indicating that structural differences between the orthologous proteins reduce inhibitor potency. The crystal structure of PfSTART1 in complex with WEHI-991 revealed the molecular basis for inhibition and provided a structural explanation for the reduced potency of this family of compounds against P. berghei. To sensitise P. berghei parasites to MMV006833 analogues, the parasites were engineered to express PfSTART1; however, these chimeric parasites remained insensitive to the compounds. This suggests that factors beyond target engagement, such as compound half-life or bioavailability, contribute to the lack of efficacy observed in the mouse malaria model.

microbiology↗

bioRxiv2025

Differential importance of MSP4 and MSP5 for infection of red blood cells between human infecting malaria parasites

Plasmodium species malaria parasites require invasion and replication within red blood cells to cause disease. Merozoite surface proteins (MSPs) are proposed to play a role in attachment of merozoites to RBCs and have long been considered as potential vaccine targets, but their functions during invasion are largely unknown. We applied targeted gene editing to investigate MSP4 and 5 function in P. falciparum, which causes most malaria mortality, and P. knowlesi, an in vitro culturable zoonotic species closely related to the widespread P. vivax. CRISPR-Cas9 gene-editing revealed that P. knowlesi MSP4 was not required for parasite growth in vitro. While P. knowlesi MSP5 could be functionally replaced by P. vivax MSP5, it was refractory to gene deletion. We confirmed the opposite for two different P. falciparum laboratory isolates where MSP4 is essential but MSP5 is dispensable. Attempts to select for reliance on the non-essential MSP (e.g. P. knowlesi MSP4 or P. falciparum MSP5) through long-term growth of inducible knock-out parasites, or via chimeric complementation of the essential MSP4 or 5 with the essential MSP from the other species, were unsuccessful. Live cell filming revealed a severe cell-entry defect with conditional knock-down of MSP5 protein expression in P. knowlesi. This study demonstrates differential importance of MSP4 and MSP5 during merozoite RBC invasion across human infecting malaria species, emphasises that vaccine candidates must be considered individually for the two most prominent human malarias and promotes MSP5 as a potential vaccine candidate for P. knowlesi and P. vivax. SignificanceFor a malaria parasite to cause disease, the merozoite form of the lifecycle has to infect and replicate within human red blood cells. Proteins on the surface of the merozoite are considered as promising vaccine candidates, but the functions of these proteins are poorly understood. Here we demonstrate that two structurally similar merozoite surface proteins (MSP), MSP4 and MSP5, have differential importance between one human infecting malaria species compared to a second. The finding that MSP4 is essential for growth in one species, and MSP5 in the other, has implications for understanding invasion biology of malaria parasites and highlights that even structurally similar vaccine targets may need to be chosen specifically for each human infecting malaria species.

microbiology↗

bioRxiv2025

An abundant merozoite surface protein of Plasmodium falciparum modulates susceptibility to inhibitory antibodies

Malaria merozoite surface proteins (MSPs), are thought to have important roles in red blood cell (RBC) invasion and their exposure on the parasite surface makes them attractive vaccine candidates. However, their role in invasion has not been directly demonstrated and their biological functions are unknown. One of the most abundant proteins is PfMSP2, which is likely an ancestral protein that has been maintained in the Plasmodium falciparum lineage and is a focus of vaccine development, whose function remains unknown. Using CRISPR-Cas9 gene-editing, we removed PfMSP2 from two different P. falciparum lines with no impact on parasite replication or phenotype in vitro, demonstrating that it is not essential for RBC invasion. However, loss of PfMSP2 led to increased inhibitory potency of antibodies targeting other merozoite proteins involved in invasion, particularly PfAMA1. In a solid-phase model, increasing concentrations of PfMSP2 protein reduced binding of different antibodies against PfAMA1 in a dose dependent manner. These data suggest that PfMSP2 can modulate the susceptibility of merozoites to protective inhibitory antibodies. The results of this study change our understanding of the potential functions of PfMSP2 and establishes a new concept in malaria where a surface protein can reduce the protective efficacy of antibodies targeting a different antigen. These findings have important implications for understanding malaria immunity and informing vaccine development.

microbiology↗

bioRxiv2024

Malaria parasite HOPS/CORVET complexes are critical for endocytosis and invasion organelles function

