Biology subjects

Claessens, A.

Publications and source records attributed to Claessens, A..

18 recordsLinked to original sources

bioRxiv2026

Genetic ablation of Sarm1 attenuates axonal degeneration in a mouse model of CMT2J

Charcot-Marie-Tooth (CMT) disease is the most common inherited neuropathy, affecting approximately 1 in 2,500 individuals, and comprises a clinically and genetically heterogeneous group of disorders involving more than 100 genes. Mutations in myelin protein zero (MPZ) typically cause the demyelinating form CMT1B, whereas the T124M substitution gives rise to CMT2J, a late-onset, predominantly axonal neuropathy with minimal myelin involvement. We previously generated a MpzT124M mouse model that recapitulates key features of the human disease, including progressive axonal degeneration and fibre loss in peripheral nerves. Because SARM1 (sterile and TIR motif-containing protein 1) is a central executioner of programmed axonal degeneration, we crossed MpzT124M mice with Sarm1-null animals and assessed electrophysiological, morphological, and molecular parameters in both heterozygous and homozygous mutants. Sarm1 deletion did not rescue electrophysiological deficits in either genotype. However, in 12-month-old homozygous MpzT124M/T124M mice, Sarm1 ablation partially reduced axonal degeneration in peripheral nerves and lowered plasma neurofilament light chain, a biomarker of neurodegeneration. In heterozygous MpzT124M/+ mice, where axonal degeneration develops more slowly, Sarm1 deletion was associated with improved motor performance and reduced morphological signs of disease by 18 months of age. Together, these findings indicate that SARM1 contributes to axonal degeneration in CMT2J and that its ablation confers partial protection, although it is insufficient to fully prevent disease progression, supporting SARM1 as a therapeutically relevant target in MPZ-associated axonal neuropathy despite incomplete disease rescue.

neuroscience↗

bioRxiv2026

Single-cell characterization of the human immune landscape reveals dysfunction in monocyte-mediated cellular interactions in symptomatic malaria

Plasmodium falciparum infection results in a wide range of phenotypes including chronic asymptomatic and symptomatic individuals. The malaria outcome depends, at least in part, on host immune responses mediated by specific cell subsets. Here, we investigated the immune landscape of chronic asymptomatic and symptomatic children using single-cell RNA sequencing. Unexpectedly, we uncovered biologically relevant immune cell complexes, which were further confirmed by imaging flow cytometry. Symptomatic infection was associated with a decrease of cell-cell interactions, particularly between non-classical monocytes and T cells. Non-classical monocytes from symptomatic children exhibited profiles with increased inflammation characterized by enhanced S100A8-S100A9 expression together with decreased expression of interaction-associated genes such as CD86 or ITGAL. Moreover, we demonstrated that inflammatory and cell interaction programs could not be activated simultaneously within the same cell. In addition, the concomitant expression of inflammatory genes and members of the LILR gene family was predominantly observed in monocytes from symptomatic individuals. These findings raise the possibility that enhanced S100A8/S100A9-LILRB1 signaling may contribute to the altered functional state of monocytes in symptomatic malaria and, potentially, may impair their interactions with T cells. Together, our results suggest that immune cell complexes may therefore reveal an additional layer of immune regulation associated with the development of clinical malaria.

genomics↗

bioRxiv2026

Large deletion variants in a Plasmodium falciparum ookinete protein gene and associations with different endemic populations and mosquito vectors

