Kathryn E. Tiedje

Epistatic Interactions between Apolipoprotein E and Hemoglobin S Genes in Regulation of Malaria Parasitemia

Apolipoprotein E is a monomeric protein secreted by the liver and responsible for the transport of plasma cholesterol and triglycerides. The APOE gene encodes 3 isoforms Ɛ4, Ɛ3 and Ɛ2 with APOE Ɛ4 associated with higher plasma cholesterol levels and increased pathogenesis in several infectious diseases (HIV, HSV). Given that cholesterol is an important nutrient for malaria parasites, we examined whether APOE Ɛ4 was a risk factor for Plasmodium infection, in terms of prevalence or parasite density. A cross sectional survey was performed in 508 children aged 1 to 12 years in Gabon during the wet season. Children were screened for Plasmodium spp. infection, APOE and hemoglobin S (HbS) polymorphisms. Median parasite densities were significantly higher in APOE Ɛ4 children for Plasmodium spp. densities compared to non-APOE Ɛ4 children. When stratified for HbS polymorphisms, median Plasmodium spp. densities were significantly higher in HbAA children if they had an APOE Ɛ4 allele compared to those without an APOE Ɛ4 allele. When considering non-APOE Ɛ4 children, there was no quantitative reduction of Plasmodium spp. parasite densities for HbAS compared to HbAA phenotypes. No influence of APOE Ɛ4 on successful Plasmodium liver cell invasion was detected by multiplicity of infection. These results show that the APOE Ɛ4 allele is associated with higher median malaria parasite densities in children likely due to the importance of cholesterol availability to parasite growth and replication. Results suggest an epistatic interaction between APOE and HbS genes such that sickle cell trait only had an effect on parasite density in APOE Ɛ4 children. This suggests a linked pathway of regulation of parasite density involving expression of these genes. These findings have significance for understanding host determinants of regulation of malaria parasite density, the design of clinical trials as well as studies of co-infection with Plasmodium and other pathogens.

Evidence of strain structure in Plasmodium falciparum var gene repertoires in children from Gabon, West Africa

Significance This paper aims to discover how diverse malaria parasites are in children from an African village. DNA sequencing shows that they are highly diverse with respect to the genes encoding the surface coat. Indeed, every child has a malaria infection with a different set of these genes. Importantly, this paper shows by computational methods that the pattern of this diversity is not random but structured to enhance the parasites’ chance to evade host immunity and has implications for the success of malaria control programs. Existing theory on competition for hosts between pathogen strains has proposed that immune selection can lead to the maintenance of strain structure consisting of discrete, weakly overlapping antigenic repertoires. This prediction of strain theory has conceptual overlap with fundamental ideas in ecology on niche partitioning and limiting similarity between coexisting species in an ecosystem, which oppose the hypothesis of neutral coexistence. For Plasmodium falciparum, strain theory has been specifically proposed in relation to the major surface antigen of the blood stage, known as PfEMP1 and encoded by the multicopy multigene family known as the var genes. Deep sampling of the DBLα domain of var genes in the local population of Bakoumba, West Africa, was completed to define whether patterns of repertoire overlap support a role of immune selection under the opposing force of high outcrossing, a characteristic of areas of intense malaria transmission. Using a 454 high-throughput sequencing protocol, we report extremely high diversity of the DBLα domain and a large parasite population with DBLα repertoires structured into nonrandom patterns of overlap. Such population structure, significant for the high diversity of var genes that compose it at a local level, supports the existence of “strains” characterized by distinct var gene repertoires. Nonneutral, frequency-dependent competition would be at play and could underlie these patterns. With a computational experiment that simulates an intervention similar to mass drug administration, we argue that the observed repertoire structure matters for the antigenic var diversity of the parasite population remaining after intervention.

Seasonal Variation in the Epidemiology of Asymptomatic Plasmodium falciparum Infections across Two Catchment Areas in Bongo District, Ghana

Understanding the epidemiology of asymptomatic Plasmodium falciparum infections is critical for countries to move toward malaria elimination. Using different methods for parasite detection, we evaluated how seasonality, spatial location, and other factors affect the age-specific epidemiology of asymptomatic malaria in Bongo District, Ghana. Asymptomatic prevalence by microscopy decreased significantly from 42.5% at the end of the wet to 27.5% at the end of the dry season (P < 0.001). Using the 18S rRNA polymerase chain reactions (PCRs), all microscopy-negative samples were screened and prevalence of submicroscopic infections also decreased significantly from the wet (55.4%) to the dry (20.7%) season (P < 0.001). Combining detection methods, 74.4% and 42.5% of the population in the wet and dry seasons, respectively, had evidence of a P. falciparum infection. Interestingly in those > 20 years of age, we found evidence of infection in 64.3% of the population in the wet and 27.0% in the dry season. Using both microscopy and PCR, the asymptomatic P. falciparum reservoir peaks at the end of the wet season and infections in all age groups constitute the reservoir of malaria infection. At the end of the wet season, spatial heterogeneity in the prevalence and density of P. falciparum infections was observed between the two catchment areas surveyed in Bongo District. These results indicate that if elimination is to succeed, interventions will need to target not just P. falciparum infections in children but also in adults, and be implemented toward the end of the dry season in this area of West Africa.

