Entering the post-genomic era of malaria research.

Entering the post-genomic era of malaria research Paul Horrocks,1 Sharen Bowman,2 Susan Kyes,3 Andrew P. Waters,4 & Alister Craig5 The sequencing of the genome of Plasmodium falciparum promises to revolutionize the way in which malaria research will be carried out. Beyond simple gene discovery, the genome sequence will facilitate the comprehensive determination of the parasite’s gene expression during its developmental phases, pathology, and in response to environmental variables, such as drug treatment and host genetic background. This article reviews the current status of the P. falciparum genome sequencing project and the unique insights it has generated. We also summarize the application of bioinformatics and analytical tools that have been developed for functional genomics. The aim of these activities is the rational, information-based identification of new therapeutic strategies and targets, based on a thorough insight into the biology of Plasmodium spp. Keywords: Plasmodium falciparum, genetics; genome, protozoan; base sequence; sequence analysis, DNA. Bulletin of the World Health Organization, 2000, 78: 1424–1437. Voir page 1434 le re´sume´ en franc¸ais. En la pa´gina 1434 figura un resumen en espan˜ol. Introduction The development of genome sequencing technolo- gies over the last five years has resulted in a wealth of sequence information, culminating in the recent announcement of a working draft of the human genome. Pathogen genomes, through their smaller size, have been even more tractable to these methodologies and are now well represented in genome science. Although not always suited as ‘‘model’’ organisms, the importance of pathogens in medicine and agriculture hasmade the exploitation of the sequence databases a high priority. But what does the production of long strings of As, Cs, Gs and Ts actually mean in terms of the alleviation of the burden of disease, particularly in developing countries? So far, genome sequencing has largely been the province of the developed world, where the resources and science infrastructure have allowed the formation of high-throughput sequencing centres. However, the use of sequencing information need not be restricted in this way, provided that resources for training can be met. Probably the most important aspect of the post-genomic era (i.e. after the sequencing has been carried out) is analysis of the primary sequence data. This is called bioinformatics and embraces a range of theoretical analyses aimed at converting the DNA sequence into biological information. Amajor part of this discipline involves the identification of genes through a number of processes, from identifying similarities with previously identified genes from other organisms, to the use of computer-derived models based on existing data. Subsequently come predictions of biological function and molecular shape (structural genomics), both of which have scope for development. Knowing the whole sequence of a pathogen genome also allows researchers to investigate the behaviour of organisms on amuch broader basis than was previously possible.Now, instead of studying the effect of drug treatment or differentiation on one or two genes, it is possible to study variation in all the genes at the same time using global transcriptional analysis. Protein patterns may also be examined, or the organism may be genetically modified (transfec- tion), providing a direct link between these biological effectors and gross phenotype. The technology for these experiments has been developed as a direct consequence of the desire to exploit the genome sequence data. It is not hard to envisage that the ability to identify and characterize the genetic blueprint of pathogens will help us recognize critical elements in the development and pathogenesis of disease- causing organisms and target our research efforts in the production of new therapies. For Plasmodium falciparum, genes and proteins acting at specific stages in the life cycle can be identified, their roles tested by 1 Post-Doctoral Research Assistant, Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, England. 2 Manager, Malaria Sequencing Project, Pathogen Sequencing Unit, Sanger Centre, Wellcome Trust Genome Campus, Hinxton, England. 3 Post-Doctoral Research Assistant, Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, England. 4 Reader, Department of Parasitology, Leiden University Medical Centre, Leiden, Netherlands. 5 Senior Lecturer, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, England (email: agcraig@liv.ac.uk). Correspondence should be addressed to this author. Ref. No. 00-0825 Special Theme – Malaria 1424 # World Health Organization 2000 Bulletin of the World Health Organization, 2000, 78 (12) genetic modification and promising candidates used in vaccine production. Parasite metabolic pathways not present in the host could also be targeted with potent inhibitors that are non-toxic to humans; and parasite drug-resistance mechanisms could also be targeted, giving existing drugs a longer effective lifetime. The sequence of the P. falciparum genome will provide many opportunities for research into malaria, but this is only a beginning, with the challenge being to turn those opportunities into effective treatments in the field. The Plasmodium falciparum genome The genome of P. falciparum consists of three discrete components: a linear repeat of a 6 kb element located within mitochondria; a 35 kb circle within a plastid- like structure (the apicoplast); and 25–30 Mb of nuclear DNA (genomic DNA). The nuclear DNA is organized into 14 chromosomes, between 0.75– 3.5 Mb in size, as determined by pulse-field gel electrophoresis (PFGE) and electron microscopic counts of kinetochore structures. Indirect evidence for the number of chromosomes also comes from genetic studies showing there are 14 linkage groups. The nuclear genome is organized in a manner typical of eukaryotes, with linear chromosomes being bounded at either end with telomeric sequences. Genome plasticity, seen in many parasite isolates and identified by size polymorphisms on PFGE, is thought to result frequently from deletions and insertions of DNA within subtelomeric sequences, a region shown to contain ordered repetitive sequence elements. The Plasmodium falciparum Genome Project Consortium Prior to the advent of yeast artificial chromosome (YAC) technology, there had been relatively little access to the parasite’s genome, as its extreme [A+T] content rendered inserts unstable in conventional bacterial plasmid clones. The estimated 80% [A+T]- rich genome could, however, be stably maintained within the pYAC4 construct, as demonstrated by the construction of a number of YAC libraries for different P. falciparum clones (1). In 1993 a con- sortium of laboratories distributed throughout the world established the Wellcome Trust Malaria Genome Mapping Project, with the aim of assem- bling YAC contiguous sequences (contigs) across each chromosome, as well as developing YAC, expressed sequence tag (EST), bioinformatic and genetic mapping