The Plasmodium falciparum PIMMS43 gene encodes an ookinete protein that is important for mosquito infection. Here, large indel variants within the coding sequence are identified, and frequencies in natural infections of humans and mosquitoes investigated. Comparing long-read genome sequences in a small panel of P. falciparum strains and related species revealed a 150 bp deletion in the central part of the gene in several strains including the standard 3D7 reference genome. Mapping of short-read genome sequence data from 524 P. falciparum infections from nine different countries to full-length PIMMS43 showed the 150 bp deletion and an alternative 90 bp deletion to be common structural variants. Across African populations, the 150 bp deletion had a mean allele frequency of 32%, and the 90 bp deletion a mean frequency of 12%, with significant geographical variation. Targeted genotyping of 30 oocysts from naturally infected mosquitoes in Tanzania by nested PCR showed overall deletion variant frequencies similar to those seen in human infections in the same country. Although sample size was limited, a difference in the variant frequencies in oocysts from Anopheles gambiae and Anopheles funestus suggests potential vector-specific selection. The findings highlight the importance of surveying structural genomic variation and its potential role in parasite adaptation. Data summaryThe long-read genome sequence data sources are listed in the Supplementary Information (Supplementary Table 1). The short-read genome sequence data from human infections were extracted as a selected subset of those in the Pf7 release of global data from the MalariaGEN Consortium as described in the Methods. The SNP and indel genotypes derived from each sample of parasites cultured in the laboratory and from natural mosquito infections are given in the Supplementary Information (Supplementary Tables 1 and 3, Supplementary Figure 1). Impact statementThis study identifies large indel polymorphisms in the malaria parasite transmission-stage gene PIMMS43, reflecting substantial structural variation previously overlooked by genome-wide SNP-focused analyses. Using long- and short-read genomic data across diverse samples followed by targeted genotyping, we show that deletion variants are frequent and geographically structured, with initial evidence consistent with potential vector-specific selection. This highlights the importance of incorporating structural variation into population genomic surveillance and studies of parasite adaptation.

genomics↗

bioRxiv2026

Tracking malaria parasite lineages through de novo mutations in highly related Plasmodium falciparum genomes

Where malaria transmission declines, the remaining infections are increasingly low-density and asymptomatic, forming a persistent reservoir that is difficult to track using conventional epidemiological approaches. However, genomic data from such community-level infections remain scarce, limiting the ability to track parasite lineages, detect clonal expansions, and identify persistent chronic infections in pre-elimination settings. Here, 78 single-genotype P. falciparum genome sequences are analysed from community infections within a small area of The Gambia, where malaria transmission has substantially declined over recent decades. Pairwise identity-by-descent (IBD) analysis revealed generally low genetic relatedness among parasites, consistent with ongoing recombination and genetic mixing at the community scale. Nevertheless, eight clusters of near-identical genomes (IBD > 0.9) were identified, enabling the inference of recent de novo mutations that differentiate these genomes. Across these clusters, 43 de novo single-nucleotide polymorphisms and 19 short indels were identified using long-read-derived reference genomes. The observed pattern of mutation in natural infections broadly resembled that previously reported from laboratory mutation-accumulation experiments, including a strong transition bias and enrichment of G:C[->]A:T substitutions. These results demonstrate that combining IBD analysis with de novo mutation detection enables fine-scale resolution of parasite relatedness and recent transmission history. As malaria transmission continues to decline, such approaches may become increasingly valuable for tracking local transmission, identify parasite lineages, and potentially distinguish persistent infections from reintroduction events.

genetics↗

bioRxiv2026

Plasmodium falciparum invasion ligand gene transcript profiles in different populations

Plasmodium falciparum merozoites invade erythrocytes using various ligand-receptor interactions. Important ligands encoded by the eba and Rh gene families have varying expression levels in different parasite isolates, affecting their vaccine candidacy. Analyses of clinical isolates from endemic areas in Africa have indicated that most variation in these expression profiles exists within each local area, and only minor differences are seen between areas, although comparisons with non-African populations have not previously been performed. To enable this, relative transcript levels of three eba genes and five Rh genes have been analysed in new population samples, Malaysian isolates sampled from Sabah State in Borneo prior to endemic malaria elimination, and Gambian isolates, cultured under the same conditions to harvest schizonts for reverse transcription quantitative PCR assays. Significant differences between these populations were seen for three of the ligand genes, levels of eba175 being higher in Malaysia, while levels of eba181 and Rh2b were lower in Malaysia. The gene transcript profiles did not differ between single genotype and or multiple-genotype isolates. The distinctness of the Malaysian population expression profile was also supported by comparing previous data on clinical isolates from Ghana. In tests for correlation with previously determined parasite multiplication rates, eba181 transcript levels correlated positively among Malaysian isolates but not among Gambian isolates. These findings suggest that expression of three P. falciparum merozoite ligands involved in invasion may be regionally differentiated, and further analysis of Asian parasite populations would be important if vaccines based on these candidates are to be considered for future use.

microbiology↗

bioRxiv2026

A partition-based spatial entropy for co-occurrence analysis with broad application.

