Molecular Approaches to Malaria

Historical records, some >3,000 years old, attest to the antiquity of the disease malaria. Using a light microscope, Alphonse Laveran noticed some crescent-shaped bodies among the red blood cells that were almost entirely transparent, save for some pigment inclusions. He recognized that these bodies were alive, and that he was looking at an animal parasite, not a bacterium or a fungus. Subsequently, he examined blood samples from 192 malaria patients: in 148 of these, he found the telltale crescents. Where there were no crescents, there were no symptoms of malaria. He named the parasite Oscillaria malariae and communicated his findings to the Societé Medicale des Hopitaux on 24 December 1880. Although malaria can be induced in a host by the introduction of parasites (called sporozoites) through the bite of an infectious female mosquito, the parasites do not immediately appear in the blood. This was surprising in view of the fact that in 1903 Fritz Schaudinn claimed to have seen sporozoites directly invade erythrocytes. All human malarial agents (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae) are transmitted through the bite of an infected female anopheline mosquito when she injects sporozoites from her salivary glands during blood feeding. Investigations at the molecular level of enzyme structure, gene sequences, chromosomal arrangements, and transcriptional control will permit an uncovering of the adhesive molecules that mediate cell-cell interactions, determine the mechanisms of protein trafficking, and identify putative drug targets and vaccine candidates.

The Plasmodium Genome Database was introduced in 2000, in response to emerging needs of the malaria research community for access to genomic-scale datasets. In its earliest manifestations, prior to completion of the Plasmodium falciparum genome sequence and curated annotation, PlasmoDB focused on automated analysis of available sequence data, enabling researchers to identify draft sequences for specific genes of interest, even in the absence of the surrounding genomic context. The availability of an effectively complete P. falciparum genome sequence has stimulated a wide range of functional genomics research, and PlasmoDB has endeavored to keep pace with these studies, providing access to the underlying datasets, and allowing a variety of integrative queries, e.g., finding all genes for which both transcript and proteomics data suggest expression in gametocyte stage parasites. PlasmoDB will continue to integrate new datasets as they emerge, developing tools of interest to the malaria researcher and facilitating the discovery of new diagnostics, drugs, and vaccines. PlasmoDB provides the user with a variety of analysis tools for examining and extracting information from the genome and predicted proteome, using BLAST, electronic PCRs, defined motif searches, and tools for the analysis of microarray and proteomics data. Gene Pages provide an encyclopedic view of the Plasmodium genome, where it is possible to look up all information about a specified gene. Plasmodium parasites may be gleaned by browsing the genome in Sequence View mode, and information on specific genes may be obtained from the various Gene Pages.

This chapter briefly outlines the cellular architecture of the blood stages of plasmodium falciparum, for which there is a considerable body of molecular data. The asexual forms, which dominate the relationship between the human host and parasite in terms of time spent and pathological impact, are traditionally classified by their detailed light microscopic features as seen in Giemsa-stained blood films. As the parasite continues to grow and differentiate, it exports membranes and other structures into the surrounding RBC. In P. falciparum, these include membranes-the clefts of Maurer, circular clefts, small vesicles, and dense protein-containing projections from the surface of the RBC, termed knobs. Similar structures exist in other species, such as Schüffner’s dots (cleft-like membranes) in Plasmodium vivax and caveolae analogous to knobs in Plasmodium knowlesi and Plasmodium cynomolgi, although these are invaginations of the membrane rather than protrusions. In this chapter, the authors follow the terminology of Atkinson and Aikawa (1990) to distinguish the circular clefts from the (short) clefts of Maurer, avoiding the name term tubulovesicular network (TVN) because of its alternative usage to describe the complete system of exported membranes. The brief description of the morphological changes occurring within the parasite during the asexual and sexual erythrocytic periods indicates the great complexity and continual modulation of cellular form typical of P. falciparum, a statement which can be repeated for the other species of malaria parasite and other stages not considered here.

