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Apicomplexan Biology in the Post-Genomic Era

Objective

A. BACKGROUND

Apicomplexan protozoa cause more human deaths (Plasmodium, Cryptosporidium, Toxoplasma) than any other group of infectious agents and are also the most significant parasites of livestock (Eimeria, Cryptosporidium, Toxoplasma, Neospora, Theileria, Babesia). Control of apicomplexan diseases may be non-existent e.g. cryptosporidiosis, or severely compromised e.g. malarial drug resistance, and there is an ongoing urgent need to develop novel, sustainable therapies. Whilst the specific biology of each parasite is unique, recent research shows that Apicomplexans share many functions related to their interaction with the host, based on homologous proteins and therefore genes that have no known counterparts in other living organisms. Several key events and biochemical pathways are conserved across the phylum and these are potentially exploitable for novel therapies.

The genome sequences of many apicomplexans will soon be known and publicly available. Sequencing is almost complete for one strain of the malarial parasite Plasmodium falciparum, and underway for additional malarial strains and species and for Babesia, Cryptosporidium, Eimeria, Theileria and Toxoplasma. This will make it possible to carry out comparisons between genomes and to begin to link gene information to specific functions. Post-genomic technologies are highly relevant for apicomplexans, which have large genomes and complex life cycles that progress through many different stages, several of which interact directly with the host. These technologies will dictate a fundamental change in the way in which many studies are carried out. Rather than studying a handful of characterised genes or gene products, global analysis of mRNAs and proteins will be possible, enabling changes in gene expression to be followed without prior knowledge of gene sequence. Moreover, once mRNAs and proteins have been identified, the encoding DNA sequences will be attainable at a speed that makes the simultaneous analysis of thousands of gene products a practical possibility. Such techniques are open to automation allowing fast and reproducible characterisation of global gene expression and combined with powerful bioinformatics methods, are among the best-placed new technologies to take advantage of the growing wealth of genome data.

The large-scale application of post-genomic techniques to the study of this important group of parasites will inevitably be multi-centred since no single institution has the resources or expertise to analyse all the available parasite systems. Common approaches must be developed for the acquisition and processing of data to ensure comparability between institutions and to avoid duplication of effort. In addition, to realise the full potential of novel post-genomic technologies it will be essential that collation and dissemination of the data generated from individual parasites be co-ordinated.

Many groups within Europe are at the leading edge of apicomplexan research. However, it is generally the case that individual scientists centre their research on one genus, often on only one or two species. Co-operation between research groups is also predominantly focused on single genera. In the future, interaction between specialists working on different parasites will be essential to maximise the enormous potential of genomic and post-genomic studies. It would be extremely short sighted not to see that uncovering therapeutic targets in one apicomplexan could have broader potential utility against other members of the phylum. Different parasites are more or less suited to specific areas of research and comparative studies, at the level of single genes, have already proved extremely valuable to aid understanding of fundamental parasitic mechanisms, such as host cell invasion, and for the evaluation of drug and vaccine candidates.

Until a few years ago drug discovery for all human and animal disease (infectious or otherwise) was concentrated on a total of only a few hundred targets. In the post-genomic era, investigation of the multitude of new targets will not be manageable unless good models are developed to validate those targets. For the Apicomplexa, such models will only be generated from intense and co-ordinated research, which focuses on the underlying mechanisms of parasite physiology, biochemistry and host-parasite interaction. There will be pressure on the scientific community to commit itself to this process in order to develop, in collaboration with industry, new drugs and vaccines.

For the above reasons, the time is now right to develop and strengthen EU-wide links with the formation of a COST action to maximise possibilities for developing novel therapies against apicomplexan parasites in the post-genomic era. It is essential that scientists working on all of the major disease-causing apicomplexans co-operate and collaborate. In particular, the integration of experts in novel technologies (bioinformatics, microarrays, proteomics, reverse genetics) with specialists working on specific biological questions will be extremely beneficial and will exploit the wealth of expertise that exists within Europe.

Relationship of COST 857 to the activities of COST 820 and other COST Actions

COST Action 820 'Vaccines against Animal Coccidioses' (1994-2000) was concerned with the development of vaccines against economically important coccidia of farm animals, principally Eimeria, Cryptosporidium, Toxoplasma and Neospora. One working group within this Action (WG6) specifically addressed the biology of host-pathogen interactions and latterly a small number of scientists who work on Plasmodium and Theileria participated in two WG6 workshops. These were very successful and provided a unique opportunity for European scientists working on all the major apicomplexan parasites to meet and discuss various aspects of conserved biology. It was agreed that the most efficient means to achieve rapid progress towards understanding these sophisticated parasites would be to co-ordinate research on different members of the phylum. This would ensure that the most appropriate experimental systems are utilised, that the wealth of genome data is exploited, that novel post-genomic technologies are successfully applied and that the results of studies are rapidly disseminated and discussed. It was also clear that there were many more scientists within Europe who would be interested in joining such a forum, if it were established. Thus the idea for forming a new COST Action, which is concerned with the elucidation of Apicomplexan biology in the post-genomic era, was born.