Lack of Geospatial Population Structure Yet Significant Linkage Disequilibrium in the Reservoir of Plasmodium falciparum in Bongo District, Ghana

Malaria control in West Africa is impeded by the large reservoir of chronic asymptomatic Plasmodium falciparum infections in the human population. This study aimed to assess the extent of diversity in the P. falciparum reservoir in Bongo District (BD), Ghana, at the end of the dry season, the lowest point in malaria transmission over the course of the year. Analysis of the variation in 12 microsatellite loci was completed for 200 P. falciparum isolates collected from a cross-sectional survey of residents of all ages from two catchment areas in BD. Analysis of the multilocus haplotypes showed high levels of genetic diversity (He = 0.74), no population differentiation yet significant linkage disequilibrium (LD) (ISA = 0.0127, P = 0.006) in BD. Multilocus LD was significant between and within catchment areas even though every haplotype in the population was unique and the majority of individuals (84.0%) harbored multiple-clone infections. The linkage structure among multilocus haplotypes was not associated with sampling location. These data provide the first study with deep sampling of the P. falciparum reservoir in an area of seasonal malaria transmission in West Africa. The co-occurrence of high multiplicity of infection (multiple-clone infections) with significant multilocus LD is surprising given the likelihood of high recombination rates in BD. The results suggest that the linkage structure among multilocus haplotypes has not been shaped by geographic separation of parasite populations. Furthermore, the observed LD levels provide a baseline population genetic metric with putatively neutral markers to evaluate the effects of seasonality and malaria control efforts in BD.

Population genomics of virulence genes of Plasmodium falciparum in clinical isolates from Uganda

Plasmodium falciparum causes a spectrum of malarial disease from asymptomatic to uncomplicated through to severe. Investigations of parasite virulence have associated the expression of distinct variants of the major surface antigen of the blood stages known as Pf EMP1 encoded by up to 60 var genes per genome. Looking at the population genomics of var genes in cases of uncomplicated malaria, we set out to determine if there was any evidence of a selective sweep of specific var genes or clonal epidemic structure related to the incidence of uncomplicated disease in children. By sequencing the conserved DBLα domain of var genes from six sentinel sites in Uganda we found that the parasites causing uncomplicated P. falciparum disease in children were highly diverse and that every child had a unique var DBLα repertoire. Despite extensive var DBLα diversity and minimal overlap between repertoires, specific DBLα types and groups were conserved at the population level across Uganda. This pattern was the same regardless of the geographic distance or malaria transmission intensity. These data lead us to propose that any parasite can cause uncomplicated malarial disease and that these diverse parasite repertoires are composed of both upsA and non-upsA var gene groups.

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 host immunity will be at a competitive advantage. The resulting frequency-dependent selection can structure parasite populations into strains of limited genetic overlap. For Plasmodium falciparum–the causative agent of malaria–in endemic regions, the high recombination rates and associated vast diversity of its highly antigenic and multicopy var genes preclude such clear clustering; 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. Significance Many pathogens, including the causative agent of malaria Plasmodium falciparum, use antigenic variation, obtained via recombination, as a strategy to evade the human immune system. The vast diversity and multiplicity of genes encoding antigenic variation in high transmission regions challenge the notion of the existence of distinct strains: temporally-persistent and specific combinations of genes relevant to epidemiology. We examine the role of human immune selection in generating such genetic population structure in the major blood-stage antigen of Plasmodium falciparum. We show, using simulated and empirical data, that immune selection generates and maintains ‘modules’ of genomes with higher genetic similarity within, than between, these groups. Selection further promotes the persistence of these modules for much longer times than those of their constituent genomes. Simulations show that the temporal modular structure reduces the speed at which hosts acquire immunity to the parasite. We argue that in P. falciparum modules can be viewed as dynamic strains occupying different niches in human immune space; they are thus relevant to formulating transmission models that encompass the antigenic diversity of the parasite. Our analyses may prove useful to understand the interplay between temporal genetic structure and epidemiology in other pathogens of human and wildlife importance.