This chapter reviews historical and technical issues, achievements to date, and future possibilities with respect to transformation of Plasmodium falciparum blood stages. Despite the relatively low efficiency of the P. falciparum transfection system, it was sufficient for both gene targeting and transgene expression approaches to be developed and utilized to analyze gene function in this organism. Given the inability to transfect linear DNA and achieve fast integration into the P. falciparum genome, a key to the success of P. falciparum transfection is the somewhat mysterious ability of transfected plasmids to replicate episomally in parasites. Transfection of P. falciparum blood stages, using transient transfection with the reporter genes chloramphenicol acetyl transferase and luciferase, has been an important tool in the identification of functional characterization of a number of transcriptional control elements from this organism. Plasmid transfection vectors designed to express transgenes have been used extensively in P. falciparum, and this has been useful in a wide variety of studies. A major advance in understanding the molecular dynamics of cellular systems has derived from elegant studies in which the gene encoding green fluorescent protein (GFP) is appended to a gene encoding a protein of interest and transfected into a living cell. Since the development of transfection for Plasmodium berghei and P. falciparum, the need for an inducible expression/conditional knockout system was recognized as a crucial next step to allow the functional analysis of many blood stage genes.

This chapter explains how such data, when combined, can give a comprehensive overview of the transcriptome of the malaria parasite throughout its life cycle. These data are expected to lead to the identification of key Plasmodium-specific regulatory targets, a fundamental step for the development of new antimalarials. Gene expression throughout the cell cycle is usually regulated by complex interactions between DNA regulatory motifs and transcription factors that bind to these motifs, as well as regulation by distinct chromatin structures. Parasite isolates possess a high degree of diversity within their var genes because of duplications, deletions, or recombination events that provide the parasite with a huge repertoire of antigenic determinants. Before the complete release of the Plasmodium falciparum genome sequence, several genomic efforts were accomplished to investigate the transcriptome of the malaria parasite. The first large-scale expression study of P. falciparum was accomplished with a DNA microarray platform constructed using a nuclease-generated genomic library. With the completion of the P. falciparum genome, two transcriptional analyses covering the entire genome were published. Both techniques allow effective and complete genome design. The 70-mer oligonucleotide array study was confined to a high-resolution analysis of the erythrocytic cell cycle. Gene expression in parasitic protozoa has shown itself to be unique when it comes to mechanisms of regulation. For instance, Leishmania and other members of the Trypanosomatidae have polycistronic transcription with maturation of their mRNA by transplicing events.

Proteome-wide studies of malaria have also lent a great deal of insight into protein-protein and protein-drug interactions, subcellular localization, functional characterization of unknown genes, and mechanisms of posttranscriptional regulation through comparisons with mRNA expression profiles. The work outlined in this chapter focuses on bottom-up approaches to study the malaria proteome. In October 2002, the genome sequence of Plasmodium falciparum was released, paving the way not only for genome-wide studies of mRNA but also for protein profiling. The accumulation of data collected from large-scale profiling experiments has also demonstrated ways that proteome data can aid the annotation of recently sequenced Plasmodium genomes. The P. falciparum genome showed markedly different properties from any other organism, namely, that it is the most A-T-rich genome sequenced to date, making the bioinformatic prediction of gene products from the genome sequence difficult based heavily on knowledge from other organisms. While proteome-wide studies of whole-cell lysates lend a great deal of information about an organism from a holistic perspective, it is equally informative to combine cell fractionation methods with high-throughput protein identifications to define the proteome of a particular subcellular location or organelle. Most significantly, correlating mRNA and protein expression profiles for individual genes revealed particular genes and families of functionally related genes that appeared to observe similar patterns of mRNA and protein accumulation.

The central theme of this chapter is the molecular evolution of species of the genus Plasmodium—the experimental methods, conclusions, and confounding elements peculiar to Plasmodium. While one section of the chapter is a brief summary of studies on the evolutionary history of Plasmodium and related organisms, another is a summary of the molecular population genetics of contemporary world populations of the two most virulent human malaria parasites. In addition to humans, Plasmodium parasites infect a range of vertebrate hosts, including birds, lizards, rodents, and nonhuman primates. Malaria has also been found in African rodents, and many of the Plasmodium species have been adapted to laboratory mice and rats and used as models for human malarias. Plasmodium ovale and P. malariae cause the rarest and least virulent forms of malaria. Population studies of P. falciparum and P. vivax, however, have already yielded unexpected results, paradoxical conclusions, and questions calling for additional research. Genome-level research has made it possible to explore malaria evolution in greater depth and rigor to help complete the picture. These changes have come through malaria parasite genome projects (for P. falciparum, P. vivax, P. yoelii, and P. reichenowi) and their associated databases, access to automated DNA sequencers in most modern laboratories, an increasingly sophisticated understanding of the unusual molecular biology and evolution of the parasites, and improvements in methods of statistical inference from DNA sequence differences within and among populations.

