Biology subjects

Cheeseman, I. H.

Publications and source records attributed to Cheeseman, I. H..

17 recordsLinked to original sources

bioRxiv2026

A Multidimensional Analysis of the Bimodal Piperaquine Response in Plasmodium falciparum

Malaria remains a pressing global health challenge, with the continued emergence of resistance threatening the long-term efficacy of artemisinin-based combination therapies (ACTs). Piperaquine (PPQ), an important partner drug in artemisinin-based combination therapies exhibits a unique bimodal dose-response phenotype associated with reduced susceptibility, yet the biological mechanism underlying this phenotype remains unknown. This phenotype is strongly associated with mutations in pfcrt and copy number amplification of plasmepsin II/III (pm II/III). Given that plasmepsins play a central role in hemoglobin degradation within the blood stage parasite digestive vacuole, and that PPQ accumulates within this compartment and perturbs heme detoxification, this phenotype likely reflects alterations in fundamental biological processes alongside drug-specific effects. We used isogenic PPQ-resistant parasite clones differing only in pm II/III copy number to integrate phenotypes with metabolic changes, and transcriptional responses to ascertain the impact of genotype combinations on parasite response to PPQ. Across increasing PPQ concentrations, parasites with elevated pm II/III copy number exhibited distinct metabolic responses compared to single-copy parasites, specifically, an altered abundance of peptides derived from hemoglobin degradation, directly implicating a core biological pathway long associated with plasmepsin function. The combination of metabolic and transcriptional data with phenotypic measurements supports a model in which increased plasmepsin expression enhances the parasites capacity to sustain hemoglobin digestion and associated metabolic activity under high PPQ concentrations. This points to a mechanistic basis for continued parasite survival, indicating that changes in hemoglobin processing within the digestive vacuole contribute to the bimodal response to PPQ. Molecular dynamics simulations further support a direct interaction between PPQ and PM II/III, as a mechanism by which these proteins impact PPQ response dynamics through both modulation of hemoglobin digestion and protein-drug interactions within the digestive vacuole.

molecular biology↗

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↗

bioRxiv2025

Advantages of outcrossing in Plasmodium falciparum: insights from genetic crosses using fluorescent labelled parasites

Malaria parasites are obligately sexual hermaphrodite protozoans with gamete fusion occurring in the mosquito midgut, followed by meiosis and recombination. Malaria parasite populations show a spectrum of populations structures ranging from predominantly selfing to highly outcrossed. However, the fitness consequences of selfing and outcrossing for malaria parasites are poorly understood. This project was designed to investigate the dynamics of gamete fusion within the mosquito midgut and the relative fitness of selfed and outcrossed zygotes. We generated florescent-labelled clones of NF54 (mCherry), an African parasite, and NHP4026 (GFP), a Thai parasite, crossed these parasites, and scored genotypes of 8540 oocysts from 435 mosquitoes sampled from 7 to 14 days post infection. We observed decreasing proportions of outcrossed oocysts and increasing levels of inbreeding over the course of the infection in two independently replicated crosses. These results are consistent with the faster maturation of transmissible sporozoites derived from outcrossed compared with selfed oocysts. Our results suggest a substantial outcrossing advantage, perhaps because this allows for the removal of deleterious mutations accumulated during asexual parasite replication in the vertebrate host. We also found that selfed NF54 oocysts were larger than outcrossed or selfed NHP4026 oocysts, which may influence production of sporozoites and onward transmission. We conclude that fluorescent labelled parasites provide clear resolution of mating patterns, temporal dynamics and transmission potential of malaria parasites in mosquitoes. Importantly, faster maturation of outcrossed parasites can maximize levels of recombination in transmitted malaria parasite populations.

genetics↗

bioRxiv2025

The genomic legacy of selectively breeding rhesus macaques for HIV/AIDS-related research

