Hyper-diverse antigenic variation and resilience to transmission-reducing intervention in falciparum malaria.

Intervention against falciparum malaria in high transmission regions remains challenging, with relaxation of control efforts typically followed by rapid resurgence. Resilience to intervention co-occurs with incomplete immunity, whereby children eventually become protected from severe disease but not infection and a large transmission reservoir results from high asymptomatic prevalence across all ages. Incomplete immunity relates to the vast antigenic variation of the parasite, with the major surface antigen of the blood stage of infection encoded by the multigene family known as var. Recent deep sampling of var sequences from individual isolates in northern Ghana showed that parasite population structure exhibited persistent features of high-transmission regions despite the considerable decrease in prevalence during transient intervention with indoor residual spraying (IRS). We ask whether despite such apparent limited impact, the transmission system had been brought close to a transition in both prevalence and resurgence ability. With a stochastic agent-based model, we investigate the existence of such a transition to pre-elimination with intervention intensity, and of molecular indicators informative of its approach. We show that resurgence ability decreases sharply and nonlinearly across a narrow region of intervention intensities in model simulations, and identify informative molecular indicators based on var gene sequences. Their application to the survey data indicates that the transmission system in northern Ghana was brought close to transition by IRS. These results suggest that sustaining and intensifying intervention would have pushed malaria dynamics to a slow-rebound regime with an increased probability of local parasite extinction.

Comparison of molecular surveillance methods to assess changes in the population genetics of Plasmodium falciparum in high transmission.

A major motivation for developing molecular methods for malaria surveillance is to measure the impact of control interventions on the population genetics of Plasmodium falciparum as a potential marker of progress towards elimination. Here we assess three established methods (i) single nucleotide polymorphism (SNP) barcoding (panel of 24-biallelic loci), (ii) microsatellite genotyping (panel of 12-multiallelic loci), and (iii) varcoding (fingerprinting var gene diversity, akin to microhaplotyping) to identify changes in parasite population genetics in response to a short-term indoor residual spraying (IRS) intervention. Typical of high seasonal transmission in Africa, multiclonal infections were found in 82.3% (median 3; range 1-18) and 57.8% (median 2; range 1-12) of asymptomatic individuals pre- and post-IRS, respectively, in Bongo District, Ghana. Since directly phasing multilocus haplotypes for population genetic analysis is not possible for biallelic SNPs and microsatellites, we chose ~200 low-complexity infections biased to single and double clone infections for analysis. Each genotyping method presented a different pattern of change in diversity and population structure as a consequence of variability in usable data and the relative polymorphism of the molecular markers (i.e., SNPs < microsatellites < var). Varcoding and microsatellite genotyping showed the overall failure of the IRS intervention to significantly change the population structure from pre-IRS characteristics (i.e., many diverse genomes of low genetic similarity). The 24-SNP barcode provided limited information for analysis, largely due to the biallelic nature of SNPs leading to a high proportion of double-allele calls and a view of more isolate relatedness compared to microsatellites and varcoding. Relative performance, suitability, and cost-effectiveness of the methods relevant to sample size and local malaria elimination in high-transmission endemic areas are discussed.

A paradoxical population structure of var DBLα types in Africa.

The var multigene family encodes the P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is important in host-parasite interaction as a virulence factor and major surface antigen of the blood stages of the parasite, responsible for maintaining chronic infection. Whilst important in the biology of P. falciparum, these genes (50 to 60 genes per parasite genome) are routinely excluded from whole genome analyses due to their hyper-diversity, achieved primarily through recombination. The PfEMP1 head structure almost always consists of a DBLα-CIDR tandem. Categorised into different groups (upsA, upsB, upsC), different head structures have been associated with different ligand-binding affinities and disease severities. We study how conserved individual DBLα types are at the country, regional, and local scales in Sub-Saharan Africa. Using publicly-available sequence datasets and a novel ups classification algorithm, cUps, we performed an in silico exploration of DBLα conservation through time and space in Africa. In all three ups groups, the population structure of DBLα types in Africa consists of variants occurring at rare, low, moderate, and high frequencies. Non-rare variants were found to be temporally stable in a local area in endemic Ghana. When inspected across different geographical scales, we report different levels of conservation; while some DBLα types were consistently found in high frequencies in multiple African countries, others were conserved only locally, signifying local preservation of specific types. Underlying this population pattern is the composition of DBLα types within each isolate DBLα repertoire, revealed to also consist of a mix of types found at rare, low, moderate, and high frequencies in the population. We further discuss the adaptive forces and balancing selection, including host genetic factors, potentially shaping the evolution and diversity of DBLα types in Africa.