Close co-operation with other COST Actions will be sought, in particular by joint workshops, exchange of publications and other common activities. In particular joint activities with the following COST Actions, both technologically and pathologically oriented, will be undertaken. This would comprise COST Action 853 on 'Agricultural Bio-Markers for Array Technology', COST Action 854 on 'Protozoal Reproduction Losses in Farm Ruminants' and COST Action 855 on 'Animal Chlamydiosis and its zoonotic implications'. Since apicomplexian pathogens affect both animals and men, also synergies with activities in the domain of medicine will be looked for and exploited.

B. OBJECTIVES AND BENEFITS

The main objective of the Action is to understand the fundamental biology of apicomplexan parasites, and the way they interact with the infected hosts, through the exploitation of genome sequence data and the application of post-genomics.

The major areas to be covered will be:

- Comparative genomics and bioinformatics
- Gene expression,
- Host-Parasite interactions such as invasion, formation of a replication-competent vacuole, parasite modulation of host cell function and survival, regulation of stage-differentiation
- Parasite biodiversity and population genetics

The driving force behind the Action will be the urgent need to identify potential drug targets and vaccine candidates. This, together with an enhanced understanding of their mode of action, should allow the development of novel, sustainable control strategies against the diseases caused by apicomplexan parasites. The benefits will include:

- Comparative analyses of apicomplexan genomes to give detailed knowledge of the classes of genes that are conserved across the phylum or restricted to certain genera.
- Analyses of apicomplexan global mRNA expression, leading to the definition of genes that are differentially regulated during parasite development or upon interaction with the host, or differentially expressed between defined parasite populations.
- The application of proteomics to apicomplexan parasites and comparison of proteomes to define proteins that are conserved across the phylum, restricted to specific parasites or localised to specialised sub-cellular organelles and compartments.
- The development of common approaches for processing and displaying post-genomic data to ensure compatibility between different research groups and thus facilitate rapid data exchange.
- The development of common approaches towards understanding apicomplexan gene function, utilising the most amenable and appropriate experimental systems.
- The identification of apicomplexan-derived proteins capable of interacting/interfering with normal host cell function (for example, signal-transduction pathways)
- The elucidation of key parasite processes and metabolic pathways.
- The identification of potential drug targets and vaccine candidates.

C. SCIENTIFIC PROGRAMME

GENE EXPRESSION

Underpinning the biology of all apicomplexans is their ability to regulate their life cycles, which demands tight control of gene expression. Analysis of gene expression is thus central to studying all fundamental biological processes in the apicomplexa. DNA microarrays and novel high-throughput, proteomic technologies are now lowering the barrier to functional genomic studies. Of equal importance is the capacity of ever more powerful bioinformatics to manipulate, store, retrieve and analyse the tremendous quantity of data generated from these studies.

Research tasks

- Analyse changes in mRNA and protein expression during stage-differentiation
- Compare gene expression between drug-resistant strains and wild-type progenitors
- Analyse changes in parasite and host gene expression during infection
- Identify potential drug targets and virulence factors

MOTILITY & THE CYTOSKELETON

Apicomplexans display a unique form of motility, which is dependent on parasite actin and myosin and which powers invasion of host-cells. Apicomplexan myosins form a unique class (myosin XIV), which may be different enough from host myosins to be drug targets. Parasites adhere to and gain purchase on substrates, such as cell surfaces, so there must be a system to transmit motion through the parasite membrane. Apicomplexans have a second cytoskeletal apparatus, the sub-pellicular microtubules, which are important in Plasmodium invasion although the nature of their role is unclear.