BackgroundRhesus macaques are the preferred non-human primate model for HIV/AIDS-related research. Specific major histocompatibility complex (MHC) haplotypes and genetic ancestry are linked to slow disease progression of Simian Immunodeficiency Virus (SIV) infections in macaques. To maximize their utility for HIV/AIDS research, the Southwest National Primate Research Center (SNPRC) has introduced targeted breeding strategies to reduce the prevalence of SIV-refractory MHC haplotypes and levels of admixture between Indian- and Chinese-origin macaques. ResultsWe characterized the MHC region in SNPRC macaques by targeted deep sequencing of 1,458 animals born over a ten-year period. Following the implementation of MHC management strategies, the prevalence of SIV-refractory MHC haplotypes reduced significantly while overall haplotype diversity was maintained. We investigated the impact of management strategies on admixture, population genetic structure, genetic diversity and inbreeding using whole exome sequencing of founding and colony-born animals (n=488). Admixture analysis of founders showed some Chinese ancestry, though population substructure more closely reflected primate research center source than geographical origin. The levels of Chinese ancestry declined significantly over time, though genetic diversity remains high. Finally, we performed genome-wide scans for genetic selection over time. We identify numerous genomic regions where allele frequencies have shifted significantly, supporting the presence of short-term adaptation within the colony. ConclusionsWe show that colony management strategies have been successful without reducing genetic diversity of the MHC or exonic regions. We also show that colony genetic substructure is related to animal colony source and that mergers and migrations have reduced inbreeding and increased overall genetic diversity.

genomics↗

bioRxiv2024

MalKinID: A Likelihood-Based Model for Identifying Malaria Parasite Genealogical Relationships Using Identity-by-Descent

Pathogen genomics is a powerful tool for tracking infectious disease transmission. In malaria, identity-by-descent (IBD) is used to assess the genetic relatedness between parasites and has been used to study transmission and importation. In theory, IBD can be used to distinguish genealogical relationships to reconstruct transmission history or identify parasites for genotype- to-phenotype quantitative-trait-locus experiments. MalKinID (Malaria Kinship Identifier) is a new likelihood-based classification model designed to identify genealogical relationships among malaria parasites based on genome-wide IBD proportions and IBD segment distributions. MalKinID was calibrated to the genomic data from three laboratory-based genetic crosses (yielding 440 parent-child and 9060 full-sibling comparisons). MalKinID identified lab generated F1 progeny with >80% sensitivity and showed that 0.39 (95% CI 0.28, 0.49) of the second- generation progeny of a NF54 and NHP4026 cross were F1s and 0.56 (0.45, 0.67) were backcrosses of an F1 with the parental NF54 strain. In simulated outcrossed importations, MalKinID accurately reconstructs genealogy history with high precision and sensitivity, with F1- scores exceeding 0.84. However, when importation involves inbreeding, such as during serial co-transmission, the precision and sensitivity of MalKinID declined, with F1-scores of 0.76 (0.56, 0.92) and 0.23 (0.0, 0.4) for PC and FS and <0.05 for second-degree and third-degree relatives. Genealogical inference is most powered 1) when outcrossing is the norm or 2) when multi- sample comparisons based on a predefined pedigree are used. MalKinID lays the foundations for using IBD to track parasite transmission history and for separating progeny for quantitative- trait-locus experiments.

genetics↗

bioRxiv2023

Genetic characterization of a captive marmoset colony using genotype-by-sequencing

The marmoset is a fundamental non-human primate model for the study of aging, neurobiology, and many other topics. Genetic management of captive marmoset colonies is complicated by frequent chimerism in the blood and other tissues, a lack of tools to enable cost-effective, genome-wide interrogation of variation, and historic mergers and migrations of animals between colonies. We implemented genotype-by-sequencing (GBS) of hair follicle derived DNA (a minimally chimeric DNA source) of 82 marmosets housed at the Southwest National Primate Research Center (SNPRC). Our primary goals were the genetic characterization of our marmoset population for pedigree verification and colony management and to inform the scientific community of the functional genetic makeup of this valuable resource. We used the GBS data to reconstruct the genetic legacy of recent mergers between colonies, to identify genetically related animals whose relationships were previously unknown due to incomplete pedigree information, and to show that animals in the SNPRC colony appear to exhibit low levels of inbreeding. Of the >99,000 single-nucleotide variants (SNVs) that we characterized, >9,800 are located within gene regions known to harbor pathogenic variants of clinical significance in humans. Overall, we show the combination of low-resolution (sparse) genotyping using hair follicle DNA is a powerful strategy for the genetic management of captive marmoset colonies and for identifying potential SNVs for the development of biomedical research models.