This chapter provides an overview of the dynamic nature of malaria from the perspective of the development of merozoites within an infected erythrocyte and the release of infectious merozoites, through the initiation and completion of the reinvasion process. The chapter encompasses discoveries or observations obtained through studies of different species of Plasmodium, which together have greatly aided and refined our understanding of these events. These species include not only the human malarias Plasmodium falciparum and P. vivax, but also the simian malaria P. knowlesi, the chimpanzee malaria P. reichenowi, bird malarias such as P. elongatum and P. gallinaceum, and the rodent malarias, principally P. yoelii and P. berghei. Malaria merozoites have a plasma membrane and the basic cellular machinery of typical eukaryotic cells, including a nucleus, endoplasmic reticulum, Golgi network, ribosomes, and mitochondria. As a merozoite begins to invade an red blood cells (RBCs), an internal membrane-lined invasion pit develops. The whole process of merozoite invasion can be divided into three or four distinct phases with a number of ultrastructural alterations and molecular events attributed to each phase, with an untold number of others likely to be discovered in the future. Merozoites must first be released from the wornout, hemoglobin-depleted, and extensively altered erythrocyte that hosted their development. Although many proteins have been identified in the spheroidal dense bodies of Toxoplasma gondii, only a few proteins besides ring-infected erythrocyte surface antigen (RESA) have been located in the dense bodies of Plasmodium.

The intense interest in sporozoite biology has been richly rewarded by the progressive unravelling of the truly remarkable journey undertaken by such a simple cell. The diversity and elegance of the strategies employed are only now being revealed. The key new comprehensive technologies underpinning these advances in our understanding includes transgenic technologies; Targeted gene disruptions; Microarray methods; Proteomic analyses. The sporozoite is one of three invasive stages in the malaria life cycle. The others are the ookinete and the merozoite. Sporozoite formation is the final step of differentiation of the oocyst. Sporozoite-infected epithelial cells can be expelled from the midgut wall into the lumen of the gut in a manner highly reminiscent of the time bomb theory of ookinete-midgut interaction. The salivary glands present a significant barrier to sporozoite development. Only sporozoites isolated from salivary glands confer significant sterile protection. Sporozoite maturation correlates with a dramatic increase of gliding motility, a unique form of substrate-dependent locomotion, and infectivity to the mammalian host. Sporozoites glide extensively through the avascular dermis until they reach a capillary. Having penetrated through the basal side of the endothelial cell layer, a proportion of sporozoites invade blood vessels and get carried away by the blood flow, whereas others actively enter lymph vessels or remain as residual sporozoites in the skin tissue. The recognition of the final target cell would then switch the parasite’s program from transmigration to productive entry by simultaneous formation of a parasitophorous vacuole.

Gametes and their precursors, the gametocytes, are the very special cell types that Plasmodium developed in the course of evolution to accomplish such key steps in its life cycle and are the subject of this chapter. In the main, cellular and molecular aspects of gametocyte and gamete formation will be covered here. Plasmodium gametocytes originate in the vertebrate host. Asexual multiplication in the bloodstream provides Plasmodium with a novel environment for its propagation but also extends the period available for sexual differentiation and transmission. In P. falciparum, sibling parasites derived from individual, isolated schizonts were analyzed in different studies by morphology and with specific antibodies distinguishing between gametocytes and asexual parasites or male and female gametocytes. Malaria parasites typically produce more female than male gametocytes, but they evolved the ability to modify gametocyte sex ratio during an infection. Sexual dimorphism clearly appears at the ultrastructural level and it is accompanied by expression of sex-specific molecular markers. Ultrastructural studies have identified secretory vesicles with an unknown osmiophilic content that are more abundant in macrogametocytes and are exocytosed within minutes of gametocyte activation, releasing their content into the parasitophorous vacuole. During microgametogenesis, the repeated rounds of replication and mitosis seem to proceed independently and uncoupled from some of the usual cell cycle checkpoints; as a result, DNA synthesis goes to completion even when mitosis is blocked by nocodazole, a microtubule-destabilizing drug, or by azadirachtin, a plant limnoid that inhibits exflagellation by interfering with mitotic spindles and axonemes.