Research Tasks

- Identify apicomplexan cytoskeletal components (actin-binding proteins, myosins, tubulins, intermediate proteins etc.) and compare with those of the host.
- Elucidate the mode of action of the motor(s) that power motility and invasion
- Identify factors that transmit the mechanical force generated by the actinomyosin system across the plasma membrane.
- Evaluate the contribution of other cytoskeletal components to the invasion process

INVASION OF HOST CELLS

Apicomplexans show wide diversity in their ability to develop within host cells in vivo. It is likely that successful invasion requires the correct combination of host cell receptor and parasite-encoded ligand. Invasion is tightly coupled to the release of proteins from parasite secretory organelles. Microneme proteins are secreted first, are crucial for parasite binding to the host cell and may also be involved in linking parasite actinomyosin across the plasma membrane to the surface. Rhoptry proteins are secreted during invasion and may contribute to the formation of pores that allow the entry of small molecules into the parasitophorous vacuole. They are also implicated in the binding of malarial merozoites to erythrocytes, and in phenotypic antigenic variation. In this last case, switching of the expression profile leads to a change in the invasion properties of merozoites, which may help the parasite adapt to changes in the host cell environment and to evade the host immune response.

Research tasks

- Identify protein repertoires of the secretory organelles using proteomics.
- Identify host cell receptors recognised by apicomplexan parasites.
- Investigate proteolytic processing of organelle proteins and characterise the proteinases responsible.
- Investigate the modulation of expression of parasite ligands in response to specific immune pressure, or to variation in the host receptors that are present.

INTRACELLULAR SURVIVAL

After invasion, the parasite resides within a replication-competent parasitophorous vacuole (PV) from which it modulates host cell components, scavenges nutrients and undergoes replication. Dense granule proteins are important in structural modification of the PV and for intracellular survival. Translocation of membrane proteins into the PV is likely to be a common feature of the Apicomplexa, and they are likely to have evolved specific machineries for protein secretion and insertion across multiple membranes. This offers a rich territory to explore novel mechanisms of protein-membrane interactions including post-translational bilayer insertion. The Plasmodium-infected erythrocyte is an excellent model because the host cell is devoid of internal compartments and incapable of de novo protein and lipid biosynthesis. Parasite proteins transported out of the PV into the erythrocyte, including the erythrocyte plasma membrane, contribute to the pathogenesis of malaria and to nutrient acquisition thus allowing growth and development of the parasite within its host cell.

Research Tasks

- Dissect the mechanism of post-secretory membrane insertion
- Investigate the role of dense granule proteins using a combination of biochemical, reverse genetic, proteomic and immunological approaches.
- Fractionate infected erythrocytes to prepare soluble and membrane fractions from the PV and the erythrocyte
- Use proteomics and reverse genetics to identify the repertoire and investigate the function of parasite proteins within each of the above fractions.

HOST CELL MODULATION

The interaction between parasite and host is crucial to the outcome of infection. Co-adaptation, generated during co-evolution has resulted in close interaction between the invader and the invaded. Thus, as well as investigating the impact that the host has on the parasite, it is equally important to analyse the extent to which host cell functions are modulated by the parasite. Many Apicomplexa

persist within the host or host cell and inhibition of host-cell death is a possible mechanism to support persistence. For Theileria and Toxoplasma, mechanisms have been identified by which apoptosis of the host cell is inhibited in a parasite-specific way. Each parasite targets different key factors in the signal transduction pathways that govern programmed cell death. Theileria goes one step further and also induces continuous proliferation, clonal expansion and spread of the leukocytes it infects. Interference with immune recognition is another mechanism by which persistence might be enhanced. Toxoplasma is able to actively down-regulate MHC class II antigen expression, thereby inhibiting antigen presentation to CD4+ T-cells.

Research tasks

- Investigate mechanisms and parasite factors involved in inhibition of apoptosis
- Investigate mechanisms and parasite factors involved in interference with immune recognition

BIODIVERSITY

A key question that underlies our understanding of the epidemiology and pathogenicity of apicomplexan parasites is the extent to which biological and genetic diversity exists within their populations. Diversity occurs at several levels, most notably within Plasmodium falciparum. Within individual cloned parasite lines differential expression from multi-gene families, leads to antigenic switching of variant antigens. Within the population, polymorphism exists at many loci and appears to be an important factor in transmission, since genetic diversity increases in areas of high parasite density. In contrast, extensive analyses of Toxoplasma isolates indicate that the genus comprises a limited number of clonal lineages that are directly correlated with their virulence in mice. For other apicomplexans, such as Neospora and Eimeria, the epidemiology is poorly understood and little is known about genetic diversity. Understanding how biodiversity may influence virulence and transmission efficiency is an important and often neglected aspect of developing control strategies. For Cryptosporidium, where there is no drug treatment available, studies into genetic diversity are crucial to identify markers for epidemiological studies in order to understand and subsequently break transmission routes since this is the only viable means of controlling the spread of disease.

