Biology subjects

Rayner, J. C.

Publications and source records attributed to Rayner, J. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Robust High-Throughput Flickering Spectroscopy for Measurements of Red Blood Cell Membrane Mechanics

The mechanical properties of red blood cell (RBC) membranes are critical to their function in oxygen delivery, and changes to these properties as RBCs age affect their journey around the circulatory system, including clearance by the spleen. Such changes can also have significant health effects,including in cardiovascular disease and on the interactions between RBCs and malaria parasites. The function of RBCs requires them to be very soft, which together with the small size of the cells brings the energy required for measurable deformation of the membrane into the range of typical thermal energies. Red Blood Cells can be observed to flicker under optical microscopy, and these shape fluctuations can be quantified and used to obtain key biophysical parameters such as the tension and bending modulus of the membrane. Typically the shape of the cell's equator is extracted, and the mean power spectrum is obtained by time-averaging the power present in the normal modes of the thermal fluctuations. Flickering spectroscopy has been used extensively on RBCs, but has so far been very low throughput and with some technical limitations. Here we address issues related to active versus passive fluctuations, focusing and optics, camera exposure and sampling, and fast contour detection. In combination with an automated imaging system it is possible to measure thousands of cells in one day, with no user input. We validate this new pipeline by chemical modification of RBC mechanics, and by comparison with simulated fluctuations including each confounding effect. The methods and codes for this robust flickering analysis will allow consistent measurements across labs.

biophysics↗

bioRxiv2026

South American Plasmodium falciparum isolates reveal new insights into the role of PfAAT1 in mediating resistance to Chloroquine

Antimalarial drug resistance remains one of the most significant challenges to global malaria control. The emergence of resistance to chloroquine, the first truly globally distributed antimalarial, has been extensively studied but is still not fully understood. While mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) primarily drive resistance, the phenotype is complex and multigenic, with mutations in the putative amino acid transporter PfAAT1 recently confirmed to play a modulatory role. To date studies have focused on PfAAT1 mutations found in African and Southeast Asian P. falciparum lineages, but chloroquine resistance emerged independently in South America, where there may be novel PfAAT1 polymorphisms that are functionally important. We used AlphaFold modelling to reveal high homology between PfAAT1 and the human lysosomal arginine transporter SLC38A9, which allows prediction of membrane orientation and identifies a partially open channel accessible from the cytoplasm. Several PfAAT1 mutations found only in South American isolates sit near the entrance of this pore, most notably V231 where mutation to aspartate is predicted to alter channel conformation and influence transport, while nearby P446A (which is always found in combination with V231D) and I248T are predicted to impact pore flexibility and local structural stability. To functionally validate these insights, we employed CRISPR/Cas9 gene editing across parasite strains with diverse geographic origins. Reverting the regional V231D mutation in the South American 7G8 strain significantly reduced CQ resistance, providing the first functional evidence that this residue modulates drug susceptibility. Furthermore, introducing the apparently Colombia-specific I248T mutation significantly enhanced parasite multiplication rates in 7G8, demonstrating complex fitness and sensitivity trade-offs. Our findings reinforce the distinct evolutionary trajectory for South American CQ resistance and highlight the necessity of including additional pfaat1 mutations in global molecular surveillance strategies. Author SummaryAntimalarial drug resistance is a major threat to global public health. Chloroquine was used widely in the 1950s-60s as part of a global malaria eradication campaign, but resistance emerged in multiple places independently and chloroquine resistant parasites directly led to the death of millions of children. Chloroquine resistance is primarily driven by mutations in the transporter PfCRT which are thought to increase export of chloroquine from the digestive vacuole, where chloroquine acts to prevent the ability of the parasite to digest haemoglobin as a source of energy. However, it is becoming increasingly clear that additional vacuolar transporters can also modulate chloroquine resistance. In this study we focused on mutations in the putative amino acid transporter 1 (PfAAT1) which are specific to South America, where chloroquine resistance emerged independently from Southeast Asia. By integrating AlphaFold structural predictions with CRISPR/Cas9 gene editing across geographically diverse parasite backgrounds we provide the first functional evidence that the region-specific V231D mutation significantly diminishes chloroquine resistance. These findings emphasise that chloroquine resistance in South America followed a unique trajectory and that PfAAT1 is involved in complex fitness and sensitivity trade-offs. This work emphasises the benefits of integrating diverse experimental and modelling approaches with global molecular surveillance.