In erythrocytes, Plasmodium falciparum has no obvious energy stores. Glucose storage forms such as amylopectin and mannitol identified in other apicomplexan parasites are not reported in P. falciparum. The P. falciparum mitochondrion maintains a transmembrane potential gradient that is essential for survival and is a target for the antimalarial atovaquone; however, it is not used for aerobic glycolysis in asexual-stage parasites. Large quantities of lactic acid produced in the vicinity of hypoxic host tissue may impair function of host cells. There are, therefore, two independent reasons for targeting glycolysis in the postgenome era: first, to kill parasites by identifying new inhibitors and eventually developing novel drugs, and second, to decrease use of glucose and output of lactic acid in those regions where there are many parasites that could compete with host tissues for glucose and add to the problem of disposal of lactate. Increased metabolic activity of Plasmodium-infected red blood cells is accompanied by the appearance of glycolytic enzymes with properties distinct from host red blood cell enzymes. The most obvious way for inhibitors to target the action of glycolytic enzymes is by blocking their catalytic sites, which allows substrate analogs to be used as probes. Glucose transport may be a promising target on theoretical grounds; Lactate dehydrogenase (LDH) has received the most attention so far in terms of rational drug development. Glycolysis in P. falciparum may contribute to disease pathogenesis by competing for glucose in host tissues, lending added impetus to discovering ways of inhibiting this key pathway.

This chapter provides an overview of the mitochondrion in malaria parasites in the light of what has been learned from the genome sequence. Historically, functions of the mitochondrion in malaria parasites were unclear because of its sac-like appearance and paucity of cristae. Prior to successful culture of Plasmodium falciparum, work on mitochondrial biochemistry also suffered from contamination with host components. The need to be aware of host mitochondrial contamination is still valid when using rodent malaria parasites as a source. Although mitochondria are called the powerhouse of cells for providing ATP, it is the generation of proton motive force across their inner membrane that justifies this moniker. Gene expression profiles of blood-stage parasites revealed an apparently coordinated expression of genes for the tricarboxylic acid (TCA) cycle enzymes. Mitochondrial ATP synthesis is carried out by a multiprotein rotary enzyme, F0F1ATP synthase (complex V), located within the inner membrane by utilizing the proton motive force. The assembly of iron-sulfur [Fe-S] clusters is a complex process requiring participation of several enzymes, chaperones, and transporters. In metazoa, mitochondria are central to the process of programmed cell death or apoptosis, releasing several of proapoptotic molecules such as cytochrome c in response to a number of different apoptotic signals, including the collapse of mitochondrial membrane potential. DNA replication, recombination, and repair would require participation of a large number of proteins in these complex processes. Mitochondrial translation machinery is necessary for the synthesis of three proteins encoded by mitochondrial DNA (mtDNA).

The discovery and characterization of the apicoplast has been one of the success stories for the growing union of molecular, cellular, and genomic biology in parasitology. The combination of these three disciplines in a short space of time has shed much light on the origin, structure, biogenesis, and metabolism of the apicoplast. The apicoplast contains visible ribosomes, distinctly smaller than the eukaryotic cytosolic ribosomes, and is surrounded by multiple membranes. The dissection of apicoplast targeting by Waller using green fluorescent protein (GFP), alongside similar constructs made in the laboratory of David Roos, inspired an ongoing series of broader targeting experiments in both Toxoplasma and Plasmodium that have revolutionized the understanding of intra- and extracellular trafficking in apicomplexans. The fluoroquinolone compound ciprofloxacin interferes with the resealing step and results in linearization of the circular DNA, and ciprofloxacin does indeed inhibit displacement-loop replication in Plasmodium falciparum. Antibiotics such as ciprofloxacin inhibit bacterial or plastid DNA replication, while other antibiotics affect transcription, translation, and posttranslational modification. An elegant analysis showed that the fusion protein is apparently trapped in the apicoplast protein-translocation machinery and somehow prevents correct division and segregation of the apicoplast. Chloroplasts are chlorophyll-containing organelles found in plants and algae. Their key function is photosynthesis, and they come in red, brown, and even colorless, nonphotosynthetic versions. Products of the isopentenyl diphosphate (IPP) pathway are presumably also used by mitochondrial ubiquinones, by dolichol in the endoplasmic reticulum (ER) and Golgi, and to prenylate proteins within the parasite’s endomembrane system.