technology (2). This consortium realized that sequencing of the entire nuclear DNA was a real possibility, yet considered the endeavour fraught with difficulties due to the extreme bias in base content. Genome sequencing has proved to be a power- ful and efficient approach in accessing the complete gene complement for organisms as diverse as Mycobacterium tuberculosis (3), Saccharomyces cerevisiae (4) and Caenorhabditis elegans (5). The advantages offered by such a tool in the investigation of human malaria eventually resulted in pilot projects being established in 1996 at three high-throughput genome centres, to establish whether sequencing the entire genome was possible: the Sanger Centre (England); The Institute for Genomic Research (TIGR) Malaria Program, Naval Medical Research Centre (NMRC) (USA); and Stanford University (USA). The Wellcome Trust, the Burroughs Wellcome Fund, the National Institute of Allergy and Infectious Diseases and the US Depart- ment of Defence provided funding for the pilot projects, and following their success these agencies agreed to fund the entire sequencing effort (6; see Table 1 for progress). Associated with this work are a number of other groups supporting the efforts of the high-throughput centres in a range of activities, including generation of chromosomal material; addi- tional mapping information; testing bacterial strains more tolerant of [A+T]-rich DNA; and the provision of a repository for P. falciparum reagents (MR4, http:// www.malaria.mr4.org/). A similar strategy is being used by all of the high- throughput sequencing centres, with individual chro- mosomes being excised from pulse-field gels, cloned as small inserts into a double-stranded vector, and sequenced in the forward and reverse directions to generate read-pair information which is used in gap filling. Sequence-tagged site and simple sequence- length polymorphism microsatellite markers from the HB3xDd2 genetic cross (7), together with the optical map (8) of ordered restriction fragments, are used to position contigs on each chromosome, or to confirm that sequence data has been assembled correctly. In addition, to help assign and order sequences originat- ing from a particular chromosome region, the groups at the Sanger Centre and Stanford University use a shotgun skim (1–2 fold coverage) of YAC clones selected from the chromosomal YAC contigs, gener- ated by the original P. falciparum mapping project. All three sequencing centres aim for an error rate of less than 1 base in every 10 000 bases. Once a section of chromosome sequence is finished it is analysed to identify a number of features, including putative protein-coding regions, tRNAs and repetitive sequences. Database searches are performed to identify similarities to protein and EST sequences. Further analyses are performed to determine whether the predictions have protein domains, signal sequences, putative membrane- spanning regions or any other distinctive features. A number of computing tools such as Hexamer/ Genefinder (R. Durbin, P. Green, L. Hillier, unpub- lished software, 1998)), and GlimmerM (9) are available to assist in the identification of protein- coding regions, but many other gene prediction programs exist or are currently being developed. These tools are efficient at identifying single, large, open reading frames (ORFs) or genes with two or three relatively large exons. However, the current 1425Bulletin of the World Health Organization, 2000, 78 (12) Entering the post-genomic era of malaria research generation of gene prediction software produces conflicting data when predicting multi-exon genes, particularly those with small exons (10, 11). These conflicting gene predictions are currently being tested experimentally by reverse-transcription polymerase chain reaction (RT–PCR) assays (Fig. 1). Annotation of current and future P. falciparum sequences is therefore an ongoing process, with predictions and annotations being refined as more information, such as the RT–PCR and EST sequencing data, becomes available. Analysis of the Plasmodium falciparum genome A total of 424 predicted protein-coding genes and 3 tRNAgenes have been identified on chromosomes 2 and 3, the two chromosomes for which sequencing has been completed (12, 13). Approximately 37% (158 genes) of these genes have a readily identifiable homologue in another species. Such similarity allows a function to be implied, with many of those identified being involved in parasite metabolism. Interestingly, a comparison of the predicted protein sequences with their homologues in other species showed that the majority of P. falciparum proteins have insertions of low complexity sequences, often runs of a single amino acid residue (typically asparagine, lysine or glutamic acid), or tandem arrays of a short peptide repeat sequence. Examples of such regions have been identified previously, and are polymorphic between different parasite isolates. P. falciparum contains two organelles thought to have arisen through endosymbiotic events: mito- chondria (14) and apicoplasts (15, 16). The unique nature of the apicoplast and the essential functions that it carries out make it a prime candidate for antimalarial drug development. Like many extra- nuclear elements, many of the genes for the organelle function have become nuclear encoded. Examina- tion of the predicted proteins encoded on chromo- somes 2 and 3 identified several that have a putative apicoplast signal sequence. This indicates that the apicoplast contains type II fatty acid synthase systems, typically associated with bacterial and plant plastids (17, 18). This supports the hypothesis that the apicoplast is algal in origin and provides a very specific target for rational drug design. Other proteins likely to play a critical role in parasite metabolism are also easily identified from searches of the P. falciparum databases. Using this approach, Jomaa et al. (19) identified two P. falciparum proteins with similarity to enzymes involved in the 1-deoxy-D-xylulose-5-phosphate pathway of isopre- noid synthesis, which is used in green algae and some bacteria, but not in animals. Antibiotics developed to inhibit this pathway have already been shown to have antimalarial activity in a rodent model system and in P. falciparum culture in vitro. Database searches permit the ‘‘virtual’’ identi- fication of P. falciparum homologues to proteins from other species, but comparative analysis of sequence organization (12, 13) has played a significant role in revealing subtelomeric coding sequences that would otherwise have gone unnoticed. A closer examination of the four available P. falciparum subtelomeric sequences shows that the order of both repetitive sequences and the variant multigene family members are conserved (Fig. 2). Members of the var, rif, stevor and Pf60 multigene families are present at all four telomeres, and new telomere-associated multigene families have alsobeen identified (conserved telomere- encoded proteins (CTPs)). While var, Pf60 and stevor had already been relatively well-characterized, rif required a more detailed analysis. Var