The P. falciparum alternative histones Pf H2A.Z and Pf H2B.Z are dynamically acetylated and antagonized by PfSir2 histone deacetylases at heterochromatin boundaries.

The Plasmodium falciparum alternative histones Pf H2A.Z and Pf H2B.Z are enriched in the same nucleosomes in intergenic euchromatin but depleted from heterochromatin. They occupy most promoters but are only dynamically associated with expression at var genes. In other organisms, acetylation of H2A.Z is important for its functions in gene expression and chromatin structure. Here, we show that acetylated Pf H2A.Z and Pf H2B.Z are dynamically associated with gene expression at promoters. In addition, acetylated Pf H2A.Z and Pf H2B.Z are antagonized by the sirtuin class III histone deacetylases (HDAC) PfSir2A and B at heterochromatin boundaries and encroach upon heterochromatin in parasites lacking PfSir2A or B. However, the majority of acetylated Pf H2A.Z and Pf H2B.Z are deacetylated by class I or II HDACs. Acetylated Pf H2A.Z and Pf H2B.Z are also dynamically associated with promoter activity of both canonical upstream var gene promoters and var gene introns. These findings suggest that both acetylated Pf H2A.Z and Pf H2B.Z play critical roles in gene expression and contribute to maintenance of chromatin structure at the boundaries of subtelomeric, facultative heterochromatin, critical for the variegated expression of genes that enable rapid adaptation to altered host environments.IMPORTANCEThe malaria parasite Plasmodium falciparum relies on variant expression of members of multi-gene families as a strategy for environmental adaptation to promote parasite survival and pathogenesis. These genes are located in transcriptionally silenced DNA regions. A limited number of these genes escape gene silencing, and switching between them confers variant fitness on parasite progeny. Here, we show that PfSir2 histone deacetylases antagonize DNA-interacting acetylated alternative histones at the boundaries between active and silent DNA. This finding implicates acetylated alternative histones in the mechanism regulating P. falciparum variant gene silencing and thus malaria pathogenesis. This work also revealed that acetylation of alternative histones at promoters is dynamically associated with promoter activity across the genome, implicating acetylation of alternative histones in gene regulation genome wide. Understanding mechanisms of gene regulation in P. falciparum may aid in the development of new therapeutic strategies for malaria, which killed 619,000 people in 2021.

Measuring changes in Plasmodium falciparum census population size in response to sequential malaria control interventions.

Here we introduce a new endpoint "census population size" to evaluate the epidemiology and control of Plasmodium falciparum infections, where the parasite, rather than the infected human host, is the unit of measurement. To calculate census population size, we rely on a definition of parasite variation known as multiplicity of infection (MOIvar), based on the hyper-diversity of the var multigene family. We present a Bayesian approach to estimate MOIvar from sequencing and counting the number of unique DBLα tags (or DBLα types) of var genes, and derive from it census population size by summation of MOIvar in the human population. We track changes in this parasite population size and structure through sequential malaria interventions by indoor residual spraying (IRS) and seasonal malaria chemoprevention (SMC) from 2012 to 2017 in an area of high-seasonal malaria transmission in northern Ghana. Following IRS, which reduced transmission intensity by > 90% and decreased parasite prevalence by ~40-50%, significant reductions in var diversity, MOIvar, and population size were observed in ~2,000 humans across all ages. These changes, consistent with the loss of diverse parasite genomes, were short lived and 32-months after IRS was discontinued and SMC was introduced, var diversity and population size rebounded in all age groups except for the younger children (1-5 years) targeted by SMC. Despite major perturbations from IRS and SMC interventions, the parasite population remained very large and retained the var population genetic characteristics of a high-transmission system (high var diversity; low var repertoire similarity) demonstrating the resilience of P. falciparum to short-term interventions in high-burden countries of sub-Saharan Africa.

