Frequent recombination events generate diversity within the multi-copy variant antigen gene families of Plasmodium falciparum

The human malaria parasite Plasmodium falciparum utilises a mechanism of antigenic variation to avoid the antibody response of its human host and thereby generates a long-term, persistent infection. This process predominantly results from systematic changes in expression of the primary erythrocyte surface antigen, a parasite-produced protein called PfEMP1 that is encoded by a repertoire of over 60 var genes in the P. falciparum genome. var genes exhibit extensive sequence diversity, both within a single parasite's genome as well as between different parasite isolates, and thus provide a large repertoire of antigenic determinants to be alternately displayed over the course of an infection. Whilst significant work has recently been published documenting the extreme level of diversity displayed by var genes found in natural parasite populations, little work has been done regarding the mechanisms that lead to sequence diversification and heterogeneity within var genes. In the course of producing transgenic lines from the original NF54 parasite isolate, we cloned and characterised a parasite line, termed E5, which is closely related to but distinct from 3D7, the parasite used for the P. falciparum genome nucleotide sequencing project. Analysis of the E5 var gene repertoire, as well as that of the surrounding rif and stevor multi-copy gene families, identified examples of frequent recombination events within these gene families, including an example of a duplicative transposition which indicates that recombination events play a significant role in the generation of diversity within the antigen encoding genes of P. falciparum.     

Evidence for the importance of genetic structuring to the structural and functional specialization of the Plasmodium falciparum var gene family

The var gene family encodes Plasmodium falciparum erythrocyte membrane 1 (PfEMP1) proteins that act as virulence factors responsible for both antigenic variation and cytoadherence of infected erythrocytes. These proteins orchestrate infected erythrocyte sequestration from blood circulation and contribute to adhesion-based complications of P. falciparum malaria infections. For this study, we analysed the genetic organization and strain structure of var genes and present evidence for three separately evolving groups that have, in part, functionally diverged and differ between subtelomeric and central chromosomal locations. Our analyses suggest that a recombination hierarchy limits reassortment between groups and may explain why some var genes are unusually conserved between parasite strains. This recombination hierarchy, coupled with binding and immune selection, shapes the variant antigen repertoire and has structural, functional and evolutionary consequences for the PfEMP1 protein family that are directly relevant to malaria pathogenesis.     

Cloning of the repertoire of individual Plasmodium falciparum var genes using transformation associated recombination (TAR)

One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.     

Allelic diversity of the Plasmodium falciparum erythrocyte membrane protein 1 entails variant-specific red cell surface epitopes

The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α(1) PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α(1) domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquisition of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.     

Structural relationships among mouse and human immunoglobulin VH genes in the subgroup III

The mouse VHIII subgroup is composed of four families which share sequence homology. We isolated a VH germ-line genomic clone, which cross hybridizes with a cDNA probe from one of these families, derived from a myeloma secreting an antigalactan antibody. We report here the nucleotide sequence of the cross hybridizing gene and show that very likely it has an anti-sheep red blood cell specificity. Comparison of its nucleotide sequence with those of the three other VHIII families shows that these genes share segmental homologies of variable lengths. This suggests that interchanges of sequence blocks between VH genes could be an important evolutionary mechanism for diversifying the germ-line repertoire. The strong homology (82%) with human VHIII genes suggests that efficient antibody sequences are strongly conserved. This conservation of homology is particularly striking when compared to the more limited homology (63%) between mouse and human C kappa genes.     

Functional Constraints on Insect Immune System Components Govern Their Evolutionary Trajectories

Roles of constraints in shaping evolutionary outcomes are often considered in the contexts of developmental biology and population genetics, in terms of capacities to generate new variants and how selection limits or promotes consequent phenotypic changes. Comparative genomics also recognizes the role of constraints, in terms of shaping evolution of gene and genome architectures, sequence evolutionary rates, and gene gains or losses, as well as on molecular phenotypes. Characterizing patterns of genomic change where putative functions and interactions of system components are relatively well described offers opportunities to explore whether genes with similar roles exhibit similar evolutionary trajectories. Using insect immunity as our test case system, we hypothesize that characterizing gene evolutionary histories can define distinct dynamics associated with different functional roles. We develop metrics that quantify gene evolutionary histories, employ these to characterize evolutionary features of immune gene repertoires, and explore relationships between gene family evolutionary profiles and their roles in immunity to understand how different constraints may relate to distinct dynamics. We identified three main axes of evolutionary trajectories characterized by gene duplication and synteny, maintenance/stability and sequence conservation, and loss and sequence divergence, highlighting similar and contrasting patterns across these axes amongst subsets of immune genes. Our results suggest that where and how genes participate in immune responses limit the range of possible evolutionary scenarios they exhibit. The test case study system of insect immunity highlights the potential of applying comparative genomics approaches to characterize how functional constraints on different components of biological systems govern their evolutionary trajectories.     