comprises a highly polymorphic gene family of roughly 50 copies per haploid genome (20–22). It codes for PfEMP-1 (P. falciparum erythrocyte membrane protein-1) variants expressed on the surface of red blood cells, from the late ring stages of infection through schizogony. Each var gene is composed of two exons (Fig. 3). Exon 1 codes for the highly variable extracellular portion of PfEMP-1, including between one and seven Duffy- binding-like (DBL) domains, with at least one cysteine-rich interdomain region (CIDR) (23). The 3’ end of exon 1 codes for the semiconserved transmembrane region, and exon 2 codes for the semiconserved cytoplasmic region of the protein. PfEMP-1 is involved in cytoadherence of infected red cells to a range of different host receptors on endothelial cells and in binding to uninfected red cells, forming rosettes. The cytoadherent or rosetting phenotype of an individual parasite-infected red cell Table 1. Progress summary for the Malaria Genome Project Chromosome Size (Mb) Statusa 14b 3.4 Late Closure 13c 3.2 Closure 12d 2.4 Late Closure 11b 2.4 Closure 10b 2.1 Closure 9c 1.8 Shotgun in progress 8c 1.7 Shotgun in progress 7c 1.7 Shotgun in progress 6c 1.6 Shotgun complete 5c 1.4 Closure 4c 1.2 Late Closure 3c 1.06 Finished 2b 0.95 Finished 1c 0.7 Late Closure a Shotgun = random phase of sequencing where no selection of clones is used. Closure = the shotgun phase leaves gaps in the chromosomal sequence and these have to be filled using combinatorial approaches based on the sequence information flanking the gaps. Sometimes the sequence data do exist but have to be extracted manually from the computer database. Late Closure = sometimes a small number of gaps remain which are refractory to high- throughput sequencing methodologies and these need highly focussed approaches to fill them. b The Institute for Genomic Research, Malaria Program, Naval Medical Research Centre. c The Sanger Centre. d Stanford University. 1426 Bulletin of the World Health Organization, 2000, 78 (12) Special Theme – Malaria has been correlated with severity of disease and disease pathology (reviewed in 24). The Pf60 family members contain a 3’ exon highly similar to the var exon 2 sequence (25, 26), and have multiple possible 5’ exon organizations. One Pf60 protein, 6.1, is expressed in the nucleus of late asexual blood stage parasites, is composed of 7 exons, and employs a mechanism for reading through an internal stop codon (27). Hybridization-based analy- sis of Pf60 suggested there were 140 copies per haploid genome, but as the sequences are highly similar to var, an adjusted estimate would be roughly 90 Pf60 genes per haploid genome. Rif was originally described as an ˜1-kb DNA fragment repeated in the P. falciparum genome, an anomalous open reading frame (ORF) with no logical methionine start site (28). There was little point in hunting for upstream exons, since the dogma at that time was that most P. falciparum genes did not have introns. The sequence was eventually spotted between two var genes (22), and from early releases of genome sequences rif was recognized as a relative of stevor (29). This analysis identified the small upstream exon for both stevor and rif sequences (Fig. 3), and showed that both sequence types contain predicted transmembrane regions, which would orient the predicted protein as a loop on the outer surface of a cell membrane. The rif sequences are distinct from, and much more polymorphic than, stevor with an estimated 200 copies of rif per haploid genome (13, 30), and roughly thirty-four members of the stevor family (29). To date, it is uncertain where and when the STEVOR proteins are expressed (31), although the relative lack of polymorphism suggests that they are not likely to be expressed on the surface of red blood cell. However, for rif the large number of highly polymorphic copies suggested a location exposed to host immune/selective pressures. Indeed, it was shown that rif sequences code for clonally variant 35–44 kDRIFIN proteins expressed on the surface of infected red blood cells (30), and that antibodies to the proteins are detectable in sera from immune indivi- duals (32). Although RIFINs and PfEMP-1 share the same cellular localization, RIFIN function remains unclear. While var genes are transcribed during all ring stages of development, and rif genes are only expressed for a short time at the late-ring/early pigmented 1427Bulletin of the World Health Organization, 2000, 78 (12) Entering the post-genomic era of malaria research trophozoite stage, both proteins are detected on the red cell surface at roughly the same time (early trophozoites) (25, 33). Further studies are necessary to determine whether these proteins are functionally linked as well as physically linked within the genome. The final gap to be sequenced on chromosome 3 covered a region of extreme [A+T] composition: 97.3% for 2.6kb. Subsequent comparison with the sequence of chromosome 2 identified a region similar in both composition and length. Both [A+T]-rich regions occur in gene-sparse areas of the chromo- somes, forming part of the longest intergenic region on chromosome 3. Closer analysis revealed the presence of chromosome-specific repetitive se- quences. Thus, their structure and extreme [A+T] composition suggest these regions are candidate centromeres. [A+T]-rich central cores are present in Saccharomyces cerevisiae and Schizosaccharomyces pombe centromeres and the latter contain complex, chro- mosome-specific sequences. Although, to date, these regions have not been demonstrated to function as P. falciparum centromeres, a third example of this structure has recently been identified on chromo- some 1. Functional genomics The availability of an increasing number of pathogen genome sequences has strengthened the impetus for ‘‘global’’ investigation. The paradigm for these studies has been set by yeast researchers, who have had access to the full genome sequence of S. cerevisiae since 1996 (4). Although implicit in genomic sequencing, the development of techniques to study functional parameters across whole genomes has evolved into a new field of research termed ‘‘functional genomics’’. For P. falciparum, a number of technical difficulties remain, but researchers are already applying functional genomics to the biology of this parasite. Bioinformatics Bioinformatics covers both sequencing and func- tional genomics in converting raw genome sequence data into useful information for the biologist. Probably the most important aspects are the search engines that can screen vast amounts of information for significant similarities between sequences, and the algorithms used to predict protein-coding regions (see above). As far as the search engines are concerned, several sites exist on the web that will allow the user to search for homology to a test sequence (see Table 2), returning the results as multiple sets of alignments. Although there are some errors in defining genes ‘‘in silico’’ (i.e, a gene model predicted by computer), it is clear that these techniques have generated an enormous number of potential