6

Parasite Biology

WHERE WE WANT TO BE IN THE YEAR 2010

For centuries, malaria parasites have successfully evaded the biological defenses of their human hosts. Researchers are perplexed by the complexity of these organisms, and many questions remain unanswered. By the year 2010, advances in the field of parasite biology will have exposed many of the complex biochemical mechanisms that allow this evasion to occur. A detailed understanding of how malaria parasites recognize and invade human liver and red blood cells, for example, how their multistage life cycle is regulated and how they rapidly become drug resistant will have provided a major boost to efforts to develop malaria vaccines and will have resulted in innovative approaches to more durable antimalarial drugs.

WHERE WE ARE TODAY

The Parasite

The human malaria parasite—actually four species of the genus Plasmodium—undergoes over a dozen distinguishable stages of development as it moves from the mosquito vector to the human host and back again. One way to conceptualize this complex life cycle is to consider it in three distinct parts: the liver phase, the blood phase, and the mosquito phase.



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MALARIA: Obstacles and Opportunities 6 Parasite Biology WHERE WE WANT TO BE IN THE YEAR 2010 For centuries, malaria parasites have successfully evaded the biological defenses of their human hosts. Researchers are perplexed by the complexity of these organisms, and many questions remain unanswered. By the year 2010, advances in the field of parasite biology will have exposed many of the complex biochemical mechanisms that allow this evasion to occur. A detailed understanding of how malaria parasites recognize and invade human liver and red blood cells, for example, how their multistage life cycle is regulated and how they rapidly become drug resistant will have provided a major boost to efforts to develop malaria vaccines and will have resulted in innovative approaches to more durable antimalarial drugs. WHERE WE ARE TODAY The Parasite The human malaria parasite—actually four species of the genus Plasmodium—undergoes over a dozen distinguishable stages of development as it moves from the mosquito vector to the human host and back again. One way to conceptualize this complex life cycle is to consider it in three distinct parts: the liver phase, the blood phase, and the mosquito phase.

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MALARIA: Obstacles and Opportunities Depending on the developmental stage and species, malaria parasites can be spherical, ring shaped, elongated, or crescent shaped, and can range in size from 1 to 20 microns in diameter (1 micron equals 1 millionth of a meter or approximately 125,000 of an inch). By comparison, a normal red blood cell has a diameter of about 7 microns. Although the four species of human malaria parasites are closely related, there are major differences among them. Plasmodium falciparum, the most pathogenic of the four species, has been found to be more closely related to avian and rodent species of Plasmodium than to the other primate and human species (McCutchan et al., 1984). The following sections of this chapter discuss general aspects of malaria parasite biology, with a focus on P. falciparum. Interspecies differences are noted where appropriate. Parasite-Host Interactions Liver Phase The liver phase of malaria begins when the female anopheline mosquito injects the sporozoite stage of the parasite into the human host during a blood meal (see Chapter 2, Figure 2-3). After just a few minutes, the sporozoites arrive at the liver and invade the liver cells (hepatocytes). Over the course of 5 to 15 days, depending on the species, the sporozoites undergo a process of asexual reproduction (known as schizogony, the “splitting process ”) that results in the production of as many as 30,000 “daughter” parasites, called merozoites. It is the merozoites that, once released from the liver, carry the malaria infection into the red blood cells (erythrocytes). The surface of the sporozoite is coated with many copies of a protein that is thought to play a key role in host cell recognition and perhaps cell invasion. Antibodies to certain portions of this circumsporozoite protein can prevent sporozoites from entering liver cells in culture and may play an important role in protecting against infection. People exposed to irradiated sporozoites are protected against infection by unaltered sporozoites. This level of immunity has not yet been obtained with use of subunit, synthetic, or gene-cloned vaccines (see Chapter 9). The circumsporozoite protein can be detected throughout the parasite 's development in the liver and may also be associated with liver-stage merozoites. The circumsporozoite protein has a unique structure of immunodominant, highly repetitive complexes of amino acids (Santoro et al., 1983). Although different Plasmodium species have distinct antigenic properties, the repetitive complexes seen in P. falciparum sporozoites are also seen in P. vivax and other parasite species, although the actual makeup of these proteins is species specific.