microbiology↗

bioRxiv2026

A combinatorial genetics approach reveals limits to redundancy within Plasmodium falciparum invasion ligand families

To maintain infection in the human bloodstream, Plasmodium falciparum parasites undergo repeated cycles of invasion of and multiplication inside red blood cells (RBCs). Two protein families, the Erythrocyte binding-like (EBA) and Reticulocyte binding-like (Rh) proteins, are known to play a key role in invasion, mediating early stages of attachment of the P. falciparum merozoite to the host RBC. There is a degree of redundancy within these families, such that disrupting the function of individual EBA/Rh proteins in vitro is not sufficient to prevent invasion. By employing a novel approach to disrupt multiple EBA and Rh genes in combination, we systematically assessed functional interdependency across these families for the first time. This analysis, and further characterisation of mutant parasites, revealed that disruption of some pairs of EBA/Rh ligands significantly impacted P. falciparum invasion, whereas others did not. Disruption of PfEBA175 in combination with either PfRh2b or PfRh4 significantly reduced parasite multiplication in vitro, indicating that the remaining EBA/Rh proteins could not fully compensate for the absence of these three key invasion ligands. PfEBA175, PfRh2b and PfRh4 may therefore represent a critical nexus of the parasite invasion machinery, and combinatorial approaches that target these specific ligands could be a beneficial therapeutic approach.

microbiology↗

bioRxiv2025

Characterisation of Two Plasmodium Virulence Factors Important for Lipid Metabolism and Disease Progression In Vivo

Malaria is caused by Plasmodium parasites and claims 610,000 deaths annually. Parasite invasion of red blood cells (RBCs) involves secretion of multiple proteins from specialised apical organelles (micronemes, rhoptries, and dense granules). These collectively facilitate host cell entry, establish a protective parasitophorous vacuole (PV), and directly and indirectly mediate interactions between the infected RBC (iRBC) and the endothelial wall. The cytoadherence of iRBCs results in sequestration within organs and plays a critical role in pathogenesis and virulence, but the function of many secreted parasite proteins remains poorly characterised. Here, we leverage spatial proteomics from Plasmodium falciparum to identify two novel Plasmodium berghei orthologue proteins containing hydrolase domains (MAP1, PBANKA_1425900, and RhoSH, PBANKA_1001500). Ultrastructure expansion microscopy reveals their localisation to the rhoptries in late schizogony, where co-immunoprecipitation assays show they interact with each other. In vivo studies demonstrate that these proteins help the parasite evade spleen-mediated clearance and contribute to disease progression. Deletion of both genes disrupts lipid metabolism and PV membrane morphology, suggesting a role in establishing or maintaining the protective PV membrane. Loss of map1 reduces sequestration of P. berghei iRBCs to adipose tissue, and conditional knockdown of the P. falciparum map1 orthologue results in reduced CD36-mediated cytoadhesion, suggesting a cross-species role in sequestration and cytoadherence. Our findings identify MAP1 and RhoSH as key mediators of Plasmodium virulence.

microbiology↗

bioRxiv2025

The spatial proteome of the Plasmodium falciparum schizont illuminates the composition and evolutionary trajectories of its organelles

Malaria is caused by apicomplexan parasites of the genus Plasmodium, with all malaria symptoms and pathology caused by parasite stages that develop within, or transit between, host erythrocytes. The ability of Plasmodium cells to parasitise erythrocytes depends on distinctive intracellular compartments associated with invasion, as well as the development of unique cellular niches within the infected host cell. However, our understanding of the biology of the malaria parasite is limited by the fact that a large proportion of the parasites proteome has no known cellular location or function. To address this problem, we have generated comprehensive high-resolution maps of protein subcellular localisation for the invasive stage of the erythrocytic life cycle of Plasmodium falciparum, the major cause of malaria mortality. Using the spatial proteomics technique hyperplexed Localisation of Organelle Proteins by Isotopic Tagging (hyperLOPIT) we generated data for 3000 P. falciparum proteins expressed in late schizont stages. Our hyperLOPIT data resolved 24 distinct cellular niches, and using supervised machine-learning we can classify 1646 proteins into one of these compartments including exported sites within the host cell. Through comparative genomic analyses our data resolve the spatial patterns of cell evolution that have shaped the development of Plasmodium species and ongoing adaptive pressures and responses that challenge our efforts to manage these major disease-causing organisms.