gene functions that can be tested in the laboratory. Perhaps the easiest forms of this are the annotations of complete chromosome sequences that are based on high-quality sequence information. The resulting tables of genes can be divided into those that have confirmed function in P. falciparum; sequences with homology to known genes in other organisms; sequences with homology to genes of unknown function in other organisms; and se- quences specific to P. falciparum of unknown function. One of the underlying assumptions is that the annotated sequence contains all the genes and that the predictions for the coding sequence are correct. While this is largely true, there has been some discussion about ‘‘missing’’ genes and the incorrect identification of splice sites. Therefore, an essential part of any bioinformatic system is the provision of software for browsing genomic sequence data so that 1428 Bulletin of the World Health Organization, 2000, 78 (12) Special Theme – Malaria independent laboratories can make their own judge- ments on coding sequences. Access to information is possible through the release of sequence data prior to publication, which has already made a significant contribution to malaria research. However, different web sites contain different parts of the sequence information from different chromosomes. The site at the National Center for Biotechnology Information (NCBI, see Table 2) has attempted to collate all the available information for P. falciparum (as well as that for other Plasmodium species and the related apicomplexan parasite Toxoplasma gondii). A recent development, funded by the Burroughs Wellcome Fund, has esta- blished a full database (http://www.PlasmoDB.org). In its current form, PlasmoDB provides: views of finished and annotated sequence in both web-based and CD format; a relational database that should facilitate entry and analysis of expression data; a ‘‘BLASTable’’ database containing the most recent genomic sequence information (finished and un- finished); text-queriable results from a complete BLAST search of all P. falciparum data against the GenBank and EMBL databases; and various other data-mining tools. In addition to the obvious interest of PlasmoDB to themalaria research community, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases has made a commitment to provide on-line ‘‘help-desk’’ support for PlasmoDB (and other parasite data- bases), through the training of scientists/bioinfor- maticians from developing countries. Microarrays Microarrays are high-density arrays of DNA targets on glass or filter supports. These have been used in several studies, most notably for S. cerevisiae. There are essentially two formats for the DNA targets: DNA fragments (usually generated by PCR) or oligonucleo- tides. PCR-derived targets for P. falciparum have been produced from genome sequence data (D. Carrucci, personal communication, 1998), or by the amplifica- tion of sequences from a mung bean nuclease library. In the mung bean nuclease library study (34), arrays were made from a random library and screened with Table 2. Plasmodium falciparum bioinformatic web-site URLs http://www.sanger.ac.uk/Projects/P_falciparum (Sequence data) http://www.tigr.org/tdb/edb/pfdb/pfdb.html (Sequence data) http://sequence-www.stanford.edu/group/malaria/index.html (Sequence data) http://www.PlasmoDB.org (‘Official’ database of the Plasmodium Genome Consortium) http://www.ncbi.nlm.nih.gov/Malaria (Genetics/bibliography/sequence data) http://www.ebi.ac.uk/parasites/parasite-genome.html (General site/proteomics) http://sites.huji.ac.il/malaria/(Metabolic pathways) 1429Bulletin of the World Health Organization, 2000, 78 (12) Entering the post-genomic era of malaria research mRNA from asexual (trophozoite) and sexual (game- tocyte) stages. The RNAs were labelled with different fluorochromes and allowed to hybridize to the arrays. Genes transcribed during asexual stages were labelled green, those transcribed during sexual stages red, and those transcribed during both stages yellow (green + red). From this single experiment the authors identified several developmentally regulated genes, including some which had been previously identified, confirming the efficacy of this approach. The potential for these types of experiments is considerable, but will require careful data handling and analysis. Hayward et al. (34) identified their candidate genes by sequencing the inserts after hybridization, but once the genome has been completely sequenced the address of each ORF will be stored as part of the array information. An array covering all of the genes in the P. falciparum genome in duplicate would have approximately 12 000– 14 000 spots; therefore every experiment would generate at least 14 000 quantitative data points per probe, in addition to the array baseline information. It is not hard to see that without a highly efficient database, the efficiency and accuracy of any analysis would be greatly reduced, particularly since transcrip- tional changes are likely to be defined by ‘‘clusters’’ of genes (35), which makes the contribution of individual genes difficult to interpret. The database must be accessible by the research community to take full advantage of the results and, as seen for C. elegans (http://www.wormbase.org), a centralized database is essential. Transcriptional analysis for P. falciparum is also being attempted by Serial Analysis of Gene Expres- sion (SAGE, 36; D. Wirth, personal communication, 1998)). This technique uses PCR amplification of mRNA to derive short sequence tags that are unique to each gene. The tags are concatenated into long strings and cloned into suitable vectors. By sequen- cing many tags, it is possible not only to identify what gene is being expressed, but also to quantify the level of expression by recording the number of times specific sequence tags are present. The handling is more complicated than hybridization to arrays, but themajor advantage of this technique is that relatively small quantities of material can be used, which is particularly important where samples may be limited (e.g. field samples). For both techniques it is important that technology transfer is carried out, including not only the mechanics such as robotics and image analysis, but also an appreciation of the assumptions and limitations. For example, neither technique can detect the products of alternative mRNA splicing events. High-density arrays are not exclusively for use in transcriptional analysis, but also have applications in genome analysis, particularly as oligonucleotide arrays. The high degree of specificity of oligonucleo- tide hybridization allows a high level of discrimina- tion, including single nucleotide substitutions. However, their use is precluded prior to the completion of the P. falciparum sequencing project due to the cost of the chemical synthesis. One example of this type of approach comes again from S. cerevisiae (37), in which allelic variation throughout the genomes of two strains of yeast was identified solely by hybridization of their genomic DNA to