Mechanisms underlying mutually exclusive expression of virulence genes by malaria parasites

A fundamental yet poorly understood aspect of gene regulation in eukaryotic organisms is the mechanisms that control allelic exclusion and mutually exclusive gene expression. In the malaria parasite Plasmodium falciparum, this process regulates expression of the var gene family--a large, hypervariable repertoire of genes that are responsible for the ability of the parasite to evade the host immune system and for pathogenesis of the disease. A central problem in understanding this process concerns the mechanisms that limit expression to a single gene at a time. Here, we describe results that provide information on the mechanisms that control silencing and single gene expression and differentiate between several models that have recently been proposed. The results provide the first evidence, to our knowledge, supporting the existence of a postulated var-specific, subnuclear expression site and also reinforce the conclusion that var gene regulation is based on cooperative interactions between the two promoters of each var gene.     

Genetic diversity of expressed Plasmodium falciparum var genes from Tanzanian children with severe malaria

ABSTRACT: BACKGROUND: Severe malaria has been attributed to the expression of a restricted subset of the var multigene family, which encodes for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 mediates cytoadherence and sequestration of infected erythrocytes into the post-capillary venules of the vital organs such as the brain, lung or placenta. Var genes are highly diverse and can be classified in three major groups (ups A, B and C) and two intermediate groups (B/A and B/C) based on the genomic location, gene orientation and upstream sequences. The genetic diversity of expressed var genes in relation to severity of disease in Tanzanian children was analysed. METHODS: Children with defined severe (SM) and asymptomatic malaria (AM) were recruited. Fulllength var mRNA was isolated and reversed transcribed into var cDNA. Subsequently, the DBL and N-terminal domains, and up-stream sequences were PCR amplified, cloned and sequenced. Sequences derived from SM and AM isolates were compared and analysed. RESULTS: The analysis confirmed that the var family is highly diverse in natural Plasmodium falciparum populations. Sequence diversity of amplified var DBL-1alpha and upstream regions showed minimal overlap among isolates, implying that the var gene repertoire is vast and most probably indefinite in endemic areas. var DBL-1alpha sequences from AM isolates were more diverse with more singletons found (p<0.05) than those from SM infections. Furthermore, few var DBL-1alpha sequences from SM patients were rare and restricted suggesting that certain PfEMP1 variants might induce severe disease. CONCLUSIONS: The genetic sequence diversity of var genes of P. falciparum isolates from Tanzanian children is large and its relationship to disease severity has been studied. Observed differences suggest that different var genes might have fundamentally different roles in the host-parasite interaction. Further research is required to examine clear disease-associations of var gene subsets in different geographical settings. The importance of very strict clinical definitions and appropriate large control groups needs to be emphasized for future studies on disease associations of PfEMP1.     

Enhanced structure prediction of gene products containing class III adenylyl cyclase domains

Domain finding algorithms are useful to understand overall domain architecture and to propose biological function to gene products. Automated methods of applying these tools to large-scale genome studies often employ stringent thresholds to recognize sequence domains. The realization of additional domains can be tedious involving manual intervention but can lead to better understanding of overall biological function. We propose a multi-step approach for the further examination of unassigned linker regions that exploits properties such as the conservation of domain architectures of homologous proteins to propose connections. Improved structure prediction is possible starting from initial domain architectures, obtained from simple 'domain finding' techniques, by concentrating on connecting unassigned regions. 254 unassigned regions have been examined in 114 gene products that potentially contain at least one class III adenylyl cyclase domain for a pilot study. Reliable structure prediction was possible for nearly 80% of unassigned regions. New connections were recognized that assign putative structure and function to these regions by indirect searches (26%). Several others (34%) could be associated with three-dimensional models that might pertain to novel folds and new functions with enough structural content and evolutionary conservation. The presence of additional domains will provide further clues to the overall function of the gene products and their recruitment in particular biochemical pathways.     