microbiology↗

bioRxiv2025

Plasmodium falciparum Myosin F with a Rab-like tail domain localises to perinuclear membranes and associates with trafficking proteins

Members of the myosin superfamily are found in all eukaryotes, including Plasmodium falciparum, the parasite that causes malaria. Plasmodium falciparum expresses six myosins, but apart from PfMyoA, these motors remain largely uncharacterised. This includes the class XXII myosin PfMyoF. Here, we characterise PfMyoF using structural prediction tools, biochemical assays, and advanced imaging. We show that PfMyoF is a plus-end directed, processive motor with a long neck domain containing six IQ motifs that bind the calmodulin homologue PfCaM. PfMyoF can dimerise and is predicted to adopt an autoinhibited conformation via tail backfolding. The PfMyoF tail contains a Rab-like domain that has not been identified in any other known class of myosin. Pull-down experiments show interactions between endogenous PfMyoF and trafficking proteins, including the vesicle marker PfRab18. Expansion and immunoelectron microscopy reveal that PfMyoF localises to a perinuclear membrane compartment. Our findings define PfMyoF as the first known myosin with a Rab-like domain and highlight its potential role in parasite membrane trafficking pathways.

molecular biology↗

bioRxiv2025

Mechanistic Insights into Dual-Active Liver and Blood-Stage Antiplasmodials

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.

microbiology↗

bioRxiv2025

Microfluidic Platform for Automatic Quantification of Malaria Parasite Invasion Under Physiological Flow Conditions

Understanding the impact of forces generated by blood flow on biological processes in the circulatory system, such as the invasion of human red blood cells by malaria parasites, is currently limited by the lack of experimental systems that integrate them. Recent systematic quantification of the growth of Plasmodium falciparum, the species that causes the majority of malaria mortality, under a range of shaking conditions has shown that parasite invasion of erythrocytes is affected by the shear stress to which the interacting P. falciparum merozoites and their target red blood cells are exposed. Blood flow could similarly impact shear stress and therefore invasion in vivo, but there is currently no method to test the impact of flow-induced forces on parasite invasion. We have developed a microfluidic device with four channels, each with dimensions similar to those of a post-capillary venule, but with different flow velocities. Highly synchronised P. falciparum parasites are injected into the device, and parasite egress and invasion rates are quantified using newly developed custom video analysis, which fully automates cell type identification and trajectory tracking. The device was tested with both wild-type P. falciparum lines and lines in which genes encoding proteins involved in parasite invasion had been deleted. Deletion of Erythrocyte Binding Antigen 175 (PfEBA175) has a significant impact on invasion under flow, but not in static culture. These findings establish for the first time that flow conditions can critically affect parasite invasion in a genotype-dependent manner. The method can be applied to other biological processes affected by fluid motion, such as cell adhesion, migration, and mechanotransduction.

microbiology↗

bioRxiv2024

Growing Malaria Parasites at a Critical Shaking Speed Mimicking Physiological Flow Reveals New Phenotypes for Invasion Ligands

Malaria kills over 600,000 people annually, with all the clinical symptoms being caused by the blood stage of the infection. Malaria parasites invade red blood cells (RBCs), where they grow and multiply until daughter parasites egress to invade new RBCs. This cycle happens primarily in the blood circulation, bone marrow, and spleen, where parasites and RBCs are exposed to flow-generated forces. Despite this, almost all in vitro growth assays are carried out in static conditions, which are a poor mimic for the conditions that malaria parasites encounter in the body. Therefore, we systematically tested the impact of dynamic conditions created by orbital shaking platforms on parasite growth and explored the link between growth and the wall shear stress forces generated by fluid motion. For several strains of the deadliest malaria species, Plasmodium falciparum, we showed that strikingly, for any given vessel, there is a critical shaking speed at which growth rates are reduced, corresponding to when the RBCs start to aggregate in the centre of the well, before growth increases again at higher shaking speeds. The force that parasites are exposed to at this critical shaking speed corresponds cloesly with previous measurements for the forces that RBCs and parasites are exposed to in the microvasculature. During invasion, the early attachment of the parasites to RBCs is dependent on two families of attachment proteins, the Erythrocyte Binding Antigen (PfEBA) family and the Reticulocyte Binding Protein Homologue (PfRH) family, which are thought to be largely redundant in function. We used a panel of PfEBA and PfRH knock-out lines to show for the first time that several of these ligands have greater importance in high wall shear stress conditions. This both adds new understanding to the function of these ligand families, and indicates that the concept of the critical shaking speed can reveal new parasite growth phenotypes.