Networks of genetic similarity reveal non-neutral processes shape strain structure in Plasmodium falciparum

Pathogens compete for hosts through patterns of cross-protection conferred by immune responses to antigens. In Plasmodium falciparum malaria, the var multigene family encoding for the major blood-stage antigen PfEMP1 has evolved enormous genetic diversity through ectopic recombination and mutation. With 50–60 var genes per genome, it is unclear whether immune selection can act as a dominant force in structuring var repertoires of local populations. The combinatorial complexity of the var system remains beyond the reach of existing strain theory and previous evidence for non-random structure cannot demonstrate immune selection without comparison with neutral models. We develop two neutral models that encompass malaria epidemiology but exclude competitive interactions between parasites. These models, combined with networks of genetic similarity, reveal non-neutral strain structure in both simulated systems and an extensively sampled population in Ghana. The unique population structure we identify underlies the large transmission reservoir characteristic of highly endemic regions in Africa.Plasmodium has evolved high genetic diversity in var genes, which encode for the major blood-stage antigen. Here, He et al. show how immune selection shapes the var gene repertoire in both simulated systems and a population in Ghana, by using neutral models and genetic similarity networks.

Signatures of competition and strain structure within the major blood-stage antigen of Plasmodium falciparum in a local community in Ghana

Abstract The concept of niche partitioning has received considerable theoretical attention at the interface of ecology and evolution of infectious diseases. Strain theory postulates that pathogen populations can be structured into distinct nonoverlapping strains by frequency‐dependent selection in response to intraspecific competition for host immune space. The malaria parasite Plasmodium falciparum presents an opportunity to investigate this phenomenon in nature, under conditions of high recombination rate and extensive antigenic diversity. The parasites major blood‐stage antigen, Pf EMP1, is encoded by the hyperdiverse var genes. With a dataset that includes thousands of var DBLα sequence types sampled from asymptomatic cases within an area of high endemicity in Ghana, we address how var diversity is distributed within isolates and compare this to the distribution of microsatellite allelic diversity within isolates to test whether antigenic and neutral regions of the genome are structured differently. With respect to var DBLα sequence types, we find that on average isolates exhibit significantly lower overlap than expected randomly, but that there also exists frequent pairs of isolates that are highly related. Furthermore, the linkage network of var DBLα sequence types reveals a pattern of nonrandom modularity unique to these antigenic genes, and we find that modules of highly linked DBLα types are not explainable by neutral forces related to var recombination constraints, microsatellite diversity, sampling location, host age, or multiplicity of infection. These findings of reduced overlap and modularity among the var antigenic genes are consistent with a role for immune selection as proposed by strain theory. Identifying the evolutionary and ecological dynamics that are responsible for the nonrandom structure in P. falciparum antigenic diversity is important for designing effective intervention in endemic areas.

Conservation of single amino-acid polymorphisms in Plasmodium falciparum erythrocyte membrane protein 1 and association with severe pathophysiology

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a parasite protein encoded by a multigene family known as var. Expressed on the surface of infected red blood cells, PfEMP1 plays a central role in parasite virulence. The DBLα domain of PfEMP1 contains short sequence motifs termed homology blocks. Variation within homology blocks, at the level of single amino-acid modifications, has not been considered before in association with severe disease. Here we identify a total of 2701 amino-acid polymorphisms within DBLα homology blocks, the majority of which are shared between two geographically distant study populations in existing transcription data from Kenya and in a new genomic dataset sampled in Ghana. Parasitemia levels and the transcription levels of specific polymorphisms are as predictive of severe disease (AUC=0.83) and of the degree of rosetting (forecast skill SS=0.45) as the transcription of classic var groups. 11 newly categorized polymorphisms were strongly correlated with grpA var gene expression (SS=0.93) and a different set of 16 polymorphisms was associated with the H3 subset (SS=0.20). These associations provide the basis for a novel method of relating pathophysiology to parasite gene expression levels—one that, being site-specific, has more molecular detail than previous models based on var groups or homology blocks. This newly described variation influences disease outcome, and can help develop anti-malarial intervention strategies such as vaccines that target severe disease. Further replication of this analysis in geographically disparate populations and for larger sample sizes can help improve the identification of the molecular causes of severe disease.

Evolutionary structure of Plasmodium falciparum major variant surface antigen genes in South America: Implications for epidemic transmission and surveillance

Abstract Strong founder effects resulting from human migration out of Africa have led to geographic variation in single nucleotide polymorphisms (SNPs) and microsatellites (MS) of the malaria parasite, Plasmodium falciparum. This is particularly striking in South America where two major founder populations of P. falciparum have been identified that are presumed to have arisen from the transatlantic slave trade. Given the importance of the major variant surface antigen of the blood stages of P. falciparum as both a virulence factor and target of immunity, we decided to investigate the population genetics of the genes encoding “Plasmodium falciparum Erythrocyte Membrane Protein 1” (Pf EMP1) among several countries in South America, in order to evaluate the transmission patterns of malaria in this continent. Deep sequencing of the DBLα domain of var genes from 128 P. falciparum isolates from five locations in South America was completed using a 454 high throughput sequencing protocol. Striking geographic variation in var DBLα sequences, similar to that seen for SNPs and MS markers, was observed. Colombia and French Guiana had distinct var DBLα sequences, whereas Peru and Venezuela showed an admixture. The importance of such geographic variation to herd immunity and malaria vaccination is discussed.