Despite the advent of spatial data science, including spatial biology, there exist few methods that study the distribution of points e.g. cells or individuals, accounting for both their own characteristics and environmental factors. We propose a new spatial entropy measure, termed the Regional Co-occurrence Entropy (RCE), that detects when categorical co-occurrences happen preferentially in specific environments. We demonstrate its use over a broad range of application fields. As examples, we study brain cell dynamics in Alzheimers Disease, identifying both known and likely novel interactions between immune cells around beta-amyloid plaques. We also investigate the diversity of buildings across a town neighborhoods, to detect potential drivers of social mixing at local scale. Finally, we dissect bird species distribution across a natural reserve, identifying potential vegetation-driven changes in community composition. Altogether, the proposed RCE enables rapid insights into interactions with an environmental component, making it a useful addition to the spatial data science toolbox. Significance StatementSpatial data is rapidly accumulating across fields as diverse as spatial biology, geography, ecology or astrophysics and promises to allow major scientific advances. For example, spatial biology is transforming our ability to study complex tissue organization and disease mechanisms. A key challenge remains the quantification of spatial relationships between individual points, such as cell types, in a statistically rigorous way. We propose a spatial entropy-based measure to quantify context-dependent interactions between categories, such as cell types across tissue subregions. Our novel method deriving from Information Science provides an efficient and versatile way to extract information from spatial datasets, with broad applicability across research fields.

bioinformatics↗

bioRxiv2026

Characterising recent antimalarial resistance in West Africa: Insights from amplicon sequencing of 17,384 Plasmodium falciparum infection samples

Plasmodium falciparum (P. falciparum) infection remains a significant public health threat in West Africa, where chemoprevention and first-line therapies are key interventions against malaria. However, the development and spread of resistance to commonly used antimalarials poses a growing threat to the efficacy of these strategies. This study characterises the recent landscape of antimalarial resistance in West Africa by analysing targeted amplicon sequences from 17,384 P. falciparum infection samples. Across countries, the prevalence of the pyrimethamine resistance-associated dhfr triple mutant allele (51I/59R/108N) exceeded 80%, while its combination with the sulphadoxine resistance-associated dhps 437G exceeded 60% of infections. Unlike the parasite genotypes in East Africa, the prevalence of the dhps 540E mutant was low (1.5%), whereas dhps 436A was common (43.8%). The chloroquine resistance marker crt 76T showed greatest geographic heterogeneity, ranging from low prevalence in Ghana (1.3%) to very common in The Gambia (64.9%). Non-synonymous mutants of kelch13 were uncommon, most with unknown relevance to artemisinin resistance and observed for the first time in Africa. However, mutants that are artemisinin resistance-associated elsewhere were detected in three infection samples from Ghana (574L, 561H, 469Y), and one in Cameroon (538V). This large-scale genomic surveillance of P. falciparum infections highlights the need for ongoing monitoring of drug resistance and for data integration throughout the region.

genomics↗

bioRxiv2025

De novo assembly of complete Plasmodium falciparum isolate genomes using PacBio HiFi sequencing technology

Plasmodium falciparum possesses a highly structured genome with extensive sequence diversity concentrated in Variant Surface Antigen (VSA) families. These genes--var, rif, and stevor--play key roles in immune evasion and pathogenesis and are difficult to assemble using short-read sequencing technologies. Here, we applied PacBio HiFi long-read sequencing to generate high-quality de novo genome assemblies from 43 P. falciparum parasite cultures originating from community cases in The Gambia. Parasites were culture-adapted, cloned by limiting dilution where possible, and sequenced using high molecular weight DNA extracts. Assemblies from single-genotype lineages were constructed using hifiasm, producing complete chromosomal-length scaffolds with high base accuracy without requiring short-read polishing. We recovered full repertoires of var, rif, and stevor genes and classified them into known subgroups. Together, our results demonstrate that PacBio HiFi sequencing enables accurate assembly of complex P. falciparum genomes from natural infections. This work provides a valuable genomic resource for future studies of parasite evolution, transmission dynamics, and antigenic diversity, and suggests that VSA repertoires can serve as reliable proxies of genetic relatedness across infections.