Performance of SNP barcodes to determine genetic diversity and population structure of Plasmodium falciparum in Africa.

Panels of informative biallelic single nucleotide polymorphisms (SNPs) have been proposed to be an economical method to fast-track the population genetic analysis of Plasmodium falciparum in malaria-endemic areas. Whilst used successfully in low-transmission areas where infections are monoclonal and highly related, we present the first study to evaluate the performance of these 24- and 96-SNP molecular barcodes in African countries, characterised by moderate-to-high transmission, where multiclonal infections are prevalent. For SNP barcodes it is generally recommended that the SNPs chosen i) are biallelic, ii) have a minor allele frequency greater than 0.10, and iii) are independently segregating, to minimise bias in the analysis of genetic diversity and population structure. Further, to be standardised and used in many population genetic studies, these barcodes should maintain characteristics i) to iii) across various iv) geographies and v) time points. Using haplotypes generated from the MalariaGEN P. falciparum Community Project version six database, we investigated the ability of these two barcodes to fulfil these criteria in moderate-to-high transmission African populations in 25 sites across 10 countries. Predominantly clinical infections were analysed, with 52.3% found to be multiclonal, generating high proportions of mixed-allele calls (MACs) per isolate thereby impeding haplotype construction. Of the 24- and 96-SNPs, loci were removed if they were not biallelic and had low minor allele frequencies in all study populations, resulting in 20- and 75-SNP barcodes respectively for downstream population genetics analysis. Both SNP barcodes had low expected heterozygosity estimates in these African settings and consequently biased analyses of similarity. Both minor and major allele frequencies were temporally unstable. These SNP barcodes were also shown to identify weak genetic differentiation across large geographic distances based on Mantel Test and DAPC. These results demonstrate that these SNP barcodes are vulnerable to ascertainment bias and as such cannot be used as a standardised approach for malaria surveillance in moderate-to-high transmission areas in Africa, where the greatest genomic diversity of P. falciparum exists at local, regional and country levels.

Neutral vs. non-neutral genetic footprints of Plasmodium falciparum multiclonal infections.

At a time when effective tools for monitoring malaria control and eradication efforts are crucial, the increasing availability of molecular data motivates their application to epidemiology. The multiplicity of infection (MOI), defined as the number of genetically distinct parasite strains co-infecting a host, is one key epidemiological parameter for evaluating malaria interventions. Estimating MOI remains a challenge for high-transmission settings where individuals typically carry multiple co-occurring infections. Several quantitative approaches have been developed to estimate MOI, including two cost-effective ones relying on molecular data: i) THE REAL McCOIL method is based on putatively neutral single nucleotide polymorphism loci, and ii) the varcoding method is a fingerprinting approach that relies on the diversity and limited repertoire overlap of the var multigene family encoding the major Plasmodium falciparum blood-stage antigen PfEMP1 and is therefore under selection. In this study, we assess the robustness of the MOI estimates generated with these two approaches by simulating P. falciparum malaria dynamics under three transmission conditions using an extension of a previously developed stochastic agent-based model. We demonstrate that these approaches are complementary and best considered across distinct transmission intensities. While varcoding can underestimate MOI, it allows robust estimation, especially under high transmission where repertoire overlap is extremely limited from frequency-dependent selection. In contrast, THE REAL McCOIL often considerably overestimates MOI, but still provides reasonable estimates for low and moderate transmission. Regardless of transmission intensity, results for THE REAL McCOIL indicate that an inaccurate tail at high MOI values is generated, and that at high transmission, an apparently reasonable estimated MOI distribution can arise from some degree of compensation between overestimation and underestimation. As many countries pursue malaria elimination targets, defining the most suitable approach to estimate MOI based on sample size and local transmission intensity is highly recommended for monitoring the impact of intervention programs.