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MALARIA: Obstacles and Opportunities It is not clear how the parasite arrives at the surface of the hepatocytes, since liver cells are not in direct contact with the blood. Also, although many mechanisms have been postulated, details of how the parasites invade liver cells remain obscure (Miller, 1977). The physiological changes that the sporozoite undergoes during the shift from a cold-blooded mosquito host to a warm-blooded human host are not well understood, nor is it understood how the parasite manages the reverse situation, as the sexual stage precursor—the gametocyte—travels from human to mosquito. In the latter case, there is evidence suggesting that changes in temperature and pH help stimulate the emergence of the gametes in the insect midgut. Schizogony in the liver had been thought to proceed without pathological manifestations, but recent evidence points to the existence of inflammatory mechanisms and cellular infiltration, including cytotoxic T cells, all of which may be immunologically important. It has been known for many years that the symptoms of malaria can recur without reexposure to the parasite, but the biological mechanisms underlying this phenomenon have only recently been determined. The reappearance of symptoms may either be due to relapse, i.e., the development of latent sporozoites, called hypnozoites, in the liver following a period during which the blood itself was parasite free, or recrudescence, a sudden upsurge of blood-stage parasites after a protracted period of very low parasite density (Krotoski et al., 1982). Relapses occur only in P. vivax and P. ovale infections, while recrudescence generally occurs in P. falciparum and P. malariae. Although the hypnozoite does not appear to undergo development during its dormant state, the fact that hypnozoites are readily eliminated by the action of the drug primaquine implies some level of metabolic activity. Blood Phase When merozoites are released from the liver into the bloodstream, asexual blood-stage reproduction, or erythrocytic schizogony, has begun. Parasite invasion of red blood cells unfolds in four steps: attachment of the merozoite to the erythrocyte, rapid deformation of the red blood cell, invagination of the erythrocyte membrane where the parasite is attached and subsequent envelopment of the merozoite, and the resealing of the erythrocyte membrane around the parasite (Aikawa et al., 1978; Hadley et al., 1986; Perkins, 1989; Bannister and Dluzewski, 1990; Wilson, 1990). Attachment, an event separate from invasion, may occur without endocytosis. There is indirect evidence that organelles at the tip of the parasite, the apical complex, are involved in invasion. For example, only invasive stages have apical organelles, and the organelles rapidly disappear after invasion. Although little is known about the biochemical functions associated with these organelles, efforts to clone proteins found in them may

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MALARIA: Obstacles and Opportunities provide some insight into their roles (Coppel et al., 1987). The attachment phase is a potential target for vaccine developers, since antibody that interferes with this process may prevent parasite invasion of red blood cells. Red blood cells have receptors for malaria parasites on their surfaces, although these receptors may be different for different parasite species. The best example of a parasite receptor on a red blood cell is the Duffy antigen, which is recognized by P. vivax; this parasite cannot invade Duffy-negative cells. Of considerable interest is the recent cloning and characterization of the P. vivax (and P. knowlesi) gene encoding the parasite protein which binds to the Duffy antigen (Adams et al., 1990; Fang et al., 1991). This protein is located in one of the apical organelles (micronemes) thought to play a role in the invasion process. Unfortunately, our understanding of the attachment process for P. falciparum is less sophisticated. Although the exact mechanism of invasion is still unresolved (see reviews cited above), it is likely that specific parasite proteases are involved (Perkins, 1989). Suggested candidates include a neutral endopeptidase (Bernard and Schrevel, 1987; Braun-Breton et al., 1988) and a chymotrypsin-like enzyme (Dejkriengkraikhul and Wilairat, 1983); selective protease inhibitors could thus be of potential interest as chemotherapeutic agents which prevent invasion. After invasion, the parasite lies within a membranous parasitophorus vacuole, where it synthesizes nucleic acids, proteins, lipids, mitochondria, and ribosomes and assembles these components into new merozoites (Ginsburg, 1990b). The entire erythrocytic asexual cycle takes between two and three days to run its course, depending on the species. Once merozoite assembly is completed, the erythrocyte ruptures and merozoites are released into the plasma, where they attach to other erythrocytes and begin the process anew. Some merozoites, for reasons not well understood, differentiate into the sexual forms of the parasite, the gametocytes. The factors that determine the sex of the gametocyte are unknown. Gametocyte development takes between 2 days (for P. vivax) and 10 days (for P. falciparum). The release of merozoites precipitates malaria's classical paroxysms of fever, chills, headache, myalgia, and malaise. Although the cause of the fevers is unknown, one theory is that endotoxin-like substances may be released during schizont rupture. The paroxysm itself may be due to transient increases in cytokines, such as interleukin-1 and tumor necrosis factor (Kwiatkowski et al., 1990). Children living in highly endemic areas often have significant parasitemias without symptoms, leading researchers to suspect the presence of “antitoxic ” immunity. Mosquito Phase When gametocytes are taken up during a mosquito's blood meal, a number of factors, including temperature, concentrations of oxygen and carbon