genomics↗

bioRxiv2025

Taurine is a Natural Host Cytoadhesion Inhibitor in Asymptomatic Malaria Cases

The prolonged dry season in malaria-endemic regions of sub-Saharan Africa can be divided into periods of high and low transmission. The majority of symptomatic malaria cases are restricted to the short high transmission period that follows the rainy season. Shortly after, persistent asymptomatic malaria cases are more prevalent throughout the prolonged low transmission period. It is still unclear whether host metabolic alterations play a role in asymptomatic infections during seasonal malaria. In this study, we analyzed the blood plasma metabolome (n=199) of individuals in The Gambia, West Africa, capturing data from both high and low malaria transmission periods. Plasma samples from individuals (n=16) were collected monthly throughout the low transmission season, enabling a longitudinal analysis of metabolic alternations over six months. Our findings reveal that significant changes in host plasma metabolite composition are associated with seasonality and malaria pathogenicity. Notably, we observed elevated levels of taurine in asymptomatic malaria infections, especially during periods of low transmission. In vitro, this naturally occurring host molecule inhibits the cytoadhesion of malaria-infected red blood cells (iRBCs), which is key to malaria disease severity and mortality. Exogenous taurine can significantly reduce or reverse binding of iRBCs to the common adhesion receptor CD36 and the endothelial protein C receptor (EPCR), the later being associated with cerebral malaria. This study uncovers a mechanism by which elevated taurine plasma levels in asymptomatic infections could reduce cytoadhesion and lead to increased splenic clearance, thereby strengthening host resistance to symptomatic infections. In the absence of health strategies targeting dry season parasite reservoirs, our findings highlight taurine as a potential prophylactic or therapeutic agent to reduce symptomatic malaria in sub-Saharan Africa. One Sentence SummaryMetabolomics uncovers taurine as a natural host inhibitor of Plasmodium falciparum cytoadhesion in asymptomatic malaria carriers, offering new insights for disease control.

microbiology↗

bioRxiv2025

upsAI: A high-accuracy machine learning classifier for predicting Plasmodium falciparum var gene upstream groups

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the hypervariable var gene family, is central to malaria pathogenesis, influencing both disease severity and immune evasion. Classifying var genes into upstream groups (upsA, upsB, upsC, upsE) is important for understanding parasite biology and clinical outcomes, but remains challenging, especially with partial sequences, such as the DBL tag or RNA-Seq assemblies. We developed upsAI, a machine learning-based classifier trained on 2,530 curated var genes, to accurately assign upstream groups using sequence features from different partial gene regions. We compared seven different methods, including support vector machines, random forest, XGB boost and HMMer models. The best model of upsAI for DBL-tags sequences achieves an overall accuracy of 83%, 92% and for full-length var genes, therefore significantly outperforming existing tools. Further, we propose a new model to distinguish between internal and subtelomeric var genes with high accuracy and scalability. upsAI is available at https://github.com/sii-scRNA-Seq/upsAI, providing a robust and efficient resource for large-scale var gene analysis. It can classify var genes from 20 genomes in under one second.

bioinformatics↗

bioRxiv2025

An acyclic nucleoside phosphonate effectively blocks the egress of the malaria parasite by inhibiting the synthesis of cyclic GMP.

The urgent need for new antimalarial therapies arises from the alarming spread of malaria parasite resistance to existing drugs. A promising candidate, UA2239, an acyclic nucleoside phosphonate with a guanine as nucleobase, demonstrates rapid and irreversible cytotoxic effects on Plasmodium parasites, both in vitro and in an animal model. It blocks the active exit process, named egress, of merozoites and gametes from infected erythrocytes. UA2239 disrupts the essential cGMP-dependent egress pathway by decreasing cGMP levels in the parasite, making guanylate cyclase (PfGC) the most likely target. We also uncovered the remarkable molecular mechanism of resistance developed by parasites after prolonged exposure to the drug, which involves mutating not the target itself, but a downstream effector. The unique mechanism action of UA2239 makes it a valuable first-in-class candidate for further development and its ability to inhibit both parasite growth and transmission highlights its therapeutic potential as a dual-stage antimalarial agent.