Mathematical assessment of the role of intervention programs for malaria control.

Malaria remains a global health problem despite the many attempts to control and eradicate it. There is an urgent need to understand the current transmission dynamics of malaria and to determine the interventions necessary to control malaria. In this paper, we seek to develop a fit-for-purpose mathematical model to assess the interventions needed to control malaria in an endemic setting. To achieve this, we formulate a malaria transmission model to analyse the spread of malaria in the presence of interventions. A sensitivity analysis of the model is performed to determine the relative impact of the model parameters on disease transmission. We explore how existing variations in the recruitment and management of intervention strategies affect malaria transmission. Results obtained from the study imply that the discontinuation of existing interventions has a significant effect on malaria prevalence. Thus, the maintenance of interventions is imperative for malaria elimination and eradication. In a scenario study aimed at assessing the impact of long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and localized individual measures, our findings indicate that increased LLINs utilization and extended IRS coverage (with longer-lasting insecticides) cause a more pronounced reduction in symptomatic malaria prevalence compared to a reduced LLINs utilization and shorter IRS coverage. Additionally, our study demonstrates the impact of localized preventive measures in mitigating the spread of malaria when compared to the absence of interventions.

Indoor residual spraying with a non-pyrethroid insecticide reduces the reservoir of Plasmodium falciparum in a high-transmission area in northern Ghana.

High-malaria burden countries in sub-Saharan Africa are shifting from malaria control towards elimination. Hence, there is need to gain a contemporary understanding of how indoor residual spraying (IRS) with non-pyrethroid insecticides when combined with long-lasting insecticidal nets (LLINs) impregnated with pyrethroid insecticides, contribute to the efforts of National Malaria Control Programmes to interrupt transmission and reduce the reservoir of Plasmodium falciparum infections across all ages. Using an interrupted time-series study design, four age-stratified malariometric surveys, each of ~2,000 participants, were undertaken pre- and post-IRS in Bongo District, Ghana. Following the application of three-rounds of IRS, P. falciparum transmission intensity declined, as measured by a >90% reduction in the monthly entomological inoculation rate. This decline was accompanied by reductions in parasitological parameters, with participants of all ages being significantly less likely to harbor P. falciparum infections at the end of the wet season post-IRS (aOR = 0.22 [95% CI: 0.19-0.26], p-value < 0.001). In addition, multiplicity of infection (MOI var ) was measured using a parasite fingerprinting tool, designed to capture within-host genome diversity. At the end of the wet season post-IRS, the prevalence of multi-genome infections declined from 75.6% to 54.1%. This study demonstrates that in areas characterized by high seasonal malaria transmission, IRS in combination with LLINs can significantly reduce the reservoir of P. falciparum infection. Nonetheless despite this success, 41.6% of the population, especially older children and adolescents, still harboured multi-genome infections. Given the persistence of this diverse reservoir across all ages, these data highlight the importance of sustaining vector control in combination with targeted chemotherapy to move high-transmission settings towards pre-elimination. This study also points to the benefits of molecular surveillance to ensure that incremental achievements are not lost and that the goals advocated for in the WHO's High Burden to High Impact strategy are realized.

Age-specific patterns of DBLα var diversity can explain why residents of high malaria transmission areas remain susceptible to Plasmodium falciparum blood stage infection throughout life.