Research Tasks

- Analyse parasite genetic diversity using tools such as random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLP).
- Determine parasite population genetic structures for Toxoplasma, Eimeria, Neospora and Cryptosporidium.
- Develop novel and discriminatory genotyping systems for these parasites and apply these to improve our understanding of their epidemiology.

D. ORGANISATION

To meet the scientific objectives and accomplish the research tasks, close collaboration and interaction between participants in the Action will be essential. Many of the research tasks will only be achieved by having input from specialists working with a specific parasite system, together with that of experts in novel technologies and bioinformatics. Co-ordination of research tasks will be channelled primarily through Working Groups (WGs), which will hold regular, specialised meetings in which participants will discuss their latest data. Short-term scientific missions (STSMs) will be an important part of the Action, especially in the early phase, as will the Action website, which will be established in the first year of the Action.

The Action will be organised according to COST400/01 'Rules and procedures for implementing COST actions'. It will be managed by a Management Committee (MC), which will convene and elect a chairperson and vice-chairperson. The MC will be responsible for ensuring the rapid dissemination of information to all participants, including the establishment and maintenance of the Action web site, the organisation of an annual workshop (to include participants from all WGs) and the facilitation of STSMs. The MC will also compile an annual report of progress achieved by the Action, which will consist of contributions from the MC, the WGs and from individual Action members.

The MC will identify and elect chairpersons and vice-chairpersons for each of 6 WGs:

- WG1: Functional genomics and bioinformatics
- WG2: Gene expression
- WG3: Motility, cytoskeleton and invasion biology
- WG4: Intracellular survival and host cell modulation
- WG5: Biodiversity and population genetics
- WG6: Development of novel targets for disease control

WG chairpersons will be responsible for ensuring that the goals of each WG are achieved and that there is appropriate interaction between the Groups. WG chairpersons will report back to the MC once each year at the MC meeting and will channel requests for holding WG meetings through the Action chairperson. Working documents from the WG, such as abstracts of papers presented, minutes, conclusions, recommendations etc., will be made available to all members of the Action through the web page and by e-mail.

E. TIMETABLE

Because of the nature and complexity of the topic, the Action lasts for a total of five years.

In the early years of the Action, whilst genome sequencing of Apicomplexan parasites is still ongoing, WG1 will be crucial to ensure good co-ordination of comparative genome research. Similarly, WG2 will be essential for establishing rapport and collaboration between laboratories investing in apicomplexan post-genomics and to ensure that data generated from different projects is compatible and can be exchanged and, where appropriate, integrated. In practice, there will be a high level of cross-fertilisation between WG1 and 2, and a number of joint meetings will be held between them, the first being within six months of the start of the Action. WG3, 4 and 5 will be operational from the beginning of the Action and will each focus on specific areas of fundamental apicomplexan biology, in order to concentrate and focus on the research tasks associated with these areas. However, all of these groups will need to interact with WG1 and 2 and exploit the data generated by genomic and post-genomic analyses. Although WG6 will be established at the outset, this will become most important later in the Action, as basic knowledge of the unique biology of apicomplexans accumulates and potential targets are identified. Membership of the WGs will be flexible, so individuals will contribute to and benefit from each WG according to their own research programmes. All WGs will meet each year at an annual workshop, which will be crucial for formulating a clear overview of progress and prioritising future research tasks.

F. ECONOMIC DIMENSION

The following COST countries have actively participated in the preparation of the Action or otherwise indicated their interest:

France
Germany
Ireland
Italy
Spain
Sweden
Switzerland
The Netherlands
UK

On the basis of national estimates provided by the representatives of these countries, the economic dimension of the activities to be carried out under the Action has been estimated, in 2002 prices, at roughly Euro 15 million.

This estimate is valid under the assumption that all the countries mentioned above, but no other countries will participate in the Action. Any departure from this will change the total cost accordingly.

G. DISSEMINATION PLAN

Abstracts of work presented at WG meetings and at the annual workshop will be published both in print (abstract books) and be made available to Action members by an e-mail list and to the rest of the scientific community via the web site. Selected papers from annual workshops will be submitted for peer-review to scientific journals, and given at other international conferences. Papers from at least one annual meeting will be submitted, for full peer review, to the International Journal of Parasitology, for consideration as a special themed issue on Apicomplexan genomics and biology. Annual reports of the Action will be prepared and submitted to the Commission in order to document progress of the Action. The web site will be an important focus for dissemination of results, posting of methods and protocols, advertising meetings, calling for abstracts and displaying abstracts & papers. It is anticipated that most of this will be done through an open web site, though the MC may consider password protection for working documents.

Coordinator

N/A