oligonucleotide arrays. The application of this technology to natural populations of parasites will be a powerful tool for molecular epidemiology. However, information from many P. falciparum genomes will need to be collated to fully cover the true extent of highly variant and recombinogenic genes such as var. Proteomics and structural genomics One of the greatest technical challenges in functional genomics concerns the analysis of protein expression (proteomics). Although genome-wide transcriptional analysis has produced very useful information, it is clear that the correlation betweenmRNA and protein levels is not perfect (38). Techniques for identifying proteins in general have developed rapidly over the last few years. However, the major advance with regard to genome sequencing has come through the use of mass spectrometry for protein analysis and characterization, by combining the mass (and there- fore amino acid composition) of peptide fragments with a database of all the available peptide combina- tions derived from the genome sequence data (see 39 for review). Using these techniques it is possible to identify individual proteins from complex mixtures resolved by two-dimensional (2-D) electrophoresis or chromatography. This, allied with improvements in the reproducibility of 2-D electrophoresis, has facilitated the differential analysis of protein compo- sition from two populations of cells. Applications of this technology, for example in the analysis of the effect of drug treatments, have an important place in malaria research. Some practical difficulties also remain, particularly for membrane proteins, which are poorly represented using classical proteomic techniques (40). However, technology continues to improve (41) and the reward will be a clear definition of the spectrum of proteins involved in critical biological processes in the parasite. Protein–protein interactions play an important role in cell biology. The tools to investigate this phenomenon, again, have been developed with yeast through the use of the ‘‘two-hybrid’’ system. In this, ORFs are fused to the Gal4 transcription-activation domain in one yeast strain and screened by mating with a second strain in which specific coding sequences have been fused with the Gal4 DNA- binding domain. Positive protein interactions are scored by the ability of the yeast progeny to grow on selective media. This procedure has recently been expanded to enable researchers to carry out a comprehensive analysis of the protein–protein inter- actions of approximately 6000 yeast ORFs (42), generating novel information as well as confirming previous specific screens. The extension of this 1430 Bulletin of the World Health Organization, 2000, 78 (12) Special Theme – Malaria technique to pathogen genome research will produce a further layer of functional information. One of the goals of computational biology is to predict the biological function of a protein from primary sequence information. Currently, the clearest manifestation of this is in structural genomics in which the three-dimensional structure of proteins is studied in the context of the amino acid sequence. While much still needs to be done, the increasing number of resolved crystal structures for biological molecules has already resulted in the generation of rules/motifs that can be applied when searching for structural information (43). As more information becomes available, including the integration of functional data in terms of active residues, these computer algorithms will improve. Metabolomics and vaccinomics One of the many outcomes from functional genomics has been the production of a new vocabulary to cover the many applications of metabolomics and vaccinomics. Thus, the entire mRNA and protein complement of an organismhave been termed the transcriptome and the proteome, respectively. In a similar vein, the use of genomic information to facilitate studies of metabolic pro- cesses has been termed metabolomics (44, 45). Clearly, the use of sequence similarities to identify components of known pathways will have a huge impact on research in this area, but functional screens have also been proposed. For example, by expressing all the predicted ORFs from a genome as hetero- logous fusion proteins it might be possible to screen for specific enzyme functions. This resource could also be used to determine host protective immune responses through immunization with protein pools. Related studies have been called vaccinomics, in which DNA vaccine plasmids containing ORFs (derived from the sequencing project) are used to immunize experimental animals and, in the case of pathogens, to screen for protection (46). Not only could this lead to the identification of vaccine candidates, but the resulting sera could also be used to determine the cellular location of each protein. As the number of sequenced genomes in- creases and researchers becomemore accustomed to thinking in ‘‘global’’ terms, the amount of informa- tion generated by functional genomics will expand rapidly. The challenges will then be to develop systems to analyse this information and to maintain a focus on the biological issues. Clearly, for malaria this would be well served through studies on the parasites themselves, facilitated by genetic crosses (47) and the technology of genetic manipulation (see below). Transfection of Plasmodium falciparum Transfection, or the introduction of exogenous DNA into the organism in vivo, potentially provides one of the most powerful tools for the analysis of the parasite’s genome, allowing us to specifically address questions relating to gene function. However, prior to the initial demonstration of luciferase expression in the chicken malaria model, P. gallinaceum, the ability to modify or disrupt components of the P. falciparum genome had eluded researchers (48). Following this report, transfection of P. falciparum was successfully carried out when a plasmid bearing the reporter gene encoding chloramphenicol acetyltransferase (CAT) was introduced into the readily cultured, intra- erythrocytic stages (49). Stable transfection of a plasmid bearing a pyrimethamine selectable marker (mdhfr-ts, a mutant dihydrofolate reductase–thymidine synthetase gene), and its subsequent integration via homologous recombination into the genome, soon followed (50). The plasmids described in these initial reports, as well as a transgene expression system (expression of a reporter gene from a plasmid stably maintained by virtue ofmdhfr-ts (51)) have provided the basis for all the subsequent studies in P. falciparum. Using both CAT and luciferase reporter genes, a series of studies have addressed the structure and function of P. falciparum transcriptional units. P. falci- parum promoters were shown to conform to a classical bipartite structure: a basal promoter regu- lated by upstream regulatory factors, with transcript stability directed by 3’ regulatory sequences (52–57). Also, the nuclear context in which promoters are placed play a key role in their function, suggesting that phenomena such as stage-specific gene expres- sion, and