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MALARIA: Obstacles and Opportunities dioxide, pH, and a mosquito exflagellation factor, are thought to contribute to the maturation of gametocytes. Male microgametes are released during a process called exflagellation. Fusion of the female macrogamete with a single microgamete results in fertilization and the formation of the ookinete. The ookinete migrates to the wall of the mosquito midgut, where it penetrates the peritrophic membrane and epithelium and comes to rest on the external surface of the stomach. Over a period of days, this stage of the parasite matures into an oocyst containing up to 10,000 motile sporozoites. When the oocyst ruptures, the sporozoites enter the mosquito circulation and travel to the salivary glands, where they are injected into the human host when the mosquito feeds. The number of sporozoites that enter the human host during a single blood meal is thought to be highly variable. Parasite Physiology and Biochemistry Feeding Malaria parasites feed by ingesting intact erythrocyte cytosol, the internal fluid portion of the cell, through an organelle, the cytostome. This process has been reconstructed recently in three dimensions from micrographs (Slomianny, 1990). When the cytostome closes around cytosol, it creates a membrane-bound vacuole. In P. falciparum, the ingested host cytosol is then exposed to a mixture of potent digestive enzymes. That digestion of hemoglobin is required for parasite survival was shown in experiments in which hemoglobin was chemically cross-linked, making it resistant to degradation by P. falciparum proteases and cathepsin D; parasites which invaded red blood cells with cross-linked hemoglobin failed to develop to trophozoites and eventually died (Geary et al., 1983). Recently, two proteases important for hemoglobin digestion in P. falciparum have been characterized. One is a cysteinyl proteinase (Rosenthal et al., 1988) and the other, which apparently initiates digestion, is an aspartyl proteinase (Goldberg et al., 1991). Reversible inhibitors of the cysteinyl protease (such as leupeptin) block hemoglobin digestion and suspend parasite growth. However, growth resumes even after prolonged incubations when the inhibitor is removed. Irreversible protease inhibitors, on the other hand, killed the parasites (Rosenthal et al., 1988). Selective protease inhibitors which block either of these two enzymes would be of considerable interest as potential antimalarial drugs. As the process of hemoglobin digestion becomes better understood (Goldberg et al., 1990), additional sites for chemotherapeutic intervention should be uncovered. During digestion, about three-fourths of the host cell hemoglobin is destroyed. The residue left from this process is an insoluble particulate complex call hemozoin; this complex contains, among other material, the