Domain rearrangements in protein evolution

Most eukaryotic proteins are multi-domain proteins that are created from fusions of genes, deletions and internal repetitions. An investigation of such evolutionary events requires a method to find the domain architecture from which each protein originates. Therefore, we defined a novel measure, domain distance, which is calculated as the number of domains that differ between two domain architectures. Using this measure the evolutionary events that distinguish a protein from its closest ancestor have been studied and it was found that indels are more common than internal repetition and that the exchange of a domain is rare. Indels and repetitions are common at both the N and C-terminals while they are rare between domains. The evolution of the majority of multi-domain proteins can be explained by the stepwise insertions of single domains, with the exception of repeats that sometimes are duplicated several domains in tandem. We show that domain distances agree with sequence similarity and semantic similarity based on gene ontology annotations. In addition, we demonstrate the use of the domain distance measure to build evolutionary trees. Finally, the evolution of multi-domain proteins is exemplified by a closer study of the evolution of two protein families, non-receptor tyrosine kinases and RhoGEFs.     

Mapping and conservation of the group-specific component gene in mouse

The group-specific component (GC), also known as the vitamin D-binding protein, transports vitamin D and its metabolites in plasma to target tissues throughout the body. The GC gene shares an evolutionary origin with genes encoding albumin (ALB) and alpha-fetoprotein (AFP). All three genes are descendants of an evolutionary ancestor that arose from an intragenic triplication. As a result, each gene is composed of three homologous domains. The study described here characterizes and compares mouse GC to the corresponding nucleotide and amino acid sequences of GC from human and rat. The deduced amino acid sequence of mouse GC was 78% identical to human and 91% identical to rat GC. The results suggest that, unlike the corresponding sequences in the ALB and AFP genes, chromosomal sequences encoding the first domain and the leader sequence of the GC gene have specifically been conserved throughout vertebrate evolution. Protection of domain I during evolution may correlate with an important functional aspect of its sequence. The mouse GC gene was mapped to chromosome 5, where the ALB and AFP genes are also located, demonstrating conservation of the three genes in vertebrate species.     

A family affair: var genes, PfEMP1 binding, and malaria disease

An immunovariant adhesion protein family in Plasmodium falciparum named erythrocyte membrane protein 1 (PfEMP1), encoded by var genes, is responsible for both antigenic variation and cytoadhesion of infected erythrocytes at blood microvasculature sites throughout the body. Elucidation of the genome sequence of P. falciparum has revealed that var genes can be classified into different groups, each with distinct 5' flanking sequences, chromosomal locations and gene orientations. Recent binding and serological comparisons suggest that this genomic organization might cause var genes to diversify into separately recombining adhesion groups that have different roles in infection and disease. Detailed understanding of PfEMP1 expression and receptor binding mechanisms during infection and of the antigenic relatedness of disease variants might lead to new approaches in prevention of malaria disease.     

Comparative Analysis of the Complete Plastid Genome Sequence of the Red Alga Gracilaria tenuistipitata var. liui Provides Insights into the Evolution of Rhodoplasts and Their Relationship to Other Plastids

We sequenced to completion the circular plastid genome of the red alga Gracilaria tenuistipitata var. liui. This is the first plastid genome sequence from the subclass Florideophycidae (Rhodophyta). The genome is composed of 183,883 bp and contains 238 predicted genes, including a single copy of the ribosomal RNA operon. Comparisons with the plastid genome of Porphyra pupurea reveal strong conservation of gene content and order, but we found major genomic rearrangements and the presence of coding regions that are specific to Gracilaria. Phylogenetic analysis of a data set of 41 concatenated proteins from 23 plastid and two cyanobacterial genomes support red algal plastid monophyly and a specific evolutionary relationship between the Florideophycidae and the Bangiales. Gracilaria maintains a surprisingly ancient gene content in its plastid genome and, together with other Rhodophyta, contains the most complete repertoire of plastid genes known in photosynthetic eukaryotes.Supplementary material () is available for this article.[Reviewing Editor: Dr. W. Ford Doolittle]