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.     

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.     

Ectopic Recombination of a Malaria var Gene during Mitosis Associated with an Altered var Switch Rate

The Plasmodium falciparum var multigene family encodes P. falciparum erythrocyte membrane protein 1, which is responsible for the pathogenic traits of antigenic variation and adhesion of infected erythrocytes to host receptors during malaria infection. Clonal antigenic variation of P. falciparum erythrocyte membrane protein 1 is controlled by the switching between exclusively transcribed var genes. The tremendous diversity of the var gene repertoire both within and between parasite strains is critical for the parasite's strategy of immune evasion. We show that ectopic recombination between var genes occurs during mitosis, providing P. falciparum with opportunities to diversify its var repertoire, even during the course of a single infection. We show that the regulation of the recombined var gene has been disrupted, resulting in its persistent activation although the regulation of most other var genes is unaffected. The var promoter and intron of the recombined var gene are not responsible for its atypically persistent activity, and we conclude that altered subtelomeric cis sequence is the most likely cause of the persistent activity of the recombined var gene.     

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

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 parasite's major blood‐stage antigen, PfEMP1, 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.     

Origins of sequence diversity in the malaria vaccine candidate merozoite surface protein-2 (MSP-2) in Amazonian isolates of Plasmodium falciparum

The recent evolution of Plasmodium falciparum is at odds with the extensive polymorphism found in most genes coding for antigens. Here, we examined the patterns and putative mechanisms of sequence diversification in the merozoite surface protein-2 (MSP-2), a major malarial repetitive surface antigen. We compared the msp-2 gene sequences from closely related clones derived from sympatric parasite isolates from Brazilian Amazonia and used microsatellite typing to examine, in these same clones, the haplotype background of chromosome 2, where msp-2 is located. We found examples of msp-2 sequence rearrangements putatively created by nonreciprocal recombinational events, such as replication slippage and gene conversion, while maintaining the chromosome haplotype. We conclude that these nonreciprocal recombination events may represent a major source of antigenic diversity in MSP-2 in P. falciparum populations with low rates of classical meiotic recombination.     

A Plasmodium whole-genome synteny map: indels and synteny breakpoints as foci for species-specific genes

Whole-genome comparisons are highly informative regarding genome evolution and can reveal the conservation of genome organization and gene content, gene regulatory elements, and presence of species-specific genes. Initial comparative genome analyses of the human malaria parasite Plasmodium falciparum and rodent malaria parasites (RMPs) revealed a core set of 4,500 Plasmodium orthologs located in the highly syntenic central regions of the chromosomes that sharply defined the boundaries of the variable subtelomeric regions. We used composite RMP contigs, based on partial DNA sequences of three RMPs, to generate a whole-genome synteny map of P. falciparum and the RMPs. The core regions of the 14 chromosomes of P. falciparum and the RMPs are organized in 36 synteny blocks, representing groups of genes that have been stably inherited since these malaria species diverged, but whose relative organization has altered as a result of a predicted minimum of 15 recombination events. P. falciparum-specific genes and gene families are found in the variable subtelomeric regions (575 genes), at synteny breakpoints (42 genes), and as intrasyntenic indels (126 genes). Of the 168 non-subtelomeric P. falciparum genes, including two newly discovered gene families, 68% are predicted to be exported to the surface of the blood stage parasite or infected erythrocyte. Chromosomal rearrangements are implicated in the generation and dispersal of P. falciparum-specific gene families, including one encoding receptor-associated protein kinases. The data show that both synteny breakpoints and intrasyntenic indels can be foci for species-specific genes with a predicted role in host-parasite interactions and suggest that, besides rearrangements in the subtelomeric regions, chromosomal rearrangements may also be involved in the generation of species-specific gene families. A majority of these genes are expressed in blood stages, suggesting that the vertebrate host exerts a greater selective pressure than the mosquito vector, resulting in the acquisition of diversity.     