This chapter briefly considers the major features of cell cycle control to provide a general context for the discussion of Plasmodium cell proliferation regulators. Progression through the cell cycle phases is controlled by the cyclin-dependent protein kinases (CDKs). These enzymes phosphorylate a number of substrates involved in processes such as in initiation of DNA synthesis or chromosome segregation. The molecular machinery controlling cell cycle progression is in essence a mere effector of signaling pathways, which are activated by a variety of intra- or extracellular stimuli. The life cycle of malaria parasites is an alternation of developmental stages where the parasite is cell cycle arrested, and stages undergoing intense cell division. The alternation of actively dividing and cell cycle- arrested developmental stages during the life cycle of malaria parasites must be associated with an efficient and versatile cell cycle control machinery, whose activity needs to be integrated with specific cell development programs. The underlying principles of cell division control at the molecular level and the identity of key players in this process such as CDKs, cyclins, and CDK inhibitors (CKIs) have been elucidated mostly through genetic analysis in yeast. The Plasmodium falciparum genome encodes a number of proteins putatively involved in calcium signaling, including calmodulin-related proteins, a calcium-transporting ATPase, and a family of CDPKs, which are composed of a protein kinase catalytic domain fused to a calcium-binding domain.

This chapter reviews the current understanding of the reasons for hemoglobin degradation, the mechanism of hemoglobin breakdown, the proteases that contribute to this process, and the potential for new antimalarial therapies that block hemoglobin hydrolysis. A large body of work suggests that a principal source of amino acids for erythrocytic parasites is the hydrolysis of globin. De novo synthesis of amino acids appears to play only a small role in supplying parasite amino acids. In Plasmodium falciparum, ring-stage parasites pinocytose the hemoglobin-rich erythrocyte cytosol into small vesicles. Upon its delivery to the P. falciparum food vacuole, and perhaps during vesicular transit to this organelle, hemoglobin is subjected to an acidic pH. Proteases of multiple catalytic classes appear to contribute to hemoglobin degradation. Biochemical characterizations of food vacuole aspartic and cysteine proteases and metalloproteases have shown that these enzymes hydrolyze hemoglobin or globin in vitro, supporting roles in hemoglobin hydrolysis. Evidence for a role for cysteine proteases in hemoglobin hydrolysis came from the observation that cysteine protease inhibitors cause a dramatic morphological abnormality in P. falciparum trophozoites, whereby food vacuoles swell and fill with undegraded hemoglobin. The processing of hemoglobin peptides in the cytosol is probably performed, at least in part, by a neutral metalloaminopeptidase. Homologs of P. falciparum proteases have been identified in other plasmodial species. Considering the selection of drug resistance, a recent study showed that parasites resistant to vinyl sulfone cysteine protease inhibitors could be selected by incubation of cultured parasites with stepwise increases in concentrations of inhibitor.

This chapter reviews recent advances in understanding the pathways for membrane biogenesis in Plasmodium, presents new information in this field learned from the available Plasmodium genome sequence and its annotation, and discusses progress in lipid-based antimalarial chemotherapy. It focuses on the pathways of synthesis of phospholipids (PLs) and neutral lipids and their importance in parasite physiology, intracellular localization and trafficking of lipids, and newly identified pharmacological targets. Studies with Plasmodium falciparum parasites grown in vitro or isolated from patients with malaria have revealed profound changes in the membrane composition and structure of surrounding uninfected red blood cells. Glycerolipid metabolism in various organisms initiates with the acylation of glycerol-3-phosphate, which can be produced by the phosphorylation of glycerol by glycerokinase or the reduction of the glycolytic intermediate dihydroxyacetone-3-phosphate by dihydroxyacetone-3-phosphate dehydrogenase. The available P. falciparum genome has revealed the presence of only one putative acyl-CoA diacylglycerol acyltransferase gene named PfDGAT1. This gene encodes a polypeptide with a molecular mass of 78.1 kDa with a broad acyl-CoA specificity, localized to the microsomes. The de novo biosynthetic pathways of phatidylethanolamine (PE) and phosphatidylcholine (PC) initiate with the phosphorylation of ethanolamine and choline, conversion of the phosphoethanolamine and phosphocholine formed into CDP-ethanolamine and CDP-choline, and DAG-dependent acylation of the latter products into PE and PC, respectively. Subcellular fractionation of the malarial parasites remains a difficult task, and cellular localization of the various lipids and mechanisms mediating their intracellular trafficking remains to be elucidated.