The tethering complexes HOPS/CORVET are central for vesicular fusion through the eukaryotic endolysosomal system, but the functions of these complexes in the intracellular development of malaria parasites are unknown. Here we show that early inactivation of core HOPS/CORVET complex subunits in Plasmodium falciparum leads to developmental arrest and accumulation of cytosolic vesicles, indicating a role of HOPS/CORVET in parasite endocytosis and fusion of endosomes to the digestive vacuole membrane. Late inactivation of the core HOPS/CORVET subunits led to the mislocalization of luminal rhoptry and microneme proteins, and to a severe defect in merozoite invasion. Ultra-expansion microscopy revealed a reduced rhoptry volume and the accumulation of numerous vesicles, further supporting a role of HOPS/CORVET in protein trafficking to the apical organelles. Malaria parasites have therefore repurposed HOPS/CORVET to perform dual functions consistent with a canonical endocytosis pathway for delivery of host cell material to the digestive vacuole in trophozoite stages and a parasite specific role in trafficking of protein cargo to the apical organelles required for invasion in schizont stages.

cell biology↗

bioRxiv2023

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample [~]4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology. IMPACT STATEMENTUsing ultrastructure-expansion microscopy we explore the fundamental cell biology of malaria parasites, providing new insights into processes including establishment of cell polarity and organelle fission.

cell biology↗

bioRxiv2022

A microtubule associated protein is essential for malaria parasite transmission

Mature gametocytes of Plasmodium (P.) falciparum display a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated to the inner membrane complex (IMC). Microtubule associated proteins (MAPs) define MT populations and modulate interaction to pellicular components. Several MAPs have been identified in Toxoplasma gondii and homologues can be found in the genome of Plasmodium species, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genus Plasmodium., especially within the Laverania subgenus, but absent in other Apicomplexa. Conditional knockdown and targeted gene disruption of Pfspm3 in P. falciparum cause severe morphological defects during gametocytogenesis leading to round, non-falciform gametocytes with an aberrant SPMT pattern. In contrast, Pbspm3 knockout in P. berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in sporozoite invasion of salivary glands leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the IMC in Pbspm3 knockout parasites suggesting a function of SPM3 in anchoring MTs to the IMC. Overall, our results highlight SPM3 as a pellicular component with essential functions for malaria parasite transmission. IMPORTANCEA key structural feature driving the transition between different life cycle stages of the malaria parasite is the unique three membrane "pellicle", consisting of the parasite plasma membrane (PPM) and a double membrane structure underlying the PPM termed the "inner membrane complex" (IMC). Additionally, there are numerous linearly arranged intramembranous particles (IMPs) linked to the IMC, which likely link the IMC to the subpellicular microtubule cytoskeleton. Here we identify, localize and characterize a novel subpellicular microtubule associated protein unique to the genus Plasmodium (P.). The knockout of this protein in the human infecting P. falciparum species result in malformed gametocytes and aberrant microtubules. We confirmed the microtubule association in the P. berghei rodent malaria homologue and show that its knockout results in a perturbated microtubule architecture, aberrant sporozoite motility and decreased transmission efficiency.

molecular biology↗

bioRxiv2022

The sulfonylpiperazine MMV020291 prevents red blood cell invasion by the malaria parasite Plasmodium falciparum through interference with actin-1/profilin dynamics

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell invasion, and have generated analogues with improved potency. Here, we identify actin and profilin as putative targets of the MMV020291 series through resistance selection and whole genome sequencing of three MMV020291 resistant populations. This revealed three non-synonymous single nucleotide polymorphisms in two genes; two in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wildtype parasites which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade red blood cells. Additionally, the series inhibits the actin-1 dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro co-sedimentation experiments using recombinant P. falciparum actin-1 and profilin proteins indicate that potent MMV020291 analogues amplify the actin-monomer sequestering effect of profilin, thereby reducing the formation of filamentous actin. Altogether, this study identifies the first compound series targeting the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

molecular biology↗

bioRxiv2021

PMRT1, a Plasmodium specific parasite plasma membrane transporter is essential for asexual and sexual blood stage development

Membrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures - the food vacuole, the apicoplast, and the parasite plasma membrane - and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1, PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions. ImportancePlasmodium falciparum-infected erythrocytes possess multiple compartments with designated membranes. Transporter proteins embedded in these membranes do not only facilitate movement of nutrients, metabolites and other molecules between these compartments, but are common therapeutic targets and can also confer antimalarial drug resistance. Orphan membrane transporter in P. falciparum without sequence homology to transporters in other evolutionary lineages and divergent to host transporters may constitute attractive targets for novel intervention approaches. Here, we localized six of these putative transporters at different subcellular compartments and probed into their importance during asexual parasite growth using reverse genetic approaches. In total, only two candidates turned out to be dispensable for the parasite, highlighting four candidates as putative targets for therapeutic interventions. This study reveals the importance of several orphan transporters to blood stage P. falciparum development.