microbiology↗

bioRxiv2024

Higher multiplication rates of Plasmodium falciparum in isolates from hospital cases compared with community infections

BackgroundParasite multiplication rates vary among Plasmodium falciparum isolates from patients with malaria, suggesting differences in virulence potential, although direct comparisons between hospital-based clinical cases and community infections are needed. MethodsCryopreserved blood samples from malaria cases presenting to a district hospital in The Gambia and infections detected in local communities were introduced to continuous culture under the same conditions. Thirty-four isolates (23 hospital-based and 11 community-based) were successfully established and then tested under exponential growth conditions over six days to derive estimated P. falciparum multiplication rates per cycle based on a 48-hour typical cycle length. ResultsA range of parasite multiplication rates in culture was seen across isolates, from 1.5-fold to 5.0-fold per cycle. Multiplication rates were significantly higher in the hospital-based isolates than the community-based isolates. There was a significantly positive correlation between parasitaemia in peripheral blood and multiplication rates in culture. There was no significant difference in multiplication rates between isolates with single or multiple parasite genotypes. ConclusionsThese findings are consistent with a hypothesis that intrinsic natural variation in parasite multiplication rate may affect levels of parasitaemia achieved during infection, and that this affects likelihood of hospital presentation. Results do not support a hypothesis that parasites modify their multiplication rates in response to competing parasites with different genotypes. SummaryRelevant to understanding parasite virulence, this study finds higher Plasmodium falciparum multiplication rates in cultured isolates from malaria cases presenting to hospital than in isolates from local community infections, and positive correlation with parasitaemia in peripheral blood of individuals.

microbiology↗

bioRxiv2024

Evolution and inhibition of the FIKK effector kinase family in P. falciparum

Among the [~]200 Plasmodium species that infect vertebrates, six infect humans. Of these, P. falciparum causes >95% of all [~]500,000 annual fatalities. Phylogenetically, P. falciparum belongs to the Laverania subgenus, a group of Plasmodium species that infect great apes. Common to Laverania species is the family of FIKK kinases. One million years ago, a single FIKK kinase conserved in all Plasmodium species gained an export element in the Laverania subgenus and expanded into the family of [~]20 atypical FIKK kinases, most of which are exported into the host cell. The fikk genes are conserved in syntenic loci across the Laverania, arguing for a rapid expansion controlling important functions in host cell remodelling and pathogenesis. We provide evidence that the FIKK paralogues evolved specific and mutually exclusive phosphorylation motif preferences, conserved across their Laverania orthologues, in a short evolutionary timeframe. Surprisingly, we find that FIKK13 has evolved exclusive tyrosine-phosphorylation preference, which was thought to be absent in Plasmodium species. Combining a crystal structure with AlphaFold2 predictions, we identify residues that determine kinase-specificity within the FIKK family in a fast-evolving flexible loop. Finally, we show that all expressed members of the FIKK kinase family can be chemically inhibited in vitro using a single compound. Such a pan-specific inhibitor of this kinase family important for virulence could reduce the ability of the parasite to gain escape-mutations and resistance.

microbiology↗

bioRxiv2024

Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B

Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1 is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 (Xbp1s). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.

neuroscience↗

bioRxiv2024

A complex Plasmodium falciparum cryptotype circulating at low frequency across the African continent