microbiology↗

bioRxiv2024

Application of barcode sequencing to increase the throughput and complexity of Plasmodium falciparum genetic screening

All the pathology and symptoms associated with malaria are caused by the growth of Plasmodium parasites inside human red blood cells. This process, which in the case of the major human malaria pathogen Plasmodium falciparum takes place over a 48-hour period, involves multiple tightly regulated developmental transitions. Understanding the P. falciparum genes that regulate these key processes could lead to the identification of targets for new drugs. However, while large-scale sequencing efforts have led to a good understanding of the P. falciparum genome and how it evolves over time and space, a disconnect remains between the amount of genome sequence data available and the amount of data describing what exactly the genes contained within it do - the phenotype. We have generated a panel of 66 P. falciparum lines carrying individual gene knockouts tagged with unique DNA barcodes. We then used these lines in a series of assays that combine flow cytometry, cell sorting and DNA barcode quantification using next generation sequencing (Barcode Sequencing or BarSeq) to phenotype key aspects of the parasite life cycle such as growth, replication capacity and cell cycle progression. This approach both yields new data about individual gene function, and outlines a new approach where barcoded P. falciparum lines are used in pooled BarSeq-based assays to generate more precise phenotype data at scale.

microbiology↗

bioRxiv2024

A novel chemogenomic screening platform for scalable antimalarial drug target identification

Large-scale chemical-genetic screening, or chemogenomics, can faciliate rapid and scalable drug target identification. To establish a chemogenomic screen for antimalarial drug target identification, we leveraged [~]600 Plasmodium berghei artificial chromosomes (PbACs) encoding potential drug targets to generate a systematic overexpression library. PbACs were engineered with DNA barcodes, enabling their quantification within mixed pools using next generation sequencing (barcode sequencing or BarSeq). Pooled transfection of PbACs into the highly genetically tractable Plasmodium knowlesi demonstrated efficient vector uptake and transcription of encoded P. berghei genes. Parasite pools were exposed to antimalarial candidates, with pilot screens probing for known gene-compound associations identifying their targets with high sensitivity. Screening antimalarial inhibitors with unknown mechanisms of action successfully identified pi4k as the target for one novel compound, which was subsequently validated using in vitro evolution in Plasmodium falciparum parasites. This sensitive and scalable chemogenomics platform therefore represents a valuable early-stage tool for antimalarial target identification.

microbiology↗

bioRxiv2024

Optical tweezers reveal that PfEBA and PfRH ligands, not PfMSP1, play a central role in Plasmodium-falciparum merozoite-erythrocyte attachment

