1. The Diversity of Plasmodium and Other Haemosporidians: The Intersection of Taxonomy, Phylogenetics and Genomics

Ellen S. Martinsen and Susan L. Perkins

As important agents of disease, a great deal of research has been focused on the malaria parasites. Yet, the species that infect humans represent only a small fraction of the diversity of the malaria parasites, and future genomics projects on closely related parasite species with diverse life histories and other key traits will likely serve as important steps to a better understanding of malaria in humans as well as the biology of the group as a whole. Before comparative studies can be performed, however, a robust phylogeny or understanding of the evolutionary history of the group must be in place. The history of the discovery and classification of the malaria parasites has been a long and sometimes circuitous one and while new species surely remain to be discovered, it is important that we continue to adhere to taxonomic principles. Recent advances in molecular systematics have both challenged and enlightened our understanding of the diversity and evolution of these organisms, though the development of new molecular markers still remains a challenge and genome sequencing faces unique hurdles.

2. The Apicomplexan Genomic Landscape - The Evolutionary Context of Plasmodium

Jeremy DeBarry, Segun Fatumo and Jessica C. Kissinger

The apicomplexan genome is quite different from the typical eukaryotic genome that graces our textbooks and the majority of literature on the topic. It is small (8.5 - 63 Mb), compact, nearly devoid of transposable elements, and lacking any significant synteny outside of genus- and in some cases, family-level classifications. The nuclear genome has experienced significant gene loss, a characteristic of parasitic organisms. Numerous genes have entered the nuclear genome via de novo creation (through recombination, accumulated mutation or gene duplication and subsequent divergence), intracellular gene transfer from organellar and algal endosymbiont genomes and lateral gene transfer from bacteria and elsewhere. Nuclear genome data are currently available for 18 species within 8 genera; namely Cryptosporidium, Eimeria, Sarcocystis, Neospora, Toxoplasma, Babesia, Theileria and Plasmodium. Analyses have revealed dynamic genomes that share a remarkably small percentage of "core" genes comprising 11-23% of total gene content, plus large repertoires of lineage- and species-specific genes. Chromosome number and the organization of genes and other genomic features along the chromosome vary across the phylum. Plasmodium, the causative agent of malaria, is typical in that genomes from the same genus are highly syntenic, but it differs in that subtelomeric regions host the majority of lineage- and species-specific genes. Tools to perform comparative analyses within Plasmodium and across the Apicomplexa are available at EuPathDB.org and other sites.

3. Plasmodium Genomics and the Art of Sequencing Malaria Parasite Genomes

Jane M. Carlton, Steven A. Sullivan and Karine G. Le Roch

It may be a cliché to state, but obtaining the genome sequence of an organism is one of the most important - if not the most important - step towards interrogating its biology. The first two malaria parasite genome sequences (Plasmodium falciparum and the rodent model Plasmodium yoelii yoelii) were published in 2002 after more than half a decade of intense sequencing, assembly gap closure, and sequence annotation. Since then, reference genomes of several more Plasmodium species have been generated, with an emphasis on malaria parasites that infect humans due to their global health importance. With the recent transformation in technologies available for the rapid and cheap production of genome sequence data, an explosion of P. falciparum genomes from a wide variety of geographical locations has started to appear, and with it all of the computational issues of large dataset manipulation, storage and analysis. We begin this chapter with a discussion of sequencing technologies, from Sanger sequencing through to current next generation sequencing platforms, to lay the foundation for many of the studies that are presented in this book. Next we describe the characteristics of a typical Plasmodium nuclear genome (with reference to those species of malaria that infect mammals), with a brief mention of the extranuclear apicoplast and mitochondrial genomes also found in the parasite. Finally, we outline how comparative genomics - literally comparing genomes within and between species - has been used as a powerful tool to elucidate malaria parasite biology and evolution..