The translation of mRNA, a fundamental property of all organisms, is carried out by the ribosome. Although the ribosomes of Plasmodium sp. are typically eukaryotic in their sedimentation properties, they do differ from the host by the rRNA having a low G+C base composition. Indeed, antibiotics that selectively disable protein synthesis over defined periods of the developmental cycle or organellar function could play an important role in a better understanding of the molecular events during the developmental cycle of the malaria parasite. Our most detailed knowledge of ribosomal chemistry comes from the study of prokaryotic ribosomes. Within the ribosomal complex lies the machinery that decodes information from the messenger RNA and catalyzes the ordered assembly of amino acids into proteins. Regulation of ribosome production is essential to all cells. The number of ribosomes present in a cell is directly related to the protein synthesizing activity and to the size of the cell. The crystal structure of Thermus thermophilus and Escherichia coli 70S ribosome, along with biochemical data, suggests that ~80% of intersubunit bridges are contributed by RNA-RNA interactions. The functional role of the pseudoknot varies considerably depending on its source. The development and spread of drug resistance are unquestionably tied to the population dynamics of parasite, host, and vector.

This chapter summarizes the currently available knowledge on sources of oxidative and nitrosative stress in malarial parasites, the different available detoxification pathways, and the impact on mechanisms of drug action. Plasmodium-infected red blood cells (Plasmodium IRBCs) appear to be under exogenous and endogenous oxidative stress. Malaria parasites induce oxidative stress in their host red blood cell. In the membrane of P. falciparum-parasitized cells, increasing amounts of hemichromes and band 3 aggregates have been demonstrated. Peroxidized IRBCs generate 4-hydroxyalk- 2-enals and alka-2,4-dienals, and these aldehydes are toxic to P. falciparum in vitro. Thus, the antioxidant capabilities of the parasite and RBC are of considerable significance. The majority of the peroxide-detoxifying capacity, however, seems to be provided by peroxiredoxins. Glutathione S-transferase exhibits glutathione peroxidase activity, which might contribute to the total peroxide-reducing capacity of the parasite, since the enzyme is present at very high concentrations. Superoxide dismutase (SOD) is the major enzyme involved in catabolizing the superoxide anion, resulting in production of molecular oxygen and hydrogen peroxide. P. falciparum possesses a classical 2-Cys glutaredoxin (PfGrx1) and a redox-active 1-Cys glutaredoxin-like protein (PfGLP-1). The glyoxalase system consists of glyoxalase I (GloI), glyoxalase II (GloII), and the coenzyme glutathione. It is a cyclic metabolic pathway removing toxic 2-oxoaldehydes like methylglyoxal by converting them to the corresponding nontoxic 2-hydroxycarboxylic acids like D-lactate. Artemisinin has been shown to react with glutathione (GSH) and to increase levels of lipid peroxidation.

The Red blood cell (RBC) membrane is naturally endowed with a variety of membrane transporters, mainly geared to optimize the respiratory function and to maintain cell homeostasis at minimal metabolic cost. Therefore, to survive within a red blood cell, the malaria parasite must alter the permeability of the host’s plasma membrane by up-regulation of existing carriers or by creation of new permeation pathways (NPP).These pathways, indispensable for parasite growth, could be possible antimalarial targets for selective inhibition, as well as routes for drug delivery. Electrophysiological techniques, such as the patch clamp, are ideal for the study of channels that are permeable to charged solutes, even though the NPP are also used for transport of electroneutral and organic osmolytes in malaria infected RBCs. Red blood cells have proven to be extremely useful as a model system to study the different membrane transport pathways, and there is a plethora of publications aimed at a detailed description of pumps, cotransporters, and specific carriers in the red blood cell membrane. In the cell-attached and excised configurations, Plasmodium falciparum-infected RBCs show a very different pattern of channel activity from uninfected cells. It has been suggested that NPP could be used as therapeutic targets. However, the exact nature of the NPP remains to be resolved; part of the present confusion, due to discrepant results, comes from the lack of background information on the channels present in noninfected red blood cell membranes.