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MALARIA: Obstacles and Opportunities heme derived from hemoglobin. Although the chemistry of this complex is becoming better understood (Goldie et al., 1990; Slater et al., 1991), its role in pathogenesis, drug action, or immunology remains undocumented. Permeability Changes in Erythrocyte Membranes Following parasite invasion, the intracellular metabolism of infected erythrocytes increases significantly. Nutrients must be brought in from outside, and waste products must be disposed of expeditiously. The cell membrane responds by increasing its capacity to transport a variety of substrates in and out of the erythrocyte, including essential amino acids, nucleosides, lactate, and fatty acids. The changes in membrane permeability allow a number of substrates, that otherwise would not be let in at all or would be let in to a limited degree, to enter the infected red blood cell. These substrates include hexitols, acidic and neutral amino acids, several small inorganic ions, and organic acids. The appearance of new erythrocyte membrane transport pathways is the result of host cell “remodeling” by the intracellular malaria parasites. It is thought that in remodeling, proteins of parasite origin become associated with host membrane components, either by adhering to the inner aspects of the membrane or by inserting themselves directly into the membrane. The experimental data strongly support this hypothesis (Haldar et al., 1986; Ginsburg and Stein, 1987; Cabantchik, 1989, 1990; Ginsburg, 1990a; Tanabe, 1990a,b). Other Parasite-Directed Changes in Erythrocyte-Membrane Structure In P. falciparum, the trophozoite stage inserts new molecules into the host erythrocyte plasma membrane. These new membrane components are responsible for the sequestration of mature parasite stages in capillaries by the process of cytoadherence. Although among the human malaria parasites only P. falciparum exhibits cytoadherence, P. vivax also induces alterations in the infected erythrocyte membrane. Ultrastructure studies have shown that the membranes of erythrocytes infected with P. vivax contain caveolar structures that appear to be connected to vesicles (Atkinson and Aikawa, 1990; Barnwell, 1990). These caveolae-vesicle complexes appear to play a role in parasite interaction with the extracellular environment. They induce antibody production and are antigenically highly variable. Nutrition and Metabolism A malaria infection initiated by a single malaria parasite may produce as many as 10 billion new organisms. Nearly all the metabolic processes of

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MALARIA: Obstacles and Opportunities the parasite are focused on supporting this enormous reproductive effort. The relatively recently acquired ability to cultivate P. falciparum in vitro has greatly expanded biochemists' ability to study parasite nutrition and metabolism. In P. falciparum, glucose can be replaced by fructose, but the parasite will not develop in vitro when another sugar, such as galactose, mannose, maltose, or ribose is substituted (Geary et al., 1985a). Although malaria parasites are capable of synthesizing the amino acids glutamate, aspartate, alanine, and leucine from glucose, they probably acquire them either through digestion of hemoglobin or from sources outside the red blood cell. Most early studies on the uptake of amino acids by malaria parasites utilized the animal parasites P. berghei, P. lophurae, P. gallinaceum, and P. knowlesi either in vivo, an approach in which experimental parameters are difficult to control, or in vitro, using less than ideal culture procedures. Observations on parasite biochemistry using cultures of P. falciparum have not always supported conclusions drawn from these flawed models. In experiments using cultured P. falciparum, it has been shown that 13 of the 20 amino acids can be obtained from the digested erythrocyte cytosol; the parasite must receive the other seven amino acids from sources outside the erythrocyte. Selective transport facilities may exist for any or all of these amino acids in P. falciparum, but evidence from other Plasmodium species suggests that this is not the case for all amino acids. Attempts to supplement glutamine, one of the amino acids, with other metabolites have been unsuccessful. Both glutamate and glutamine are required for continuous cultivation, indicating that interconversion is limited at best. There appears to be only one vitamin, calcium pantothenate, that is not provided by the erythrocyte but is needed by the parasite for survival. Evidence for this comes from in vitro studies using culture medium containing this vitamin (Divo et al., 1985a). The malaria parasite's requirement for para-aminobenzoic acid and folic acid is well documented. The requirement for these vitamins, found in red blood cells, is probably strain specific. Sulfonamides, which inhibit folic acid synthesis, have been used as antimalarial drugs for years. The story of folic acid metabolism is complicated, however, and not all sulfonamides are equally potent as antimalarials. Surprisingly, unlike most organisms, P. falciparum does not seem to require biotin. The ability to develop in the absence of this vitamin was demonstrated by growing the parasites in the presence of several biotin antagonists, including avidin (Geary et al., 1985b). Pyrimidines and purines are the two main building blocks of DNA. Malaria parasites can synthesize the former de novo, but purines are a required nutrient (Gero and O'Sullivan, 1990). Hypoxanthine is the preferred purine source, but other purines readily substitute for hypoxanthine. Studies of the kinetics of DNA synthesis in P. falciparum have revealed