Immunity to Plasmodium falciparum is non-sterilising, thus individuals residing in malaria-endemic areas are at risk of infection throughout their lifetime. Here we seek to find a genomic epidemiological explanation for why residents of all ages harbour blood stage infections despite lifelong exposure to P. falciparum in areas of high transmission. We do this by exploring, for the first known time, the age-specific patterns of diversity of variant antigen encoding (var) genes in the reservoir of infection. Microscopic and submicroscopic P. falciparum infections were analysed at the end of the wet and dry seasons in 2012-2013 for a cohort of 1541 residents aged from 1 to 91 years in an area characterised by high seasonal malaria transmission in Ghana. By sequencing the near ubiquitous Duffy-binding-like alpha domain (DBLα) that encodes immunogenic domains, we defined var gene diversity in an estimated 1096 genomes detected in sequential wet and dry season sampling of this cohort. Unprecedented var (DBLα) diversity was observed in all ages with 42,399 unique var types detected. There was a high degree of maintenance of types between seasons (>40% seen more than once), with many of the same types, especially upsA, appearing multiple times in isolates from different individuals. Children and adolescents were found to be significant reservoirs of var DBLα diversity compared with adults. Var repertoires within individuals were highly variable, with children having more related var repertoires compared to adolescents and adults. Individuals of all ages harboured multiple genomes with var repertoires unrelated to those infecting other hosts. High turnover of parasites with diverse isolate var repertoires was also observed in all ages. These age-specific patterns are best explained by variant-specific immune selection. The observed level of var diversity for the population was then used to simulate the development of variant-specific immunity to the diverse var types under conservative assumptions. Simulations showed that the extent of observed var diversity with limited repertoire relatedness was sufficient to explain why adolescents and adults in this community remain susceptible to blood stage infection, even with multiple genomes.

An accurate method for identifying recent recombinants from unaligned sequences.

MOTIVATION: Recombination is a fundamental process in molecular evolution, and the identification of recombinant sequences is thus of major interest. However, current methods for detecting recombinants are primarily designed for aligned sequences. Thus, they struggle with analyses of highly diverse genes, such as the var genes of the malaria parasite Plasmodium falciparum, which are known to diversify primarily through recombination. RESULTS: We introduce an algorithm to detect recent recombinant sequences from a dataset without a full multiple alignment. Our algorithm can handle thousands of gene-length sequences without the need for a reference panel. We demonstrate the accuracy of our algorithm through extensive numerical simulations; in particular, it maintains its effectiveness in the presence of insertions and deletions. We apply our algorithm to a dataset of 17 335 DBLα types in var genes from Ghana, observing that sequences belonging to the same ups group or domain subclass recombine amongst themselves more frequently, and that non-recombinant DBLα types are more conserved than recombinant ones. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at https://github.com/qianfeng2/detREC_program. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Unravelling var complexity: Relationship between DBLα types and var genes in Plasmodium falciparum.

The enormous diversity and complexity of var genes that diversify rapidly by recombination has led to the exclusion of assembly of these genes from major genome initiatives (e.g., Pf6). A scalable solution in epidemiological surveillance of var genes is to use a small 'tag' region encoding the immunogenic DBLα domain as a marker to estimate var diversity. As var genes diversify by recombination, it is not clear the extent to which the same tag can appear in multiple var genes. This relationship between marker and gene has not been investigated in natural populations. Analyses of in vitro recombination within and between var genes have suggested that this relationship would not be exclusive. Using a dataset of publicly-available assembled var sequences, we test this hypothesis by studying DBLα-var relationships for four study sites in four countries: Pursat (Cambodia) and Mae Sot (Thailand), representing low malaria transmission, and Navrongo (Ghana) and Chikwawa (Malawi), representing high malaria transmission. In all study sites, DBLα-var relationships were shown to be predominantly 1-to-1, followed by a second largest proportion of 1-to-2 DBLα-var relationships. This finding indicates that DBLα tags can be used to estimate not just DBLα diversity but var gene diversity when applied in a local endemic area. Epidemiological applications of this result are discussed.

The impact of indoor residual spraying on Plasmodium falciparum microsatellite variation in an area of high seasonal malaria transmission in Ghana, West Africa.