4. Genome Diversity and Applications in Genetic Studies of the Human Malaria Parasites Plasmodium falciparum and Plasmodium vivax

Sittiporn Pattaradilokrat, Jianbing Mu, Philip Awadalla, and Xin-zhuan Su

The publication of the genomes of the human malaria parasites Plasmodium falciparum and Plasmodium vivax has provided the foundation for developing high-throughput methods for systematic investigation into genomic variability in parasite populations. Various tools for discovering genome-wide polymorphisms and genotyping, including DNA microarrays and high-throughput sequencing, are readily available for the malaria research community. Elucidating the genetic diversity within and between populations forms the basis of population genetic analysis that not only will aid in deciphering the evolution and adaptation of the parasites but also has important implications for the development and implementation of therapeutic interventions. Here we summarize the recent advances in the studies of genome variation, population genetics, and the development of high-throughput genetic tools for investigating gene function in P. falciparum and P. vivax.

5. Functional Genomics of Plasmodium Parasites

Zbynek Bozdech and Peter R. Preiser

Over the last decade, functional genomics of Plasmodium species uncovered many new insights into gene and protein expression that characterizes both the growth and multiplication of malaria parasites in their natural hosts. Genome- and proteome-wide technologies provided new insights into the physiology of all major developmental stages as well as many regulatory mechanisms that control progression of the complex Plasmodium lifecycle. It is now clear that the morphological transition between the developmental stages is linked to broad changes in gene expression that are regulated by several types of regulatory mechanisms including specific transcription factors and chromatin remodeling machinery as well as global DNA/RNA metabolic processes. At the protein level, translation rate, protein turnover and posttranslational modifications appeared to be important contributing factors to the lifecycle regulation. In addition, it is clear that Plasmodium parasites are able to transcriptionally and post-transcriptionally respond to some external stimuli. Both the broad regulation of gene/protein expression during the life cycle and the transcriptional/translational plasticity likely reflect a robust adaptation of malaria parasites to their natural hosts and thus their ability to spread amongst many populations throughput the world. Better understanding for these dynamic properties of the Plasmodium genome will provide the tools for the design and development of new malaria control strategies.

6. Plasmodium Experimental Genetic Crosses

Lisa C. Ranford-Cartwright, Karen l. Hayton and Michael T. Ferdig

Experimental genetic crosses mimic the sexual reproduction process, and accompanying genetic recombination, that occurs between individuals of the same species during natural transmission. Experimental crosses performed using rodent and human species of Plasmodium have been used to link phenotype and genotype for a variety of traits, and have been particularly useful for understanding phenotypes for which no obvious candidate genes are known. In addition, analysis of experimental crosses has provided insights into the frequency and types of recombination that occur. Some biological traits are explained by inheritance of single genes, whereas several loci, known as quantitative trait loci (qtl), contribute to a "complex trait". Linkage analysis of experimental genetic crosses of Plasmodium falciparum have identified parasite loci contributing to resistance to a number of antimalarial drugs such as chloroquine and quinine, as well as loci controlling the ability of parasites to invade erythrocytes of different primate species, the ability to infect mosquitoes, and intraerythrocytic growth rates. Genetic mapping can also identify genomic regions associated with the control of gene expression (expression qtl or eQTL). Genetic crosses and genetic mapping continue to play a significant role in our understanding of malaria parasite biology, transmission, and drug resistance.

7. Regulation of Gene Expression

Kirk W. Deitsch and Ron Dzikowski

Malaria parasites represent an evolutionary lineage quite distant from the model organisms within the crown group of eukaryotes on which much of our current knowledge of basic biological mechanisms is based. With regard to regulation of gene expression, studies in Plasmodium have identified numerous aspects that are conserved in higher eukaryotic organisms; however, there are also several characteristics that appear to be quite different, and others that remain very poorly understood. In this chapter we review what is known about the many steps involved in regulating gene expression, from transcription initiation, through mRNA processing to protein synthesis. A better understanding of how specific gene expression patterns are controlled will shed light on such important features of parasite biology as antigenic variation, sexual differentiation, and cell cycle progression, to name a few.