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MALARIA: Obstacles and Opportunities that incorporation of labeled purines into DNA begins approximately 30 hours after merozoite invasion and increases logarithmically for another 14 to 18 hours, when schizogony is completed. Because erythrocytes contain considerable concentrations of amino acids and vitamins that may be important to parasite development, it is difficult to determine in experimental settings whether decreased parasite viability due to nutritional factors is a result of effects on the parasite itself or on the red blood cell. Thus, it is not known whether nutrients identified as crucial for malaria parasite cultivation are required for erythrocytes only, parasites only, or both. For example, the amino acids glutamine, glycine, and cysteine, while necessary for long-term survival of erythrocytes in culture, may not be required parasite nutrients. Energy Transformations and Mitochondria There has been considerable debate about whether the erythrocytic stages of mammalian malaria parasites possess mitochondria, the energy-producing organelles essential for all life forms. The falciparum parasite uses glucose as its primary energy source. In fact, glucose utilization is significantly greater in the infected erythrocyte than in the uninfected cell. Progress is being made in the characterization of the enzymes involved in glycolysis in P. falciparum (Roth et al., 1988; Roth, 1990). However, there is no evidence supporting the presence of a Krebs cycle, a key energy-producing process of the mitochondria. The presence of mitochondria in the erythrocytic asexual stages of P. falciparum has recently been shown, but their actual function is not well understood (Divo et al., 1985b). Recent advances in the molecular biology of the mitochondrial DNA of malaria parasites may help to unravel the role of the mitochondrion (Gardner et al., 1988). The importance of this organelle to the parasite is underscored by the fact that mitochondrial toxins are highly lethal. Antibiotics used to treat falciparum infection, such as the tetracyclines, clindamycin, and erythromycin, appear to work by blocking the development of parasite mitochondria (Prapunwattana et al., 1988). Of great interest in this regard is the recent finding that mitochondrial DNA of P. falciparum encodes an RNA polymerase which is closely related to prokaryotic polymerases and is sensitive to rifampicin, potentially explaining the antimalarial activity of this drug (Gardner et al., 1991). The erythrocytic stages of many mammalian malaria parasites appear not to derive their metabolic energy through classical electron transport. The mitochondria may participate in ion transport, but the role this plays in metabolism is unclear. It is not known whether components analogous to those present in the mammalian terminal electron transport system function in the malaria parasite, and for what purpose, since the organism, like many

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MALARIA: Obstacles and Opportunities other parasitic protozoans and all parasitic worms so far studied, has rather limited terminal respiration (Scheibel, 1988). Mammalian malaria parasites are aerobic fermenters, capable of partially decomposing metabolic substrates to fermentation products but unable to oxidize them completely to carbon dioxide and water. Since the substrates are not completely metabolized, it would appear that terminal respiration is either absent or rate limiting in the parasite. Available evidence supports the view that malaria parasites are microaerophilic, homolactate fermenters (Scheibel et al., 1979). Parasite Lipids Bloodstage malaria parasites contain no lipid reserves (all lipids of intracellular plasmodia are membrane associated), and they lack the means for degrading lipids. Plasmodia also appear to lack the capacity to synthesize fatty acids de novo from acetate, but they can and do incorporate exogenous fatty acids into their phospholipids, thereby maintaining a lipid-fatty acid composition distinct from that of the host cell (Vial et al., 1990). Because malaria parasites lack lipid reserves, they are incapable of fabricating fatty acids and cholesterol, and they have limited capacity for saturation and desaturation as well as for chain-elongating and chain-shortening reactions. The blood stages of Plasmodium species must satisfy their lipid requirements by relying on dynamic exchanges with plasma, an activity akin to that of the red blood cell itself but at a much higher rate (Scheibel and Sherman, 1988). Evasion of Host Defenses Cytoadherence The greater virulence of P. falciparum than of other human malaria parasites is due to the sequestration of parasitized erythrocytes into the deep vasculature. The attachment of infected red blood cells to the vascular endothelium in P. falciparum malaria occludes capillaries in the brain, heart, and other vital organs and causes tissue anoxia. Cerebral malaria, the most serious and often fatal consequence of falciparum malaria, is thought to be caused in part by the cytoadherence of infected erythrocytes, although changes in vascular permeability and cytokine-related cerebral inflammation may also be important (MacPherson et al., 1985). Cytoadherence in other tissues causes serious, but rarely fatal, anoxia. Recent evidence suggests that the cytoadherence properties of parasite strains is correlated with the severity of disease but not with cerebral malaria (Ho et al., 1991). Cytoadherence sequesters infected red blood cells in the venous circulation 's lower oxygen environment, an important consideration for an organism