Here, we report the first population genetic study to examine the impact of indoor residual spraying (IRS) on Plasmodium falciparum in humans. This study was conducted in an area of high seasonal malaria transmission in Bongo District, Ghana. IRS was implemented during the dry season (November-May) in three consecutive years between 2013 and 2015 to reduce transmission and attempt to bottleneck the parasite population in humans towards lower diversity with greater linkage disequilibrium. The study was done against a background of widespread use of long-lasting insecticidal nets, typical for contemporary malaria control in West Africa. Microsatellite genotyping with 10 loci was used to construct 392 P. falciparum multilocus infection haplotypes collected from two age-stratified cross-sectional surveys at the end of the wet seasons pre- and post-IRS. Three-rounds of IRS, under operational conditions, led to a >90% reduction in transmission intensity and a 35.7% reduction in the P. falciparum prevalence (p < .001). Despite these declines, population genetic analysis of the infection haplotypes revealed no dramatic changes with only a slight, but significant increase in genetic diversity (He : pre-IRS = 0.79 vs. post-IRS = 0.81, p = .048). Reduced relatedness of the parasite population (p < .001) was observed post-IRS, probably due to decreased opportunities for outcrossing. Spatiotemporal genetic differentiation between the pre- and post-IRS surveys (D = 0.0329 [95% CI: 0.0209 - 0.0473], p = .034) was identified. These data provide a genetic explanation for the resilience of P. falciparum to short-term IRS programmes in high-transmission settings in sub-Saharan Africa.

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents.

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.

Frequency-Dependent Competition Between Strains Imparts Persistence to Perturbations in a Model of Plasmodium falciparum Malaria Transmission.

In high-transmission endemic regions, local populations of Plasmodium falciparum exhibit vast diversity of the var genes encoding its major surface antigen, with each parasite comprising multiple copies from this diverse gene pool. This strategy to evade the immune system through large combinatorial antigenic diversity is common to other hyperdiverse pathogens. It underlies a series of fundamental epidemiological characteristics, including large reservoirs of transmission from high prevalence of asymptomatics and long-lasting infections. Previous theory has shown that negative frequency-dependent selection (NFDS) mediated by the acquisition of specific immunity by hosts structures the diversity of var gene repertoires, or strains, in a pattern of limiting similarity that is both non-random and non-neutral. A combination of stochastic agent-based models and network analyses has enabled the development and testing of theory in these complex adaptive systems, where assembly of local parasite diversity occurs under frequency-dependent selection and large pools of variation. We show here the application of these approaches to theory comparing the response of the malaria transmission system to intervention when strain diversity is assembled under (competition-based) selection vs. a form of neutrality, where immunity depends only on the number but not the genetic identity of previous infections. The transmission system is considerably more persistent under NFDS, exhibiting a lower extinction probability despite comparable prevalence during intervention. We explain this pattern on the basis of the structure of strain diversity, in particular the more pronounced fraction of highly dissimilar parasites. For simulations that survive intervention, prevalence under specific immunity is lower than under neutrality, because the recovery of diversity is considerably slower than that of prevalence and decreased var gene diversity reduces parasite transmission. A Principal Component Analysis of network features describing parasite similarity reveals that despite lower overall diversity, NFDS is quickly restored after intervention constraining strain structure and maintaining patterns of limiting similarity important to parasite persistence. Given the described enhanced persistence under perturbation, intervention efforts will likely require longer times than the usual practice to eliminate P. falciparum populations. We discuss implications of our findings and potential analogies for ecological communities with non-neutral assembly processes involving frequency-dependence.

Histone modifications associated with gene expression and genome accessibility are dynamically enriched at Plasmodium falciparum regulatory sequences.

BACKGROUND: The malaria parasite Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear. RESULTS: Here, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity. CONCLUSIONS: The dynamic H3 acetylations define P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.

Evolution of Antimalarial Drug Resistance Markers in the Reservoir of Plasmodium falciparum Infections in the Upper East Region of Ghana.