switching of expression between members of the var multigene family, may rely on epigenetic factors, such as chromatin assembly (58, 59). More advanced work, utilizing gene disruption and allelic replacement, has formed the basis of a series of recent reports that examined aspects of P. falciparum biology, such as cytoadhesion, gameto- genesis and drug resistance (Table 3). Disruption of the gene encoding the knob-associated histidine-rich protein (KAHRP), located within electron-dense knob structures on the surface of infected erythro- cytes, showed that this protein is important in their formation (51). Follow-up studies demonstrated that the knob structure is critical in supporting PfEMP-1 protein during its interaction with host receptors under fluid flow conditions. Pfg27, a protein expressed early following a parasite’s commitment to sexual differentiation, is also essential since disruption of this gene resulted in the abortion of transfectants early in gametogenesis, and resulted in highly vacuolated and morphologically disrupted parasites (60). The roles of mutations within genes conferring resistance to a wide range of antimalarial drugs have also been investigated by altering ORFs rather than disrupting them (61). And the roles of dihydropteroate synthase in sulfadoxine resistance, and P-glycoprotein homologue 1 (Pgh-1) in multidrug resistance, were elegantly demonstrated by introducing mutations associated with drug resistance in epidemiological studies into parasites bearing a drug-sensitive back- 1431Bulletin of the World Health Organization, 2000, 78 (12) Entering the post-genomic era of malaria research ground (62). Moreover, these studies have also been used to demonstrate the reverse situation, where mutations of cg2, a candidate chloroquine resistance gene, did not confer resistance in a sensitive parasite background, prompting investigations that identified a more promising candidate (D. Fiddock, T. Wellems, personal communication, 1999). The primary limitation with this type of experimental strategy is that the intraerythrocytic stage parasites investigated are haploid. Targeted disruption of essential genes inevitably result in the death of the parasite; moreover, any effect on parasite viability will place that proportion of the population at a growth disadvantage. Thismay be partially overcome by selection, or by the presence of alternative pathways that can overcome the growth disadvantage. Proposals for a consortium of laboratories, similar to the EUROFAN network for S. cerevisiae, to systematically ‘‘knock-out’’ every gene identified by the genome project have considered a wide variety of issues, such as poor parasite viability, low transfection efficiency, the numbers of laboratories able to create mutants, and the unit cost per gene knock-out (63). Although there are still many issues to be resolved, it has been agreed that some form of systematic analysis of the parasite’s gene complement should take place. However, whether each laboratory would be allocated a share of the genome, or whether a thematic approach is used (based on a laboratory’s interest in a particular subject, such as erythrocyte invasion, cytoadhesion or gametocytogenesis) re- mains to be determined. Malaria model systems The lack of in vitro culture systems for most P. falciparum developmental stages and the ethical considerations necessary for the use of New World monkeys and chimpanzees highlight the advantages offered by model malaria parasite systems. Transfec- tion of animal malaria models has opened up the entire parasite’s life cycle, including those in the invertebrate mosquito host, for critical analysis in a manner not possible with human parasites (64). Moreover, the efficiency of the transfection process, facilitating double-cross over gene replacements, and the rapid selection of transfectants in vivo, have resulted in the rapid accumulation of new insights into Plasmodium spp. biology. Following the first reports of transfection in the rodent malarial model P. berghei in 1995 (65), disruptions of the genes encoding circumsporozoite protein (CS) (66) and thrombospondin-related anonymous protein (TRAP) (67) demonstrated the value of this approach in the investigation of function: apart from their roles in host cell recogni- tion and invasion, new roles in sporozoite formation and gliding motility were attributed to CS and TRAP, respectively. Recurrent biological themes have Table 3. Genes modified or ‘‘knocked out’’ in Plasmodium Plasmodium gene targeteda Phenotype Reference Pb CS Inhibition of sporozoite formation, loss of infectivity 66 Pb TRAP Sporozoites fail to glide and show reduced infectivity 67 Pb, Pf CTRP Ookinetes non-motile and fail to invade midgut epithelia and develop into oocysts 83–85 Pf KAHRP Infected-red blood cells ‘‘knobless’’ and have reduced binding to CD36 under 51 flow conditions PF G27 Total (3’ disruption) or significant (5’ disruption) inhibition of gametocytogenesis 60 Plasmodium gene modified Manipulation and phenotype Reference Pf DHPS mutation Introduction of field-observed mutations give rise to sulfadoxine 61 resistance when introduced Pf pgh1 replacement Introduction of field observed mutations give rise to mefloquine, halofantrine 62 and quinine resistance. Also resistance to chloroquine in a strain-specific manner. Pb TRAP Replacement with Pf TRAP. Sporozoites can glide, invade salivary glands 86 and are infectious Pb TRAP Replacement with Pf TRAP mutants. TRM- sporozoites do not glide or invade 86 salivary glands, but remain infectious. Amut sporozoites do not invade salivary glands, but remain infectious Pb TRAP Replacement with Pb TRAP mutant. Cyt ~S and ~L sporozoites do not glide 87 normally, cannot invade salivary glands and are not infectious. Cyt/MIC-2 sporozoites can glide, invade salivary glands and are infectious Pf Acyl Carrier Protein Green fluorescent protein tag demonstrates need for bipartite leader 80 sequence to correctly target protein to apicoplast and conservation of leader within apicoplasts. Pf MSP1 Replaced C terminal with that of P. chabaudi MSP-1 demonstrates conservation 88 of function and potential for immune variation. a Except where indicated, the targeted loci have been disrupted after integration. Genes targeted in P. berghei are indicated by Pb or PB and genes targeted in P. falciparum are indicated by Pf or PF. 1432 Bulletin of the World Health Organization, 2000, 78 (12) Special Theme – Malaria already been demonstrated. For example, CTRP, a TRAP homologue, has been shown to have a role in ookinete motility and thus in the ability to infect mosquitoes. The readily available and immunologically well- characterized animal hosts allow a full range of host parasite interactions to be investigated. Following the success of this work, transfection of two primate malaria models has been established at the Biochem- ical Primate Research Centre in the Netherlands. Both P. knowlesi (68) and P. cynomolgi (69) have been transfected with entirely heterologous plasmid con- structs bearing a Toxoplasma gondii DHFR-TS drug selectable