8. Invasion of Host Red Blood Cells by Malaria Parasites

Amy Kristine Bei and Manoj Theodore Duraisingh

The first challenge faced by the invasive merozoite form of the Plasmodium parasite when released into the bloodstream is to invade and establish itself within a host erythrocyte. Following hepatic schizont rupture, merozoites are released into the circulation and must successfully locate, bind to, and invade an erythrocyte lest they are cleared by the immune system. Subsequent cycles of erythrocytic egress, invasion and growth result in the escalation of parasitemia, often associated with clinical symptoms. The parasite has developed molecular strategies that allow it to invade the host cell while avoiding the attack of the immune system and in response to diversity of the receptor repertoire of its human host. Depending on the species, there is a limited window of time in which the merozoite remains viable for invasion. The invasive potential of the merozoite is influenced by three levels of host cell selection: 1) selection between host species (species specific tropism), 2) selection among individuals within a host species (erythrocyte receptor diversity), and 3) selection of subpopulations within an individual (age dependent invasion). The host-parasite interactions that allow for efficient host cell selection and invasion of erythrocytes will be the topic addressed by this chapter.

9. Host Cell Remodelling and Protein Trafficking

Silvia Haase, Hayley E. Bullen, Sarah C. Charnaud, Brendan S. Crabb, Paul R. Gilson and Tania F. de Koning Ward

Inside their respective vertebrate hosts, Plasmodium spp spend most of their life residing within hepatocytes and erythrocytes, with large-scale infection of the latter responsible for the clinical symptoms associated with malaria. These parasites extensively remodel these host cells for a variety of purposes relating to both pathogenesis and maintaining growth. Remodelling of the erythrocytic stage has been most intensively studied in P. falciparum and is the subject of this chapter. To help remodel their hosts these parasites export hundreds of proteins into the erythrocytic compartment. This principally alters the architecture of the erythrocyte, rendering the host membrane more permeable to solutes and nutrients, and also increasing the rigidity and adhesiveness of the infected erythrocyte. Moreover, because erythrocytes lack a secretory apparatus, the parasite must also export many additional proteins to help traffic other proteins to their correct destination within the host cell. The functions of some of these exported proteins will be discussed as will recent progress that has been made in unravelling how exported proteins gain access to the host compartment.

10. Dissecting Mosquito-parasite Interactions Through Molecular Biology and Biochemistry: Genomic, Proteomic and Glycomic Analyses

Lindsay A. Parish, Lindsey S. Garver, David R. Colquhoun, Ceereena Ubaida Mohien, Elizabeth Weissbrod and Rhoel R. Dinglasan

Our understanding of the malaria parasite-mosquito vector host interactions has grown significantly in the post-genomic era. The sequencing of the Anopheles gambiae genome and functional genomics has revolutionized our approaches at examining the role of the mosquito vector host in malaria transmission. Thirteen other anopheline species (including the major vectors for Plasmodium vivax) are hoped to be completely sequenced within the next decade. Now, more than ever, there is a heightened research focus on Vector Biology and the transmission stages of the malaria parasite through the mosquito, from gametocytes to sporozoites, with the underlying aim of developing novel transmission-blocking vaccines/interventions. This chapter provides an overview of what is currently known at the genomic, proteomic and glycomic levels, about the role of different molecules, tissue compartments and molecular pathways in the mosquito vector and the ensuing strategies that have evolved, which can potentially impact malaria parasite transmission.

11. The Malariologist's Molecular Toolbox

Alexander G. Maier

This chapter will summarize many of the recent advances in experimental techniques that have greatly increased our ability to address fundamental questions of parasite biology. Many of these advances are improvements on basic transfection techniques through new generation vectors and regulatable expression systems; however, advances in the analysis of gene expression, microscopy and the analysis of field samples have also proven very fruitful.