Evolution of the multi-domain structures of virulence genes in the human malaria parasite, Plasmodium falciparum

The var gene family of Plasmodium falciparum encodes the immunodominant variant surface antigens PfEMP1. These highly polymorphic proteins are important virulence factors that mediate cytoadhesion to a variety of host tissues, causing sequestration of parasitized red blood cells in vital organs, including the brain or placenta. Acquisition of variant-specific antibodies correlates with protection against severe malarial infections; however, understanding the relationship between gene expression and infection outcome is complicated by the modular genetic architectures of var genes that encode varying numbers of antigenic domains with differential binding specificities. By analyzing the domain architectures of fully sequenced var gene repertoires we reveal a significant, non-random association between the number of domains comprising a var gene and their sequence conservation. As such, var genes can be grouped into those that are short and diverse and genes that are long and conserved, suggesting gene length as an important characteristic in the classification of var genes. We then use an evolutionary framework to demonstrate how the same evolutionary forces acting on the level of an individual gene may have also shaped the parasite's gene repertoire. The observed associations between sequence conservation, gene architecture and repertoire structure can thus be explained by a trade-off between optimizing within-host fitness and minimizing between-host immune selection pressure. Our results demonstrate how simple evolutionary mechanisms can explain var gene structuring on multiple levels and have important implications for understanding the multifaceted epidemiology of P. falciparum malaria.     

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.     

Population genomics of the immune evasion (var) genes of Plasmodium falciparum

Var genes encode the major surface antigen (PfEMP1) of the blood stages of the human malaria parasite Plasmodium falciparum. Differential expression of up to 60 diverse var genes in each parasite genome underlies immune evasion. We compared the diversity of the DBLalpha domain of var genes sampled from 30 parasite isolates from a malaria endemic area of Papua New Guinea (PNG) and 59 from widespread geographic origins (global). Overall, we obtained over 8,000 quality-controlled DBLalpha sequences. Within our sampling frame, the global population had a total of 895 distinct DBLalpha "types" and negligible overlap among repertoires. This indicated that var gene diversity on a global scale is so immense that many genomes would need to be sequenced to capture its true extent. In contrast, we found a much lower diversity in PNG of 185 DBLalpha types, with an average of approximately 7% overlap among repertoires. While we identify marked geographic structuring, nearly 40% of types identified in PNG were also found in samples from different countries showing a cosmopolitan distribution for much of the diversity. We also present evidence to suggest that recombination plays a key role in maintaining the unprecedented levels of polymorphism found in these immune evasion genes. This population genomic framework provides a cost effective molecular epidemiological tool to rapidly explore the geographic diversity of var genes.     

Plasmodium falciparum antigenic variation. Mapping mosaic var gene sequences onto a network of shared, highly polymorphic sequence blocks

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, encoded by an extremely diverse gene family called var . Understanding of the genetic organization of var genes is hampered by sequence mosaicism that results from a long history of non-homologous recombination. Here we have used software designed to analyse social networks to visualize the relationships between large collections of short var sequences tags sampled from clinical parasite isolates. In this approach, two sequences are connected if they share one or more highly polymorphic sequence blocks. The results show that the majority of analysed sequences including several var -like sequences from the chimpanzee parasite Plasmodium reichenowi can be either directly or indirectly linked together in a single unbroken network. However, the network is highly structured and contains putative subgroups of recombining sequences. The major subgroup contains the previously described group A var genes, previously proposed to be genetically distinct. Another subgroup contains sequences found to be associated with rosetting, a parasite virulence phenotype. The mosaic structure of the sequences and their division into subgroups may reflect the conflicting problems of maximizing antigenic diversity and minimizing epitope sharing between variants while maintaining their host cell binding functions.     

The malaria genome sequencing project: complete sequence of Plasmodium falciparum chromosome 2

An international consortium has been formed to sequence the entire genome of the human malaria parasite Plasmodium falciparum. We sequenced chromosome 2 of clone 3D7 using a shotgun sequencing strategy. Chromosome 2 is 947 kb in length, has a base composition of 80.2% A + T, and contains 210 predicted genes. In comparison to the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, a greater proportion of genes containing introns, and nearly twice as many proteins containing predicted non-globular domains. A group of putative surface proteins was identified, rifins, which are encoded by a gene family comprising up to 7% of the protein-encoding gene in the genome. The rifins exhibit considerable sequence diversity and may play an important role in antigenic variation. Sixteen genes encoded on chromosome 2 showed signs of a plastid or mitochondrial origin, including several genes involved in fatty acid biosynthesis. Completion of the chromosome 2 sequence demonstrated that the A + T-rich genome of P. falciparum can be sequenced by the shotgun approach. Within 2-3 years, the sequence of almost all P. falciparum genes will have been determined, paving the way for genetic, biochemical, and immunological research aimed at developing new drugs and vaccines against malaria.     

Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.     

Plasmodium falciparum variant surface antigen expression patterns during malaria

The variant surface antigens expressed on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence "tags" to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite "rosetting" phenotype, a well established virulence determinant. Our results suggest that information on the state of the host-parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.