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MALARIA: Obstacles and Opportunities known to be microaerophilic and highly susceptible to oxidant stress, and prevents the cells from circulating through the spleen, where they would be destroyed. Infections with P. falciparum parasites that have lost the ability to cytoadhere are invariably attenuated, and parasite reproduction rates are readily controlled by the spleen (Langreth and Peterson, 1985). One reason P. falciparum has such an explosive reproduction rate may be due in part to the fact that it escapes the role that the spleen seems to play in limiting infections by other malaria species. The ability to cytoadhere is tied to the appearance of knobs, conical protrusions about 100 nanometers by 30 nanometers, on the host erythrocyte membrane. A considerable understanding of the biochemistry of knobs has been gained, extending to the cloning of the most important protein associated with them (Sharma and Kilejian, 1987; Ellis et al., 1987; Pologe et al., 1987; Triglia et al., 1987). Although some parasite-infected red blood cells that form knobs do not adhere, all natural infections with P. falciparum are both K+ (indicating knob formation) and C+ (indicating cytoadherence). When parasites have been cultured for a period of time, they lose their ability to produce knobs and to cytoadhere (K−,C−). Some cultured parasites form knobs but do not adhere (K+,C−). When clones of K−,C− parasites (which have never been found in natural infections) are injected into aotus monkeys, the resulting infections are mild with low parasite densities. Cultured parasites without knobs but with cytoadherent capability (K−,C+) have been observed, which raises questions about the relationship between knob formation and cytoadherence (Udomsangpetch et al., 1989). Whether K−,C+ parasites occur naturally or are simply an artifact of in vitro experimental manipulations is unknown (Trager, 1989). Just as there is an incomplete picture of the parasite-derived molecules necessary for cytoadherence, so too are the relevant receptors on endothelial cells incompletely defined. One cell surface protein that appears to have a role in cytoadherence is an 88 kilodalton antigen known as CD-36. This glycoprotein is expressed on the surface of cells that mediate cytoadherence, such as endothelial cells, monocytes, and C32 melanoma cells, but not on related cells to which infected erythrocytes do not adhere. Antibody to CD-36 readily reverses cytoadherence. Other proteins thought to serve as cell surface receptors during cytoadherence are thrombospondin and ICAM-1 (CD-54) (Howard and Gilladoga, 1989; Chulay and Ockenhouse, 1990). Neoantigens There are several additional parasite-induced antigens that appear on the surface of infected red blood cells and may be immunologically important. Some of these “neoantigens” appear to be linked to knob formation and are responsible for cytoadherence, while for others no clear role has