genetics↗

bioRxiv2023

Using a P. falciparum genetic cross to dissect the relative contributions of pfcrt and plasmepsin II/III to piperaquine response-related traits

Piperaquine (PPQ) is widely used in combination with dihydroartemisinin (DHA) as a first-line treatment against malaria parasites. Multiple genetic drivers of PPQ resistance have been reported, including mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and increased copies of plasmepsin II/III (pm2/3). We generated a cross between a Cambodia-derived multi-drug resistant KEL1/PLA1 lineage isolate (KH004) and a drug susceptible parasite isolated in Malawi (Mal31). Mal31 harbors a wild-type (3D7-like) pfcrt allele and a single copy of pm2/3, while KH004 has a chloroquine-resistant (Dd2-like) pfcrt allele with an additional G367C substitution and four copies of pm2/3. We recovered 104 unique recombinant progeny and examined a targeted set of progeny representing all possible combinations of variants at pfcrt and pm2/3 for detailed analysis of competitive fitness and a range of PPQ susceptibility phenotypes, including PPQ survival assay (PSA), area under the dose-response curve (AUC), and a limited point IC50 (LP-IC50). We find that inheritance of the KH004 pfcrt allele is required for PPQ resistance, whereas copy number variation in pm2/3 further enhances resistance but does not confer resistance in the absence of PPQ-R-associated mutations in pfcrt. Deeper investigation of genotype-phenotype relationships demonstrates that progeny clones from experimental crosses can be used to understand the relative contributions of pfcrt, pm2/3, and parasite genetic background, to a range of PPQ-related traits and confirm the critical role of the PfCRT G367C substitution in PPQ resistance. ImportanceResistance to PPQ used in combination with DHA has emerged in Cambodia and threatens to spread to other malaria-endemic regions. Understanding the causal mutations of drug resistance and their impact on parasite fitness is critical for surveillance and intervention, and can also reveal new avenues to limiting the evolution and spread of drug resistance. An experimental genetic cross is a powerful tool for pinpointing the genetic determinants of key drug resistance and fitness phenotypes and have the distinct advantage of assaying the effects of naturally evolved genetic variation. Our study was significantly strengthened because the full a range of copies of KH004 pm2/3 was inherited among the progeny clones, allowing us to directly test the role of pm2/3 copy number on resistance-related phenotypes in the context of a unique pfcrt allele. Our multi-gene model suggests an important role for both loci in the evolution of this ACT resistant parasite lineage.

genomics↗

bioRxiv2022

The amino acid transporter pfaat1 modulates chloroquine resistance and fitness in malaria parasites

Malaria parasites break down host hemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, but typically affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on an amino acid transporter (pfaat1) S258L variant which increased from 0-87% in frequency between 1984 and 2014 in parallel with pfcrt1 K76T. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ-resistance but at a cost of reduced fitness, while pfaat1 F313S, a common Southeast Asian polymorphism, reduces CQ-resistance while restoring fitness. Our analyses reveal hidden complexity in CQ-resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport.

genomics↗

bioRxiv2022

Human alveolar macrophage response to Mycobacterium tuberculosis: immune characteristics underlying large inter-individual variability

Mycobacterium tuberculosis (M.tb) establishes residence and growth in human alveolar macrophages (AMs). Large inter-individual variation in M.tb-AM interactions is a potential early indicator of TB risk and efficacy of therapies and vaccines. Herein, we systematically analyze interactions of a virulent M.tb strain with freshly isolated human AMs from 28 healthy adult donors, measuring host RNA expression and secreted candidate proteins associated with TB pathogenesis over 72h. We observe large inter-individual differences in bacterial uptake and growth, with tenfold variation in M.tb load at 72h, reflected by large variation of gene expression programs. Systems analysis of differential and variable RNA and protein expression identifies TB-associated genes and networks (e.g., IL1B and IDO1). RNA time profiles document early stimulation of M1-type macrophage gene expression followed by emergence of an M2-type profile. The fine-scale resolution of this work enables the separation of genes and networks regulating early M.tb growth dynamics, and development of potential markers of individual susceptibility to M.tb infection and response to therapies.

systems biology↗

bioRxiv2021

Optimizing bulk segregant analysis of drug resistance using Plasmodium falciparum genetic crosses conducted in humanized mice