Malaria pathogenesis and parasite multiplication both depend on the ability of Plasmodium falciparum merozoites to invade human erythrocytes. Invasion is a complex multi-step process that is known to involve multiple P. falciparum proteins but dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified P. falciparum strains, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment phenotypes for the first time. Most of the interactions investigated did not affect the force needed to pull merozoites and erythrocytes apart, including loss of the major P. falciparum merozoite surface protein PfMSP1 and PfGAP45, part of the glideosome actinomyosin motor complex. The only factors that significantly reduced the strength of merozoite-erythrocyte attachment were ones that disrupted the function of members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families. While these assays also reinforced the known redundancy within these families, with the deletion of some ligands not impacting detachment force, it appears that the PfEBA/PfRH families play a central role in merozoite attachment, not the major merozoite surface protein PfMSP1. Author summaryMalaria is a devastating disease caused by a parasitic infection. The deadliest species is Plasmodium falciparum, which causes more than 600,000 deaths annually. The parasites life cycle is complex, but all the symptoms of malaria are caused when the parasites replicate in human red blood cells. Replication depends on the invasion of the red blood cells by the parasites which is a complex process involving multiple molecular interactions and multiple steps. Invasion begins with the attachment of the parasite to the red blood cell, making this step of particular interest in the development of new therapeutics. We assessed which interactions are key to the strength of attachment using an optical tweezer assay, which allowed us to directly measure the binding force between individual parasites and red blood cells whilst using a range of molecular and genetic tools that target specific interactions known to have a role in invasion. This showed that loss of a protein commonly thought to be critical to the early stages of binding (PfMSP1) had no effect on attachment strength, whereas disruptions of several members from two families of proteins (the Erythrocyte Binding Like protein family and the reticulocyte binding-like protein family) affect attachment strength.

molecular biology↗

bioRxiv2022

The structure of a Plasmodium vivax Tryptophan Rich Antigen suggests a lipid binding function for a pan-Plasmodium multi-gene family

Tryptophan Rich Antigens (TRAgs) are encoded by a multi-gene family in all Plasmodium species, significantly expanded in P. vivax, but their function is not currently known. We show that multiple P. vivax TRAgs are expressed on the merozoite surface and that one, PVP01_0000100 binds red blood cells with a strong preference for reticulocytes. Solving the structure of the C-terminal tryptophan rich domain that defines the TRAg family revealed a three-helical bundle that is conserved across Plasmodium and has homology with lipid-binding BAR domains involved in membrane remodelling. Biochemical assays confirmed that this domain has lipid binding activity with preference for sulfatide, a glycosphingolipid present in the outer leaflet of plasma membranes. Deletion of the putative orthologue in P. knowlesi, PKNH_1300500, impacts invasion in reticulocytes, suggesting a role for membrane remodelling during this essential process. Together, this work suggests a molecular function for the TRAg family for the first time.

microbiology↗

bioRxiv2022

Barcoding genetically distinct Plasmodium falciparum strains for comparative assessment of fitness and antimalarial drug resistance

The repeated emergence of antimalarial drug resistance in Plasmodium falciparum, including to the current frontline antimalarial artemisinin, is a perennial problem for malaria control. Nextgeneration sequencing has greatly accelerated the identification of polymorphisms in resistance-associated genes, but has also highlighted the need for more sensitive and accurate laboratory tools to profile current and future antimalarials, and to quantify the impact of drug resistance acquisition on parasite fitness. The interplay of fitness and drug response is of fundamental importance in understanding why particular genetic backgrounds are better at driving the evolution of drug resistance in natural populations, but the impact of parasite fitness landscapes on the epidemiology of drug resistance has typically been laborious to accurately quantify in the lab, with assays being limited in accuracy and throughput. Here we present a scalable method to profile fitness and drug response of genetically distinct P. falciparum strains with well-described sensitivities to several antimalarials. We leverage CRISPR/Cas9 genome-editing and barcode sequencing to track unique barcodes integrated into a non-essential gene (pfrh3). We validate this approach in multiplex competitive growth assays of three strains with distinct geographical origins. Furthermore, we demonstrate that this method can be a powerful approach for tracking artemisinin response as it can identify an artemisinin resistant strain within a mix of multiple parasite lines, suggesting an approach for scaling the laborious ring-stage survival assay (RSA) across libraries of barcoded parasite lines. Overall, we present a novel high-throughput method for multiplexed competitive growth assays to evaluate parasite fitness and drug response

microbiology↗

bioRxiv2022

Resolving drug selection and migration in an inbred South American Plasmodium falciparum population with identity-by-descent analysis