molecular biology↗

bioRxiv2021

Experimental re-infected cats do not transmit SARS-CoV-2

SARS-CoV-2 is the causative agent of COVID-19 and responsible for the current global pandemic. We and others have previously demonstrated that cats are susceptible to SARS-CoV-2 infection and can efficiently transmit the virus to naive cats. Here, we address whether cats previously exposed to SARS-CoV-2 can be re-infected with SARS-CoV-2. In two independent studies, SARS-CoV-2-infected cats were re-challenged with SARS-CoV-2 at 21 days post primary challenge (DPC) and necropsies performed at 4, 7 and 14 days post-secondary challenge (DP2C). Sentinels were co-mingled with the re-challenged cats at 1 DP2C. Clinical signs were recorded, and nasal, oropharyngeal, and rectal swabs, blood, and serum were collected and tissues examined for histologic lesions. Viral RNA was transiently shed via the nasal, oropharyngeal and rectal cavities of the re-challenged cats. Viral RNA was detected in various tissues of re-challenged cats euthanized at 4 DP2C, mainly in the upper respiratory tract and lymphoid tissues, but less frequently and at lower levels in the lower respiratory tract when compared to primary SARS-CoV-2 challenged cats at 4 DPC. Histologic lesions that characterized primary SARS-CoV-2 infected cats at 4 DPC were absent in the re-challenged cats. Naive sentinels co-housed with the re-challenged cats did not shed virus or seroconvert. Together, our results indicate that cats previously infected with SARS-CoV-2 can be experimentally re-infected with SARS-CoV-2; however, the levels of virus shed was insufficient for transmission to co-housed naive sentinels. We conclude that SARS-CoV-2 infection in cats induces immune responses that provide partial, non-sterilizing immune protection against reinfection.

microbiology↗

bioRxiv2020

Pfcerli2, a duplicated gene in the malaria parasite Plasmodium falciparum essential for invasion of erythrocytes as revealed by phylogenetic and cell biological analysis.

Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1, a related cytosolically exposed rhoptry bulb protein. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to an inhibition of merozoite invasion after tight junction formation. PfCERLI2 knockdown was associated with inhibition of rhoptry antigen processing and a significant elongation of the rhoptries, suggesting that the inability of merozoites to invade is caused by aberrant rhoptry function due to PfCERLI2 deficiency. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.

microbiology↗

bioRxiv2020

Structure-based identification and functional characterization of an essential lipocalin in the malaria parasite Plasmodium falciparum

Proteins of the lipocalin family are known to bind small hydrophobic ligands and are involved in various physiological processes ranging from lipid transport to oxidative stress responses. The genome of the malaria parasite Plasmodium falciparum contains a single protein PF3D7_0925900 with a lipocalin signature. Using crystallography and small-angle X-ray scattering, we show that the protein has a tetrameric structure of typical lipocalin monomers, hence we name it P. falciparum lipocalin (PfLCN), the first lipocalin structurally and functionally characterized in a single-celled eukaryote. We show that PfLCN is expressed in the intraerythrocytic stages of the parasite and localizes to the parasitophorous and food vacuoles. Conditional knockdown of PfLCN impairs parasite development, which can be rescued by treatment with the radical scavenger Trolox or by temporal inhibition of hemoglobin digestion. This suggests a key function of PfLCN in counteracting oxidative stress induced cell damage during multiplication of parasites within red blood cells.

cell biology↗

bioRxiv2019

Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

To identify potential inhibitors of egress and invasion in the asexual blood stage of Plasmodium falciparum, we screened the Medicines for Malaria Venture (MMV) Pathogen Box. This compound library comprises of 400 drugs against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, Nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 {micro}M, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has greater implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.\n\nImportancePlasmodium falciparum causes the most severe form of malaria and with emerging resistance to frontline treatments, there is the need to identify new drug targets in the parasite. One of the most critical processes during the asexual blood stage in the parasites lifecycle is the egress from old red blood cells (RBCs) and subsequent invasion of new RBCs. Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. Identifying novel compounds that inhibit these essential processes would further their development into possible antimalarials that would be highly effective at killing asexual RBC stage parasites when used in combination with drugs that target the intraerythrocytic growth phase. These compounds potentially may also be used as novel tools to study the complex biology of parasites to gain further insight into the mechanisms behind egress and invasion.

cell biology↗

bioRxiv2019

PfCERLI1, a conserved rhoptry associated protein essential for invasion by Plasmodium falciparum merozoites.

The disease-causing blood stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. We show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, biochemical techniques and semi-quantitative super-resolution microscopy show that PfCERLI1 knockdown prevents secretion of key rhoptry antigens that coordinate merozoite invasion. PfCERLI1 is the first rhoptry associated protein identified to have a direct role in function of this essential malaria invasion organelle which has broader implications for understanding apicomplexan invasion biology.

microbiology↗