BackgroundClassical genetic crosses in malaria parasites involve isolation, genotyping, and phenotyping of multiple progeny parasites, which is time consuming and laborious. Bulk segregant analysis (BSA) offers a powerful and efficient alternative to identify loci underlying complex traits in the human malaria parasite, Plasmodium falciparum. MethodsWe have used BSA, which combines genetic crosses using humanized mice with pooled sequencing of progeny populations to measure changes in allele frequency following selection with antimalarial drugs. We used dihydroartemisinin (DHA) drug selection in two genetic crosses (Mal31xKH004 and NF54xNHP1337). We specifically investigated how synchronization, cryopreservation, and the drug selection regimen of progeny pools impacted the success of BSA experiments. FindingsWe detected a strong and repeatable quantitative trait locus (QTL) at chr13 kelch13 locus in both crosses, but did not detect QTLs at ferredoxin (fd), the apicoplast ribosomal protein S10 (arps10), multidrug resistance protein 2 (mdr2). QTLs were detected using synchronized, but not unsynchronized pools, consistent with the stage-specific action of DHA. We also successfully applied BSA to cryopreserved progeny pools. InterpretationOur results provide proof-of-principal of the utility of BSA for rapid, robust genetic mapping of drug resistance loci. Use of cryopreserved progeny pools expands the utility of BSA because we can conduct experiments using archived progeny pools from previous genetic crosses. BSA provides a powerful approach that complements traditional QTL methods for investigating the genetic architecture of resistance to antimalarials, and to reveal new or accessory loci contributing to artemisinin resistance. FundingNational Institutes of Health (NIH); Wellcome trust. O_TEXTBOXResearch in contextO_ST_ABSEvidence before this studyC_ST_ABSGenetic crosses have been immensely successful for determining the genetic basis of drug resistance in malaria parasites, but require laborious cloning, characterization of drug resistance and genome-wide genotyping of individual progeny. This is a major limitation given that genetic crosses can now be conducted efficiently using humanized mice, rather than chimpanzees. Bulk segregant analysis (BSA) provides an attractive alternative approach because (i) large numbers of uncloned recombinant progeny can be analyzed, increasing statistical power (ii) phenotyping is not required, because we identify QTLs by treatment of progeny bulks, and identifying genome regions that show skews in allele frequency after treatment (iii) genome sequencing of bulk samples provides a rapid, accurate readout of genome-wide allele frequencies. This approach has been effectively leveraged in yeast, rodent malaria and several model organisms. Added value of this studyHere we validate and optimize this approach for P. falciparum genetic crosses, focusing on resistance to dihydroartemisin (DHA) a central component of the first-line antimalarial combination. Mutations in kelch13 are known to confer resistance to DHA, but several additional candidate loci have also been suggested to contribute. Our results confirm involvement of kelch13, but did not identify linkage with other putative candidate loci. We optimized methodology, showing that synchronization is critical, and that BSA can be successfully applied to cryopreserved progeny pools. Implications of all the available evidenceBSA combined with recent advances in rapidly generating genetic crosses provides a powerful approach to investigate the genetic basis of drug resistance in P. falciparum. C_TEXTBOX

genomics↗

bioRxiv2020

Efficient transcriptome profiling across the malaria parasite erythrocytic cycle by flow sorting

Plasmodium falciparum is the most virulent and widespread of the human malaria parasite species. This parasite has a complex life cycle that involves sexual replication in a mosquito vector and asexual replication in a human host. During the 48-hour intraerythrocytic developmental cycle (IDC), parasites develop and multiply through the morphologically distinct ring, trophozoite and schizont stages. Stage-specific transcriptomic approaches have shown gene expression profiles continually change throughout the IDC. Cultures of tightly synchronized parasites are required to capture the transcriptome specific to a developmental stage. However, the most commonly used synchronization methods require lysis of late stages, potentially perturbing transcription, and often do not result in tightly synchronized cultures. To produce complete transcriptome profiles of the IDC a synchronous culture requires frequent sampling over a 48-hour period, this is both time consuming and labor intensive. Here we develop a method to sample the IDC densely by isolating parasites from an asynchronous culture with fluorescence activated cell sorting (FACS). We sort parasites in tight windows of IDC progression based on their DNA/RNA abundance. We confirmed the tight synchronization and stage specificity by light microscopy and RNAseq profiling. We optimized our protocol for low numbers of sorted cells allowing us to rapidly capture transcriptome profiles across the entire IDC from a single culture flask. This methodology will allow malaria stage-specific studies to perform experiments directly from asynchronous cultures with high accuracy and without the need for labor-intensive time-course experiments.