The population structure of the malaria parasite Plasmodium falciparum can reveal underlying demographic and adaptive evolutionary processes. Here, we analyse population structure in 4,376 P. falciparum genomes from 21 countries across Africa. We identified a strongly differentiated cluster of parasites, comprising [~]1.2% of samples analysed, geographically distributed over 13 countries across the continent. Members of this cluster, named AF1, carry a genetic background consisting of a large number of highly differentiated variants, rarely observed outside this cluster, at a multitude of genomic loci distributed across most chromosomes. At these loci, the AF1 haplotypes appear to have common ancestry, irrespective of the sampling location; outside the shared loci, however, AF1 members are genetically similar to their sympatric parasites. AF1 parasites sharing up to 23 genomic co-inherited regions were found in all major regions of Africa, at locations over 7,000 km apart. We coined the term cryptotype to describe a complex common background which is geographically widespread, but concealed by genomic regions of local origin. Most AF1 differentiated variants are functionally related, comprising structural variations and single nucleotide polymorphisms in components of the MSP1 complex and several other genes involved in interactions with red blood cells, including invasion and erythrocyte antigen export. We propose that AF1 parasites have adapted to some as yet unidentified evolutionary niche, by acquiring a complex compendium of interacting variants that rarely circulate separately in Africa. As the cryptotype spread across the continent, it appears to have been maintained mostly intact in spite of recombination events, suggesting a selective advantage. It is possible that other cryptotypes circulate in Africa, and new analysis methods may be needed to identify them.

genomics↗

bioRxiv2023

RNA polymerase III is involved in regulating Plasmodium falciparum virulence

While often undetected and untreated, persistent seasonal asymptomatic malaria infections remain a global public health problem. Despite the presence of parasites in the peripheral blood, no symptoms develop. Disease severity is correlated with the levels of infected red blood cells (iRBCs) adhering within blood vessels. Changes in iRBC adhesion capacity have been linked to seasonal asymptomatic malaria infections, however how this is occurring is still unknown. Here we present evidence that RNA polymerase III (RNA Pol III) transcription in Plasmodium falciparum is downregulated in field isolates obtained from asymptomatic individuals during the dry season. Through experiments with in vitro cultured parasites, we have uncovered an RNA Pol III-dependent mechanism that controls pathogen proliferation and expression of a major virulence factor in response to external stimuli. Our findings establish a connection between P. falciparum cytoadhesion and a non-coding RNA family transcribed by Pol III. Additionally, we have identified P. falciparum Maf1 as a pivotal regulator of Pol III transcription, both for maintaining cellular homeostasis and responding adaptively to external signals. These results introduce a novel perspective that contributes to our understanding of P. falciparum virulence. Furthermore, they establish a connection between this regulatory process and the occurrence of seasonal asymptomatic malaria infections.

microbiology↗

bioRxiv2022

Genomic variation during culture-adaptation of genetically complex Plasmodium falciparum clinical isolates

Experimental studies on the biology of malaria parasites have been mostly based on laboratory-adapted lines, but there is limited understanding of how these may differ from parasites in natural infections. Loss-of-function mutants have previously been shown to emerge during culture of some Plasmodium falciparum clinical isolates, in analyses that focused on single-genotype infections. The present study included a broader array of isolates, mostly representing multiple-genotype infections which are more typical in areas where malaria is highly endemic. Genome sequence data from multiple time points during several months of culture adaptation of 28 West African isolates were analysed, including previously available sequences along with new genome sequences from additional isolates and timepoints. Some genetically complex isolates eventually became fixed over time to single surviving genotypes in culture, whereas others retained diversity although proportions of genotypes varied over time. Drug-resistance allele frequencies did not show overall directional changes, suggesting that resistance-associated costs are not the main causes of fitness differences among parasites in culture. Loss-of-function mutants emerged during culture in several of the multiple-genotype isolates, affecting genes (including AP2-HS, EPAC and SRPK1) for which loss-of-function mutants were previously seen to emerge in single-genotype isolates. Parasite clones were derived by limiting dilution from six of the isolates, and sequencing identified de novo variants not detected in the bulk isolate sequences. Interestingly, most of these were nonsense mutants and frameshifts disrupting the coding sequence of EPAC, the gene with the largest number of independent nonsense mutants previously identified in laboratory-adapted lines. Analysis of Identity-By-Descent to explore relatedness among clones revealed co-occurring non-identical sibling parasites, illustrative of the natural genetic structure within parasite populations.

microbiology↗

bioRxiv2020

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood. We show intrinsic multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation at each of the cultured timepoints robustly correlated with parasite levels in patients at clinical presentation, indicating parasite control of multiplication that contributes to virulence.

microbiology↗