marker controlled by P. berghei regulatory sequences. One particular advantage of primate malaria models is that they allow investigation of transfected parasites within both their natural and artificial hosts. Within strict ethical limitations, the use of these systems facilitatesmeaningful analyses of host–parasite interactions and will allow the mecha- nisms governing the host immune response to be examined, aswell as providing an experimentalmodel for evaluating vaccines. Animal models share many features with their humanmalarial counterparts, such as life cycles, host- cell restrictions and immune responses. For example, P. cynomolgi infection of macaques is a particularly good model for P. vivax infection, sharing preference for reticulocytes and hypnozoite formation (69). Although we have a substantial pool of knowledge for animalmalariamodels, particularly with respect to homologous vaccine antigens, we understand com- paratively little about these systems at the molecular level. Software for sequencing EST libraries, as well as low-coverage genomic shotgun sequencing, have already provided a fundamental boost to the application of animal models to human malaria biology and should be supported with larger-scale sequencing activities. Use of animal models would allow intensive programmatic approaches to gene function to be considered through the use of knock- out/tagging methods. Where species ofPlasmodium, such as P. knowlesi and P. cynomolgi, provide models for malaria infection and immunity, the closely related apicomplexan T. gondii provides a model for many aspects of Plasmodium biology (70). Transfection systems are well developed in this parasite, with high-efficiency transformation and a number of markers for both positive and negative selection (71). This experimen- tally tractable system has been instrumental in tackling issues such as plastid structure and function (72), host cell invasion, drug resistance, virulence and gene expression. Unlike Plasmodium spp., which exclusively integrateDNAmolecules through homo- logous recombination, T. gondii also permits non- homologous integration, allowing the entire genome to be tagged for a phenotype-based approach to gene function.For this,Plasmodiumwill require a transposon- based system of simple recognition site specificity, such as the Drosophila mariner element, which efficiently transfers into a number of eukaryotic genomes including Leishmania major (73). Both low- and high-technology developments are needed to continue a systematic assault on the Plasmodium genome, such as improved culture meth- ods for certain human andmodel bloodstage parasites, gamete development and efficient sporozoite culture. These, together with advances in transfection tech- nology, such as new markers, methods to increase transfection efficiencies and adoption of the Tet- repressor system, will combine to make transfection a more reliable and sensitive tool. However, only the coupling of these advances with the knowledge of the parasite‘s entire gene complement will allow the advances to be fully exploited. Comparative genomics Mention has already beenmade of the difficulties that can be encountered when annotating a complete genome, particularly assigning function to unknown genes and correctly identifying genes that contain numerous exons. Ultimately, the availability of a fully or extensively sequenced genome from a related species will be an invaluable tool that will facilitate an accurate assessment of the coding potential of a genome (74). Studies on the chromosomes of rodent malaria species demonstrated that there was little, if any, difference in gene linkage (75), and that large conserved linkage groups could be detected between these rodent malarias and P. falciparum (76). Limited, but more detailed, analysis revealed that genome organization is highly conserved in the internal non- subtelomeric regions of the chromosome (77, 78). Thus, the expectation is that alignment of orthologous chromosome regions will reveal detailed intron–exon boundaries, and help identify ortholo- gous, but polymorphic, antigen-coding genes, cen- tromeres and species-specific genes. In this last regard, comparison with a more distant apicomplexan genome may also prove valuable. Although gene order may not be well conserved, many salient features of apicomplexans will be revealed through genome comparisons, for example the apicoplast (79–81). The ease of transformation of Toxoplasma gondii and its suitability for cell biological studies, combined with comparative genomics, provides an excellent opportunity to illuminate malaria biology, develop drug targets (19, 82) and possibly vaccines. Conclusion The ability to completely sequence genomes has had a huge impact on the way in which scientific research is conducted. As the number of pathogen genomes that have been completed increases, the impetus to develop techniques that utilize this information has increased. In parallel with the P. falciparum genome project, techniques such as transfection, microarrays and proteomics, as well as bioinformatic analysis, are 1433Bulletin of the World Health Organization, 2000, 78 (12) Entering the post-genomic era of malaria research already being developed and applied to fundamental biological questions. The combination of these tools is expected to provide a predictive platform from which to launch hypothesis-driven biological research. Central to this expectation is continuing financial support for the further development of techniques and resources to exploit the genome sequence. Developments that will provide insight into parasite biology are publicly accessible relational databases, and detailed comparative and survey analyses. It must be emphasized that the overall goal of such costly enterprises is to work towards a cure for malaria, and the contribution of this technological approach could be immense. The current challenge to the research community is to continue the dissemination of this technology and to focus efforts on the development of new drugs and vaccines. n Re´sume´ La recherche sur le paludisme s’engage dans l’e`re postge´nomique Le de´veloppement des techniques de se´quenc¸age du ge´nome a permis de recueillir au cours de ces cinq dernie`res anne´es une masse d’informations sur ces se´quences, qui ont abouti a` l’annonce faite re´cemment qu’on e´tait parvenu a` obtenir une premie`re version de travail du ge´nome humain. Les ge´nomes des germes pathoge`nes, du fait de leur taille re´duite, ont e´te´ encore plus faciles a` analyser avec ces techniques et sont de´sormais bien repre´sente´s dans la ge´nomique. Bien qu’ils ne puissent pas toujours servir de micro- organismes « mode`les », l’importance des germes pathoge`nes en me´decine et en agriculture ont fait de l’exploitation des bases de donne´es relatives aux se´quences dont ils sont constitue´s une priorite´ de tout premier ordre. Mais que signifie re´ellement la production de ces longues se´ries d’A-C-G-T sur le plan de la re´duction de la charge de morbidite´, en particulier dans les pays en de´veloppement ? Jusqu’ici, le