genomics↗

bioRxiv2020

Rapid emergence of clonal interference during malaria parasite cultivation

Laboratory cultivation of the malaria parasite Plasmodium falciparum has underpinned nearly all advances in malariology in the past 30 years. When freshly isolated clinical isolates are adapted to in vitro culture mutations rapidly fix increasing the parasite growth rate and stability. While the dynamics of culture adaptation are increasingly well characterized, we know little about the extent of genomic variation that arises and spreads during long term culture. To address this we cloned the 3D7 reference strain and maintained a culture for ~84 asexual cycles (167 days). Growth rate of the culture population increased 1.14-fold over this timeframe. We used single cell genome sequencing of parasites at cycles 21 and 84 to measure the accumulation of diversity in vitro. This parasite population showed strong signals of adaptation across this time frame. By cycle 84 two dominant clades had arisen and were segregating with the dynamics of clonal interference. This highlights the continual process of adaptation in malaria parasites, even in parasites which have been extensively adapted to long term culture.

genomics↗

bioRxiv2019

Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, when parental alleles segregate evenly and, for hermaphroditic organisms, when numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. Here we examine the statistical power of two different genetic crosses - (1) between a laboratory parasite (NF54) of African origin and a patient-derived Asian parasite, and (2) between two sympatric patient-derived Asian parasites. We generated >140 unique recombinant clones over a 12-month period from the four parental genotypes, doubling the number of unique recombinant progeny generated in the previous 30 years. Both crosses show bi-parental inheritance of plastid markers amongst recombinant progeny, in contrast to previous crosses (conducted using chimpanzee hosts) which carried single dominant plastid genotypes. Both crosses show distinctive segregation patterns. The allopatric African/Asian cross has minimal levels of inbreeding (2% of clonal progeny are inbred) and extreme skews in marker segregation, while in the sympatric Asian cross, inbred progeny predominate (66% of clonal progeny are inbred) and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny arrays (particularly the sympatric Asian cross) have excellent power to map large-effect mutations to a 31 kb interval and can capture complex, epistatic interactions that were far beyond the capacity of previous malaria crosses to detect. The extreme segregation distortion in the allopatric African/Asian cross erodes power to detect linkage in several genome regions, but the repeatable distortions observed offer promising alternative approaches to identifying genes underlying traits of interest. These crosses show surprising variation in marker segregation, nevertheless, the increased progeny numbers improve our ability to rapidly map biomedically important parasite traits. Author SummaryUnderstanding how genome mutations contribute to newly emerging drug resistance in parasites like Plasmodium falciparum is important to monitor the spread of drug resistance. This scenario has been playing out in Southeast Asia with the emergence and spread of artemisinin resistance. Here we show that new P. falciparum genetic crosses, using mice carrying human liver cells and infused with human red blood cells (the human liver-chimeric FRG NOD huHep/huRBC mouse), provide an important new tool for understanding complex interactions underlying drug resistance phenotypes. We report two new genetic maps with 84 and 60 unique recombinant progeny, which doubles the number of progeny available from 4 previous P. falciparum genetic crosses. Through extensive simulations we show that with 84 progeny we can find association for a gene that controls only 20% of the variation in a phenotype. We also show that a cross generated from Southeast Asian parasites collected from the same geographic region have unique characteristics not previously observed in P. falciparum genetic crosses. This Southeast Asian cross exhibits even segregation across the genome, unbiased inheritance of mitochondria and apicoplast and higher levels of inbreeding than previously observed.

genetics↗

bioRxiv2019

Genetic mapping of fitness determinants across the malaria parasite Plasmodium falciparum life cycle

