The genome of model malaria parasites, and comparative genomics

The field of comparative genomics of malaria parasites has recently come of age with the completion of the whole genome sequences of the human malaria parasite Plasmodium falciparum and a rodent malaria model, Plasmodium yoelii yoelii. With several other genome sequencing projects of different model and human malaria parasite species underway, comparing genomes from multiple species has necessitated the development of improved informatics tools and analyses. Results from initial comparative analyses reveal striking conservation of gene synteny between malaria species within conserved chromosome cores, in contrast to reduced homology within subtelomeric regions, in line with previous findings on a smaller scale. Genes that elicit a host immune response are frequently found to be species-specific, although a large variant multigene family is common to many rodent malaria species and Plasmodium vivax. Sequence alignment of syntenic regions from multiple species has revealed the similarity between species in coding regions to be high relative to non-coding regions, and phylogenetic footprinting studies promise to reveal conserved motifs in the latter. Comparison of non-synonymous substitution rates between orthologous genes is proving a powerful technique for identifying genes under selection pressure, and may be useful for vaccine design. This is a stimulating time for comparative genomics of model and human malaria parasites, which promises to produce useful results for the development of antimalarial drugs and vaccines.     

The Plasmodium Apicoplast Genome: Conserved Structure and Close Relationship of P. ovale to Rodent Malaria Parasites

Apicoplast, a nonphotosynthetic plastid derived from secondary symbiotic origin, is essential for the survival of malaria parasites of the genus Plasmodium. Elucidation of the evolution of the apicoplast genome in Plasmodium species is important to better understand the functions of the organelle. However, the complete apicoplast genome is available for only the most virulent human malaria parasite, Plasmodium falciparum. Here, we obtained the near-complete apicoplast genome sequences from eight Plasmodium species that infect a wide variety of vertebrate hosts and performed structural and phylogenetic analyses. We found that gene repertoire, gene arrangement, and other structural attributes were highly conserved. Phylogenetic reconstruction using 30 protein-coding genes of the apicoplast genome inferred, for the first time, a close relationship between P. ovale and rodent parasites. This close relatedness was robustly supported using multiple evolutionary assumptions and models. The finding suggests that an ancestral host switch occurred between rodent and human Plasmodium parasites.     

The conserved genome organisation of non-falciparum malaria species: the need to know more

The current knowledge on genomes of non-falciparum malaria species and the potential of model malaria parasites for functional analyses are reviewed and compared with those of the most pathogenic human parasite, Plasmodium falciparum. There are remarkable similarities in overall genome composition among the different species at the level of chromosome organisation and chromosome number, conserved order of individual genes, and even conserved functions of specific gene domains and regulatory control elements. With the initiative taken to sequence the genome of P. falciparum, a wealth of information is already becoming available to the scientific community. In order to exploit the biological information content of a complete genome sequence, simple storage of the bulk of sequence data will be inadequate. The requirement for functional analyses to determine the biological role of the open reading frames is commonly accepted and knowledge of the genomes of the animal model malaria species will facilitate these analyses. Detailed comparative genome information and sequencing of additional Plasmodium genomes will provide a deeper insight into the evolutionary history of the species, the biology of the parasite, and its interactions with the mammalian host and mosquito vector. Therefore, an extended and integrated approach will enhance our knowledge of malaria and will ultimately lead to a more rational approach that identifies and evaluates new targets for anti-malarial drug and vaccine development.     

Highly conserved gene arrangement of the mitochondrial genomes of 23 Plasmodium species

Mitochondrial (mt) genomes from diverse phylogenetic groups vary considerably in size, structure and organization. The genus Plasmodium, the causative agent of malaria, has the smallest mt genome in the form of a tandemly repeated, linear element of 6 kb. The Plasmodium mt genome encodes only three protein genes (cox1, cox3 and cob) and large- and small-subunit ribosomal RNA (rRNA) genes, which are highly fragmented with 19 identified rRNA pieces. The complete mt genome sequences of 21 Plasmodium species have been published but a thorough investigation of the arrangement of rRNA gene fragments has been undertaken for only Plasmodium falciparum, the human malaria parasite. In this study, we determined the arrangement of mt rRNA gene fragments in 23 Plasmodium species, including two newly determined mt genome sequences from P. gallinaceum and P. vinckei vinckei, as well as Leucocytozoon caulleryi, an outgroup of Plasmodium. Comparative analysis reveals complete conservation of the arrangement of rRNA gene fragments in the mt genomes of all the 23 Plasmodium species and L. caulleryi. Surveys for a new rRNA gene fragment using hidden Markov models enriched with recent mt genome sequences led us to suggest the mtR-26 sequence as a novel candidate LSU rRNA fragment in the mt genomes of the 24 species. Additionally, we found 22-25 bp-inverted repeat sequences, which may be involved in the generation of lineage-specific mt genome arrangements after divergence from a common ancestor of the genera Eimeria and Plasmodium/Leucocytozoon.     