se´quenc¸age du ge´nome a surtout e´te´ la spe´cialite´ du monde de´veloppe´, ou` les ressources et les infrastructures scientifiques en place ont permis la cre´ation de centres de se´quenc¸age a` haut rendement. Toutefois, l’utilisation des re´sultats de ce se´quenc¸age n’a aucune raison d’eˆtre ainsi limite´e, pour autant qu’on puisse disposer de ressources pour la formation. L’analyse des donne´es des se´quences primaires est probablement l’aspect le plus important de l’e`re postge´nomique (c’est-a`-dire de celle qui va succe´der au se´quenc¸age proprement dit). C’est ce qu’on appelle la bio-informatique, qui englobe toute une se´rie d’analyses the´oriques visant a` convertir les se´quences d’ADN en informations biologiques. Une part importante de cette discipline a trait a` l’identification des ge`nes par le biais d’un certain nombre de processus, depuis l’analyse des similitudes qu’ils pre´sentent avec des ge`nes de´ja` connus d’autres organismes, jusqu’a` l’emploi de mode`les informatise´s base´s sur les donne´es existantes. Viennent ensuite les pre´visions que l’on peut faire en matie`re de fonction biologique et de forme mole´culaire (ge´nomique structurelle), qui toutes deux ont un potentiel de de´veloppement important. Le fait de connaıˆtre l’ensemble des se´quences du ge´nome d’un germe pathoge`ne permet e´galement aux chercheurs d’e´tendre beaucoup plus qu’auparavant leur champ d’investigation du comportement de ces germes. De´sormais, au lieu d’e´tudier l’effet d’un traitement me´dicamenteux ou la diffe´renciation sur un ou deux ge`nes, il est possible d’e´tudier simultane´ment les variations pre´sente´es par l’ensemble des ge`nes au moyen d’une analyse transcriptionnelle globale. On peut e´galement examiner les protidogrammes, ou modifier ge´ne´tiquement le micro-organisme (transfection), of- frant ainsi un lien direct entre ces effecteurs biologiques et le phe´notype macroscopique. La mise au point des techniques ne´cessaires a` ces expe´riences est une conse´quence directe du de´sir d’exploiter les donne´es du se´quenc¸age ge´nomique. Il n’est pas difficile de pre´voir que la possibilite´ d’identifier et de caracte´riser le sche´ma d’organisation ge´ne´tique des germes pathoge`nes nous permettra d’identifier les e´le´ments essentiels a` leur de´veloppement et a` leur pathogene`se et de cibler nos efforts de recherche sur la production de nouveaux traitements. En ce qui concerne Plasmodium falciparum, on peut identifier les ge`nes et les prote´ines qui agissent lors de stades particuliers du cycle e´volutif de l’he´matozoaire, de´terminer leur roˆle au moyen de modifications ge´ne´tiques et utiliser ceux qui sont prometteurs pour la production de vaccins. Les voies me´taboliques parasitaires, absentes chez l’hoˆte, pourraient e´galement servir de cible a` des inhibiteurs puissants qui ne soient pas toxiques pour l’homme ; enfin, on pourrait cibler les me´canismes de la pharmacore´sis- tance des plasmodies et allonger ainsi la dure´e d’efficacite´ des me´dicaments existants. Le se´quenc¸age du ge´nome de P. falciparum offrira de nombreuses possibilite´s de recherche sur le paludisme, mais il ne s’agit la` que d’un de´but, l’objectif e´tant de transformer ces possibilite´s en traitements efficaces sur le terrain. Resumen La investigacio´n del paludismo en la era posgeno´mica El desarrollo de las tecnologı´as de secuenciacio´n del genoma a lo largo de los u´ltimos cinco an˜os ha generado una gran cantidad de informacio´n sobre secuencias, que ha culminado en el reciente anuncio de un borrador del genoma humano. Debido a su menor taman˜o, los genomas de microorganismos pato´genos se han prestado au´n ma´s a esas metodologı´as, y actualmente esta´n bien representados en el mundo de la geno´mica. 1434 Bulletin of the World Health Organization, 2000, 78 (12) Special Theme – Malaria Aunque no siempre sirven como microorganismos «modelo», la trascendencia de esos pato´genos para la medicina y la agricultura ha hecho que la explotacio´n de las bases de datos de secuencias se convierta en una gran prioridad. ¿Pero co´mo puede repercutir realmente la identificacio´n de largas cadenas de A, C, G y T en el objetivo de reducir la carga de morbilidad, en particular en los paı´ses en desarrollo? Hasta el momento, la secuenciacio´n de genomas se ha circunscrito en su gran mayorı´a al mundo desarrollado, donde los recursos y la infraestructura cientı´fica han permitido crear centros de secuenciacio´n muy productivos. No obstante, el uso de la informacio´n sobre las secuencias no tiene por que´ quedar limitado de ese modo, siempre y cuando haya recursos para formacio´n. El aspecto ma´s importante de la era posgeno´mica, es decir, la que comienza al completarse la secuenciacio´n, es probablemente el ana´lisis de las secuencias. Este campo, lo que se conoce como bioinforma´tica, comprende una serie de ana´lisis teo´ricos cuyo fin es convertir la secuencia de ADN en informacio´n biolo´gica. Una parte importante de esta disciplina implica la identificacio´n de genes mediante una serie de procedimientos, que van desde la determinacio´n de las similitudes con otros genes descritos anteriormente hasta el uso de modelos informa´ticos basados en los datos existentes. Posterior- mente se hacen predicciones sobre la funcio´n biolo´gica y la forma molecular (geno´mica estructural), cosas ambas que abren amplias perspectivas de desarrollo. El conocimiento de la secuencia completa del genoma de un pato´geno tambie´n permite a los cientı´ficos investigar el comportamiento de los micro- organismos con una base ma´s amplia. Ahora, en lugar de estudiar el efecto de un tratamiento farmacolo´gico o de la diferenciacio´n en uno o dos genes, es posible estudiar la variacio´n simulta´nea de todos los genes utilizando el ana´lisis transcripcional global. Se puede asimismo examinar la distribucio´n de las proteı´nas o modificar gene´ticamente el microorganismo (transfeccio´n), y establecer ası´ una relacio´n directa entre esos efectores biolo´gicos y el fenotipo general. La tecnologı´a para llevar a cabo esos experimentos es consecuencia directa del intere´s por explotar las secuencias geno´micas. Cabe prever que la capacidad para identificar y caracterizar la huella gene´tica de los agentes pato´genos ayudara´ a reconocer elementos clave del desarrollo y los mecanismos patoge´nicos de los microorganismos causantes de enfermedades, y a centrar nuestras investigaciones en el desarrollo de nuevos tratamientos. En el caso de Plasmodium falciparum, es posible identificar los genes y las proteı´nas que actu´an en determinadas etapas del ciclo de vida, verificar su funcio´n mediante te´cnicas de modificacio´n gene´tica, y utilizar candidatos prometedores en la produccio´n de vacunas. Las vı´as metabo´licas del para´sito que no esta´n presentes en el hue´sped son otra posible diana de inhibidores potentes no to´xicos para el ser humano; adema´s, se podrı´a interferir en los mecanismos de farmacorresistencia del para´sito, y alargar ası´ la vida eficaz de los medicamentos actuales. 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