Malaria is transmitted through female Anopheline mosquitoes where gamete fusion and meiosis occurs, and humans where parasites proliferate asexually. We describe a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages in human malaria parasite Plasmodium falciparum using bulk segregant analysis (BSA). We combined experimental genetic crosses using humanized mice, with selective whole genome amplification and BSA at multiple developmental stages in both mosquito and vertebrate host to examine parasite competition and identify genomic regions under selection. We generated crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasite clones recently isolated from Southeast Asian patients. We then quantified genome-wide changes in allele frequency in the parasite progeny population from infected midgut and salivary glands of Anopheles stephensi mosquitoes, infected livers, emerging merozoites and aliquots of in vitro cultured progeny parasites at intervals over 30 days. Three striking results emerge: we observed (i) a strong skew (>80%) towards alleles from the ART-R parent in the mosquito stage, that dropped to [~]50% in the blood stage as selfed ART-R parasites were selected against; (ii) highly repeatable skews in allele frequencies across the genome in blood stage parasites; (iii) particularly strong selection (selection coefficient (s) [≤] 0.18/asexual cycle) against alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 containing ARPS10. This approach robustly identifies selected loci and has strong potential for identifying parasite genes that interact with the mosquito vector or compensatory loci involved in drug resistance.

evolutionary biology↗

bioRxiv2018

Resolving within-host malaria parasite diversity using single-cell sequencing

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum, but the composition of these infections cannot be directly inferred from bulk sequence data. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Estimation of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of new infections in malaria-infected patients and allows high resolution dissection of intra-host variation in malaria parasites.

genetics↗

bioRxiv2017

High-resolution single-cell sequencing of malaria parasites

Single-cell genomics is a powerful tool for determining the genetic structure of complex communities of unicellular organisms. Patients infected with the malaria-causing parasite, Plasmodium falciparum, often carry multiple, genetically distinct parasites. Little is known about the diversity and relatedness of these lineages. We have developed an improved single-cell genomics protocol to reconstruct individual haplotypes from infections, a necessary step in uncovering parasite ecology within the host. This approach captures singly-infected red blood cells (iRBCs) by fluorescence-activated cell sorting (FACS) prior to whole genome amplification (WGA) and whole genome sequencing (WGS). Here, we demonstrate that parasites in late cell cycle stages, which contain increased DNA content, are far superior templates for generating high quality genomic data. Targeting of these cells routinely generates near-complete capture of the 23Mb P. falciparum genome (mean breadth of coverage 90.7%) at high efficiency. We used this approach to analyze the genomes of 48 individual cells from a polyclonal malaria infection sampled in Chikhwawa, Malawi. This comprehensive dataset enabled high-resolution estimation of the clonality and the relatedness of parasite haplotypes within the infection, long-standing problems in malaria biology.

genomics↗

bioRxiv2016

Longitudinal genomic surveillance of Plasmodium falciparum malaria parasites reveals complex genomic architecture of emerging artemisinin resistance in western Thailand

BackgroundArtemisinin-based combination therapies are the first line of treatment for Plasmodium falciparum infections worldwide, but artemisinin resistance (ART-R) has risen rapidly in in Southeast Asia over the last decade. Mutations in kelch13 have been associated with artemisinin (ART) resistance in this region. To explore the power of longitudinal genomic surveillance to detect signals in kelch13 and other loci that contribute to ART or partner drug resistance, we retrospectively sequenced the genomes of 194 P. falciparum isolates from five sites in Northwest Thailand, bracketing the era in which there was a rapid increase in ART-R in this region (2001-2014).\n\nResultsWe evaluated statistical metrics for temporal change in the frequency of individual SNPs, assuming that SNPs associated with resistance should increase frequency over this period. After Kelch13-C580Y, the strongest temporal change was seen at a SNP in phosphatidylinositol 4-kinase (PI4K), situated in a pathway recently implicated in the ART-R mechanism. However, other loci exhibit temporal signatures nearly as strong, and warrant further investigation for involvement in ART-R evolution. Through genome-wide association analysis we also identified a variant in a kelch-domain-containing gene on chromosome 10 that may epistatically modulate ART-R.\n\nConclusionsThis analysis demonstrates the potential of a longitudinal genomic surveillance approach to detect resistance-associated loci and improve our mechanistic understanding of how resistance develops. Evidence for additional genomic regions outside of the kelch13 locus associated with ART-R parasites may yield new molecular markers for resistance surveillance and may retard the emergence or spread of ART-R in African parasite populations.

genomics↗