The human malaria parasite Plasmodium falciparum is globally widespread, but its prevalence varies significantly between and even within countries. Most population genetic studies in P. falciparum focus on regions of high transmission where parasite populations are large and genetically diverse, such as sub-Saharan Africa. Understanding population dynamics in low transmission settings, however, is of particular importance as these are often where drug resistance first evolves. Here, we use the Pacific Coast of Colombia and Ecuador as a model for understanding the population structure and evolution of Plasmodium parasites in small populations harboring low genetic diversity. The combination of low transmission and a high proportion of monoclonal infections means there are few outcrossing events and clonal lineages persist for long periods of time. Yet despite this, the population is evolutionarily labile and has successfully adapted to multiple drug regimes. Using 166 newly sequenced whole genomes, we measure relatedness between parasites, calculated as identity by descent (IBD), and find 17 distinct but highly related clonal lineages, six of which have persisted in the region for at least a decade. This inbred population structure is captured in more detail with IBD than with other common population structure analyses like PCA, ADMIXTURE, and distance-based trees. We additionally use patterns of intra-chromosomal IBD and an analysis of haplotypic variation to explore the role of recombination in spreading drug resistance mutations throughout the region. Two genes associated with chloroquine resistance, crt and aat1, show evidence of hard selective sweeps, while selection appears soft and/or incomplete at three other key resistance loci (dhps, mdr1, and dhfr). Overall, this work highlights the strength of IBD analyses for studying parasite population structure and resistance evolution in regions of low transmission, and emphasizes that drug resistance can evolve and spread in extremely small populations, as will occur in any region nearing malaria elimination.

evolutionary biology↗

bioRxiv2022

The erythrocyte membrane properties of beta thalassaemia heterozygotes and their consequences for Plasmodium falciparum invasion

Malaria parasites such as Plasmodium falciparum have exerted formidable selective pressures on the human genome. Of the human genetic variants associated with malaria protection, beta thalassaemia (a haemoglobinopathy) was the earliest to be associated with malaria prevalence. However, the malaria protective properties of beta thalassaemic erythrocytes remain unclear. Here we studied the mechanics and surface protein expression of beta thalassaemia heterozygous erythrocytes, measured their susceptibility to P. falciparum invasion, and calculated the energy required for merozoites to invade them. We found invasion-relevant differences in beta thalassaemic cells versus matched controls, specifically: elevated membrane tension, reduced bending modulus, and higher levels of expression of the major invasion receptor basigin. However, these differences acted in opposition to each other with respect to their likely impact on invasion, and overall we did not observe beta thalassaemic cells to have lower P. falciparum invasion efficiency for any of the strains tested.

microbiology↗

bioRxiv2021

Regulators of male and female sexual development critical for transmission of a malaria parasite

The transmission of malaria parasites from vertebrate host to mosquito vector requires a developmental switch in asexually dividing blood-stage parasites to sexual reproduction. In Plasmodium berghei the transcription factor AP2-G is required and sufficient for this switch, but how a particular sex is determined in a haploid parasite remains unknown. Using a global screen of barcoded mutants, we here identify ten genes essential for the formation of either male or female sexual forms and validate their importance for transmission. High-resolution single-cell transcriptomics of wild-type and mutant parasites portrays the developmental bifurcation and reveals a regulatory cascade of putative gene functions in determination and subsequent differentiation of each sex. A male-determining gene with a LOTUS/OST-HTH domain points towards unexpected conservation of molecular mechanisms of gametogenesis in animals and a distantly related eukaryotic parasite.

genomics↗

bioRxiv2021

STEM CELL TECHNOLOGY PROVIDES NOVEL TOOLS TO UNDERSTAND HUMAN VARIATION IN Plasmodium falciparum MALARIA

Plasmodium falciparum interacts with several human cell types during their complex life cycle, including erythrocytes and hepatocytes. The enuclated nature of erythrocytes makes them inaccessible to genetic tools, which in turn makes studying erythrocyte proteins involved in malaria invasion and development particularly difficult. Here we overcome this limitation using stem cell technology to develop a universal differentiation protocol for in vitro derivation of erythrocytes from a variety of stem cell lines of diverse origin. This allows manipulation of erythrocytic genes and examination of their impact on the parasite by flow cytometric detection of parasite haemozoin. Deletion of Basigin, the essential receptor for P. falciparum, abrogates invasion, while other less studied proteins such as ATP2B4 have a minor effect. Reprogramming of induced pluripotent stem cells from -thalassemia primary samples shows reduced infection levels, demonstrating this approach is useful for understanding the effect of natural human polymorphisms on the disease.

cell biology↗