BACKGROUND: The majority of Plasmodium falciparum infections, constituting the reservoir in all ages, are asymptomatic in high-transmission settings in Africa. The role of this reservoir in the evolution and spread of drug resistance was explored. METHODS: Population genetic analyses of the key drug resistance-mediating polymorphisms were analyzed in a cross-sectional survey of asymptomatic P. falciparum infections across all ages in Bongo District, Ghana. RESULTS: Seven years after the policy change to artemisinin-based combination therapies in 2005, the pfcrt K76 and pfmdr1 N86 wild-type alleles have nearly reached fixation and have expanded via soft selective sweeps on multiple genetic backgrounds. By constructing the pfcrt-pfmdr1-pfdhfr-pfdhps multilocus haplotypes, we found that the alleles at these loci were in linkage equilibrium and that multidrug-resistant parasites have not expanded in this reservoir. For pfk13, 32 nonsynonymous mutations were identified; however, none were associated with artemisinin-based combination therapy resistance. CONCLUSIONS: The prevalence and selection of alleles/haplotypes by antimalarials were similar to that observed among clinical cases in Ghana, indicating that they do not represent 2 subpopulations with respect to these markers. Thus, the P. falciparum reservoir in all ages can contribute to the maintenance and spread of antimalarial resistance.

Competition for hosts modulates vast antigenic diversity to generate persistent strain structure in Plasmodium falciparum.

In their competition for hosts, parasites with antigens that are novel to the host immune system will be at a competitive advantage. The resulting frequency-dependent selection can structure parasite populations into strains of limited genetic overlap. For the causative agent of malaria, Plasmodium falciparum, the high recombination rates and associated vast diversity of its highly antigenic and multicopy var genes preclude such clear clustering in endemic regions. This undermines the definition of strains as specific, temporally persisting gene variant combinations. We use temporal multilayer networks to analyze the genetic similarity of parasites in both simulated data and in an extensively and longitudinally sampled population in Ghana. When viewed over time, populations are structured into modules (i.e., groups) of parasite genomes whose var gene combinations are more similar within than between the modules and whose persistence is much longer than that of the individual genomes that compose them. Comparison to neutral models that retain parasite population dynamics but lack competition reveals that the selection imposed by host immunity promotes the persistence of these modules. The modular structure is, in turn, associated with a slower acquisition of immunity by individual hosts. Modules thus represent dynamically generated niches in host immune space, which can be interpreted as strains. Negative frequency-dependent selection therefore shapes the organization of the var diversity into parasite genomes, leaving a persistence signature over ecological time scales. Multilayer networks extend the scope of phylodynamics analyses by allowing quantification of temporal genetic structure in organisms that generate variation via recombination or other non-bifurcating processes. A strain structure similar to the one described here should apply to other pathogens with large antigenic spaces that evolve via recombination. For malaria, the temporal modular structure should enable the formulation of tractable epidemiological models that account for parasite antigenic diversity and its influence on intervention outcomes.

Identifying functional groups among the diverse, recombining antigenic var genes of the malaria parasite Plasmodium falciparum from a local community in Ghana.

A challenge in studying diverse multi-copy gene families is deciphering distinct functional types within immense sequence variation. Functional changes can in some cases be tracked through the evolutionary history of a gene family; however phylogenetic approaches are not possible in cases where gene families diversify primarily by recombination. We take a network theoretical approach to functionally classify the highly recombining var antigenic gene family of the malaria parasite Plasmodium falciparum. We sample var DBLα sequence types from a local population in Ghana, and classify 9,276 of these variants into just 48 functional types. Our approach is to first decompose each sequence type into its constituent, recombining parts; we then use a stochastic block model to identify functional groups among the parts; finally, we classify the sequence types based on which functional groups they contain. This method for functional classification does not rely on an inferred phylogenetic history, nor does it rely on inferring function based on conserved sequence features. Instead, it infers functional similarity among recombining parts based on the sharing of similar co-occurrence interactions with other parts. This method can therefore group sequences that have undetectable sequence homology or even distinct origination. Describing these 48 var functional types allows us to simplify the antigenic diversity within our dataset by over two orders of magnitude. We consider how the var functional types are distributed in isolates, and find a nonrandom pattern reflecting that common var functional types are non-randomly distinct from one another in terms of their functional composition. The coarse-graining of var gene diversity into biologically meaningful functional groups has important implications for understanding the disease ecology and evolution of this system, as well as for designing effective epidemiological monitoring and intervention.