Antigenic variation is a key survival strategy employed by a wide range of infectious organisms, allowing them to colonize and persist in a vertebrate host in the face of an evolving immune response. Variant surface antigens (VSA) of the parasite responsible for the most severe form of human malaria, Plasmodium falciparum, also contribute significantly to the pathology of disease. Characterization of var gene coding sequences is difficult due to their extreme diversity, but there are certain key conserved features in var gene structure. The conservation in upstream sequences, in terms of both sequence and organization, may indicate some evolutionary pressure that restricts recombination between limited subsets of var genes. The adhesion of different Duffy-binding-like (DBL) and cysteine-rich interdomain region (CIDR) domain types to host cell-surface molecules has been investigated in heterologous expression studies, establishing domains responsible for particular adhesive phenotypes and pinpointing regions critical for binding. Clonal switching of var gene expression during chronic infection reflects the sum of several molecular processes, including temporal regulation of var gene expression during intraerythrocytic development, mutually exclusive expression of only one var variant per infected erythrocytes (IE), and the ability to switch var expression in progeny parasites. Chronic infection with malaria is characterized by periodic peaks of parasitemia. A consensus model for variant expression during chronic infections can now be proposed. Following merozoite release from the liver, an initial P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant dominates the first cycle of intraerythrocytic infection.

The discovery that Plasmodium falciparum-infected erythrocytes can bind to uninfected erythrocytes to form rosette-like clumps of cells was first made in the late 1980s. Some of the parasite ligands and host uninfected erythrocyte receptors that mediate rosette formation have been identified, and work has begun to determine the potential for a rosette-inhibiting antidisease vaccine. Despite this progress, the function of rosetting remains unknown, and the exact role of rosetting in the pathogenesis of severe malaria remains controversial. In falciparum malaria it may be the combination of rosetting and cytoadherence, together with high parasite burdens, that is particularly obstructive to microvascular blood flow and could lead to hypoxia, tissue damage, and severe malaria. Skeptics of rosetting claim that there is no evidence that rosettes form in vivo. A number of different red blood cell rosetting receptors have been described, including CR1, heparan sulfate-like molecules, ABO blood group sugars, and CD36. The CD36 glycoprotein, which is an important endothelial receptor for cytoadherence, is expressed at low levels on red blood cells but only rarely acts as a rosetting receptor in field isolates. The development of rosette-inhibiting immune responses in natural malaria infections has received relatively little attention. There is lack of proof that rosetting causes severe malaria. However, evidence does support a direct role for rosetting in the pathogenesis of some cases of life-threatening malaria.

This chapter talks about the modes of action and mechanisms of Plasmodium falciparum resistance to the antifolate drugs sulfadoxine-pyrimethamine (SP), pyrimethamine, and cycloguanil, as well as the quinoline-based drugs, notably chloroquine (CQ), mefloquine (MFQ), and quinine (QN). Antifolates comprise a group of drugs that work through inhibition of folate metabolism of various organisms, including malaria parasites. In all Plasmodium species, as well as other protozoa and some plants, dihydrofolate reductase (DHFR) exists as a bifunctional enzyme that includes thymidylate synthase (TS), which forms deoxythymidylate (dTMP) from deoxyuridylate, while another substrate, methylenetetrahydrofolate, is converted to dihydrofolate (DHF). Resistance to DHFR inhibitors, including pyrimethamine and cycloguanil, arose soon after their deployment as antimalarials. As an alternative to SP, a combination of dapsone with chlorcycloguanil, administered as its prodrug chlorproguanil, has recently been developed. Chloroquine use began worldwide in the late 1940s, and for several decades, this drug remained the gold standard in the prevention and treatment of uncomplicated malaria. Mechanistic models relating pH-dependent physiological changes in relation to CQ resistance (CQR) have recently seen intriguing yet contradictory developments. Direct evidence in support of a determining role for pfcrt in CQR first came from transfection studies showing that coexpression of mutant pfcrt in CQ-sensitive (CQS) parasites produced low-level, VP-reversible CQR. Focused and applied efforts from the academic, industrial, funding, and health care sectors on a significantly greater scale are vital to achieving any success in reducing the devastating impact that malaria maintains on the poorest nations of this world.

Well-controlled genetic studies are easier to conduct with mice than with humans, and it has long been known that certain strains of mice consistently show a greater degree of resistance to infection than other strains. It is clear that these differences are primarily genetic, rather than environmental or acquired. This chapter discusses specific genetic loci that have been identified as responsible for some of these differences. Genetic factors also appear to underlie some striking differences in resistance to malaria that have been observed between ethnic groups who live in the same area. The chapter deals with the growing list of genes that show some evidence of influencing resistance to malaria. Glycophorins A and B, encoded by the homologous genes GYPA and GYPB, are major sialoglycoproteins of the erythrocyte membrane, which carries the antigenic determinants for various blood groups. Hemoglobin S (HbS) homozygotes have sickle cell disease, a debilitating and often fatal disorder caused by the red blood cell deformities that result from this structural defect, particularly at low oxygen concentrations. The thalassemias are a group of genetic disorders due to defective production of α- or β- globin chains, arising from a diverse set of deletions and other disruptions of the globin gene clusters on chromosomes 11 and 16. Haptoglobin, encoded by the HP gene, is a protein found in plasma. Parasite sequestration in cerebral capillaries, a hallmark of human cerebral malaria, is notable by its absence in the Plasmodium berghei ANKA (PBA) experimental model.

