Mary M. Rorick

Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001

Homology blocks of Plasmodium falciparum var genes and clinically distinct forms of severe malaria in a local population

BackgroundThe primary target of the human immune response to the malaria parasite Plasmodium falciparum, P. falciparum erythrocyte membrane protein 1 (PfEMP1), is encoded by the members of the hyper-diverse var gene family. The parasite exhibits antigenic variation via mutually exclusive expression (switching) of the ~60 var genes within its genome. It is thought that different variants exhibit different host endothelial binding preferences that in turn result in different manifestations of disease.ResultsVar sequences comprise ancient sequence fragments, termed homology blocks (HBs), that recombine at exceedingly high rates. We use HBs to define distinct var types within a local population. We then reanalyze a dataset that contains clinical and var expression data to investigate whether the HBs allow for a description of sequence diversity corresponding to biological function, such that it improves our ability to predict disease phenotype from parasite genetics. We find that even a generic set of HBs, which are defined for a small number of non-local parasites: capture the majority of local sequence diversity; improve our ability to predict disease severity from parasite genetics; and reveal a previously hypothesized yet previously unobserved parasite genetic basis for two forms of severe disease. We find that the expression rates of some HBs correlate more strongly with severe disease phenotypes than the expression rates of classic var DBLα tag types, and principal components of HB expression rate profiles further improve genotype-phenotype models. More specifically, within the large Kenyan dataset that is the focus of this study, we observe that HB expression differs significantly for severe versus mild disease, and for rosetting versus impaired consciousness associated severe disease. The analysis of a second much smaller dataset from Mali suggests that these HB-phenotype associations are consistent across geographically distant populations, since we find evidence suggesting that the same HB-phenotype associations characterize this population as well.ConclusionsThe distinction between rosetting versus impaired consciousness associated var genes has not been described previously, and it could have important implications for monitoring, intervention and diagnosis. Moreover, our results have the potential to illuminate the molecular mechanisms underlying the complex spectrum of severe disease phenotypes associated with malaria—an important objective given that only about 1% of P. falciparum infections result in severe disease.

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.

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.

Dynamic landscapes: A model of context and contingency in evolution

Although the basic mechanics of evolution have been understood since Darwin, debate continues over whether macroevolutionary phenomena are driven by the fitness structure of genotype space or by ecological interaction. In this paper we propose a simple model capturing key features of fitness-landscape and ecological models of evolution. Our model describes evolutionary dynamics in a high-dimensional, structured genotype space with interspecies interaction. We find promising qualitative similarity with the empirical facts about macroevolution, including broadly distributed extinction sizes and realistic exploration of the genotype space. The abstraction of our model permits numerous applications beyond macroevolution, including protein and RNA evolution.

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.

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.

Influenza evolution navigates stability valleys.

By reconstructing how an influenza protein collected in 1968 might have evolved into one collected in 2007, researchers have obtained new insights into the interactions between genetic mutations.