A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell

Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasite-induced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures.     

The Unique Structure of the Apicoplast Genome of the Rodent Malaria Parasite Plasmodium chabaudi chabaudi

The apicoplast, a non-photosynthetic plastid of apicomplexan species, has an extremely reduced but highly conserved genome. Here, the apicoplast genome of the rodent malaria parasite Plasmodium chabaudi chabaudi (Pcc) isolate CB was characterized. Although the set of genes in the genome is identical, the copy number of some tRNA genes differs between Pcc and other Plasmodium species because the Pcc DNA has only one rRNA/tRNA gene cluster, which is normally duplicated in other species. The location of the duplicated trnR(ACG) and trnM implies that one of the duplicated clusters in the ancestral molecule has been lost due to an intramolecular recombination event. The Pcc DNA occurs in two isoforms with an internal inversion between them. The presence of a unique variant in the duplicated trnT gene suggests that the two isoforms are interconvertible. This is the first report of the complete nucleotide sequence of a Plasmodium apicoplast DNA.     

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.     

Structural RNAs of known and unknown function identified in malaria parasites by comparative genomics and RNA analysis

As the genomes of more eukaryotic pathogens are sequenced, understanding how molecular differences between parasite and host might be exploited to provide new therapies has become a major focus. Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. In this article we identify by comparative genomics and validate by RNA analysis numerous previously unknown structural RNAs encoded by the Plasmodium falciparum genome, including the telomerase RNA, U3, 31 snoRNAs, as well as previously predicted spliceosomal snRNAs, SRP RNA, MRP RNA, and RNAse P RNA. Furthermore, we identify six new RNA coding genes of unknown function. To investigate the relationships of the RNA coding genes to other genomic features in related parasites, we developed a genome browser for P. falciparum (http://areslab.ucsc.edu/cgi-bin/hgGateway). Additional experiments provide evidence supporting the prediction that snoRNAs guide methylation of a specific position on U4 snRNA, as well as predicting an snRNA promoter element particular to Plasmodium sp. These findings should allow detailed structural comparisons between the RNA components of the gene expression machinery of the parasite and its vertebrate hosts.     

Genome comparison of human and non-human malaria parasites reveals species subset-specific genes potentially linked to human disease

Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.     

Genome-wide identification of molecular mimicry candidates in parasites

Among the many strategies employed by parasites for immune evasion and host manipulation, one of the most fascinating is molecular mimicry. With genome sequences available for host and parasite, mimicry of linear amino acid epitopes can be investigated by comparative genomics. Here we developed an in silico pipeline for genome-wide identification of molecular mimicry candidate proteins or epitopes. The predicted proteome of a given parasite was broken down into overlapping fragments, each of which was screened for close hits in the human proteome. Control searches were carried out against unrelated, free-living eukaryotes to eliminate the generally conserved proteins, and with randomized versions of the parasite proteins to get an estimate of statistical significance. This simple but computation-intensive approach yielded interesting candidates from human-pathogenic parasites. From Plasmodium falciparum, it returned a 14 amino acid motif in several of the PfEMP1 variants identical to part of the heparin-binding domain in the immunosuppressive serum protein vitronectin. And in Brugia malayi, fragments were detected that matched to periphilin-1, a protein of cell-cell junctions involved in barrier formation. All the results are publicly available by means of mimicDB, a searchable online database for molecular mimicry candidates from pathogens. To our knowledge, this is the first genome-wide survey for molecular mimicry proteins in parasites. The strategy can be adopted to any pair of host and pathogen, once appropriate negative control organisms are chosen. MimicDB provides a host of new starting points to gain insights into the molecular nature of host-pathogen interactions.     

Co-ordinated programme of gene expression during asexual intraerythrocytic development of the human malaria parasite Plasmodium falciparum revealed by microarray analysis

Plasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.     

Circumsporozoite protein gene from Plasmodium reichenowi, a chimpanzee malaria parasite evolutionarily related to the human malaria parasite Plasmodium falciparum

We have cloned and sequenced the gene encoding the circumsporozoite (CS) protein of Plasmodium reichenowi a Plasmodium falciparum-like malaria parasite of chimpanzees. Comparison of the two CS proteins reveals both similarities and differences in these two evolutionarily related parasites that have adapted to different hosts. The P. reichenowi CS protein has a new repeat sequence, NVNP, in addition to the P. falciparum-like NANP and NVDP repeats. In the immunodominant TH2R and TH3R regions of the CS protein, the amino acid sequences are similar in both parasite proteins. The differences in the two proteins exist in domains around the conserved regions, Region I and Region II, which are otherwise conserved in the CS proteins of P. falciparum analyzed to date. Studies of parasite protein genes of evolutionarily related malaria parasites, together with other immunologic and biologic characteristics, will help better understand the evolution and host parasite relationship of malaria parasites and may provide a tool for identifying protein determinants for malaria vaccine development.     

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.