The resurgence of malaria morbidity and mortality underlines the failure of hopes for the eradication of malaria of the middle part of the 20th century. By the 1950s, control measures based on chemotherapy with chloroquine and use of dichlorodiphenyltrichloroethane (DDT) had reduced the disease burden of malaria so substantially that many policymakers believed the task of malaria control was close to being completed. DNA sequences from Plasmodium falciparum parasites can be delivered as DNA molecules (DNA vaccines) or various recombinant attenuated DNA viruses to generate candidate DNA-based vaccines. There is an unimaginably large number of possible malaria constructs for inclusion in a malaria vaccine. In general, recombinant protein-based platforms are chosen where antibody induction is thought to be the most important effector mechanism. Adjuvants are chemicals which, when administered in combination with an antigen, augment the immune response to the antigen. All vaccines, including malaria vaccines, are evaluated in animal models to assess their safety and immunogenicity. For a vaccine to have any public health impact it must be licensed, manufactured, promoted, and distributed. Once a vaccine that prevents clinical disease is licensed by a regulatory authority, it may yet be many years before the vaccine is added to local immunization programs in countries where malaria is endemic. Inclusion of sexual-stage vaccine components in multistage vaccines is theoretically attractive.

Anopheles gambiae is the most important vector of malaria in sub-Saharan Africa, where most of the world’s human malaria cases and deaths occur each year. Genetically, A. gambiae sensu stricto is a very polymorphic taxon. A. gambiae is clearly a species with a high degree of genetic population structure, particularly in west and central Africa. A. gambiae has three pairs of chromosomes, an X and Y sex-determining pair and two autosomes, chromosomes 2 and 3. The shotgun sequences from Celera and Genoscope were assembled with the Celera assembler into 8,987 scaffolds (ordered and oriented sets of contigs with gaps) that constitute the assembled genome. The A. gambiae genome is currently undergoing a major update of the assembly, which will then be followed by an entirely new annotation. A major milestone for the VectorBase developers will be the completion of the first draft of the Aedes aegypti genome and close comparison between the Anopheles and Aedes genomes. Postgenome studies using large-scale data sets involving expressed sequence tags (ESTs), microarray expression analysis, single nucleotide polymorphisms, and proteomics data, in addition to third-party annotations, are essential to provide information on the annotation of the genome and to pinpoint unique and fundamental aspects of mosquito biology that could be exploited for control. Insecticides are an essential component to most malaria control programs. Our future challenge will be to determine the function of gene products and to establish precisely how they interact in time and space to affect vector biology and disease transmission.

This chapter traces the development of gene expression studies in mosquitoes from a historical perspective. In addition, it reviews the relevant technologies that made the advances possible. While the principal focus is on the anopheline vectors of malaria, Anopheles gambiae in particular, the authors intend to cite relevant advances in other insects, most notably the yellow fever mosquito, Aedes aegypti. The rapid development of molecular techniques and their applications in model organisms such as the vinegar fly, Drosophila melanogaster, fostered the reemergence of interest in whether genetics bolstered by molecular biology could provide useful tools in combating malaria transmission. Extensive expressed sequence tag (EST) studies designed to dissect the molecular components of innate immunity in mosquitoes have generated large amounts of information on the mosquito transcriptome. Extensive cDNA sequencing also has been used to identify genes differentially expressed in insecticide-resistant and -susceptible mosquitoes. Serial analysis of gene expression (SAGE) is based on the sequential analysis in large quantities of short cDNA sequence tags. Genes activated by both bacterial and malaria infection include those encoding a peptidoglycan recognition protein LB receptor, the gram-negative bacteria-binding protein opsonin, an fibrinogen- like lectin, a thioester-containing putative opsonin, the 14-D serine protease, the CED-6-like phagocytic adaptor,and the leucinerich repeat putative receptor. Monitoring genome-wide changes in gene expression patterns in whole A. gambiae specimens is now feasible, and it is expected that this will be possible with other vectors in the near future.