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MALARIA: Obstacles and Opportunities TABLE 6-1 Well-Characterized P. falciparum Proteins Associated with Infected Erythrocyte Membranes Name Synonym(s) MW (kDa) Comments PfEMP-1 IP 265-285a Believed to mediate cytoadherence; trypsin sensitive; antigenically variable; selection for cytoadherence selects for larger PfEMP-1 MESAb ,c PfEMP-2 250-300a Phosphoprotein; binds to band 4.1; located at knobs KAHRPb ,c KP PfHRP-I, 80-109a Present at knobs; isolates lacking this protein do not have knob structures detectable by electron microscopy PfHRP-IIb ,c   Secreted from intact red blood cells 11-1b ,c   >1,200 Associated with knobs 41-2b   29   RESAb ,c Pf155 155 Phosphoprotein; present in ring stage; minor variability; binds to spectrin TR   105 Putative receptor for transferrin a Varies in size between isolates. b Nucleotide sequence data available. c Blocks of tandem repeats in protein. Abbreviations: PfEMP-1, P. falciparum erythrocyte membrane protein-1; PfEMP-2, P. falciparum erythrocyte membrane protein 2; IP, iodinatable protein; MESA, mature-parasite-infected erythrocyte surface antigen; KAHRP, knob-associated histidine-rich protein; PfHRP-I, P. falciparum histidine-rich protein I; PfHRP-II, P. falciparum histidine-rich protein II; KP, knob protein; 11-1 and 41-2, individual recombinant clones; RESA, ring-infected erythrocyte surface antigen; TR, transferrin receptor; MW, molecular weight; kDa, kilodaltons. been determined. Table 6-1 outlines several important erythrocyte membrane alterations caused by parasite-derived antigens (Howard, 1988; Petersen et al., 1990). Molecular Biology Most work on malaria parasite molecular biology has been devoted to the identification and cloning of genes which encode potentially protective antigens. As noted above, however, the use of molecular biology techniques has opened new avenues for research on the basic biology of these organisms. This section describes advances in other areas of parasite molecular biology which indicate the enormous potential of the technology in pursuing questions of fundamental relevance. Several recent reviews can be consulted for more detail (Walliker et al., 1987; Braun, 1988; Weber et al., 1988). It should be noted that the unusual codon bias found in P. falciparum, which is highly skewed toward adenine-thymidine richness, generates some fundamental difficulties for the use of typical gene expression systems (Saul and Battistutta, 1988).

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MALARIA: Obstacles and Opportunities Physiology and Biochemistry The mitochondrion, an organelle essential to parasite survival, is the target of a number of antimalarial antibiotics. These antibiotics, while clinically useful, were formulated for use against bacterial infections but could be better focused to kill malaria parasites if more were known about the biochemistry of the parasite mitochondrion. Drugs that impede mitochondrial function and that work more rapidly than the presently used antibacterial antibiotics would be a useful addition to the current chemotherapeutic armamentarium. RESEARCH FOCUS: Mitochondrial biochemistry, with the goal of better targeting malaria antibiotics. Malaria parasites have an absolute requirement for purines, one of the principal building blocks of DNA. The purine salvage pathways used by the parasite appear to be distinct from those of the erythrocyte. RESEARCH FOCUS: Parasite enzymes in the purine salvage pathway that could serve as targets for drug therapy. Developmental Biology A remarkable progression of development is observed in nearly all stages of the malaria parasite, but we know almost nothing of the bases for the unfolding of these programmed changes. A greater understanding of the mechanisms that control the developmental progression of sporozoites to exoerythrocytic schizonts and of ring stages to trophozoites and erythrocytic schizonts, of the developmental regulation of gametocytogenesis, and of fertilization and development in the mosquito is expected to lead to the identification of a number of attractive targets for chemotherapeutic intervention. RESEARCH FOCUS: The cell biology of development in the various stages of the life cycle of malaria parasites. With the availability of a convenient culture system, the blood stage parasites represent a useful subject for initial studies. Drug Action and Resistance Little is known about the mode of action of chloroquine and other quinoline-containing antimalarial drugs. This lack of knowledge has hindered rational drug design. In addition, studies of apparent resistance

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MALARIA: Obstacles and Opportunities genes, and of chloroquine resistance genes in particular, do not explain the apparent lack of resistance seen with many other quinoline-containing anti-malarial drugs. The danger is that newly developed antimalarial drugs may be clinically useless by the time their mechanism of action is discovered. RESEARCH FOCUS: The mode of action of antimalarial drugs, especially chloroquine, and the genetic and biological components of antimalarial drug resistance. Clinical Immunity In the optimistic rush to develop a malaria vaccine, the concept of clinical immunity (premunition or concomitant immunity) has received little attention. Clinically immune individuals are believed by some investigators to be protected from disease because they maintain a low-grade infection that continually stimulates an otherwise lethargic immune response, thereby preventing a serious rise in parasite numbers. This phenomenon is different from sterilizing immunity, which controls infection by eliminating parasitemia or by preventing infection altogether. RESEARCH FOCUS: Factors that determine clinical immunity. Cytoadherence Parasite cytoadherence antigens on the surface of infected erythrocytes may be the most important antigens associated with the immunological clearance of a natural infection. Antibodies to these cytoadherence antigens free schizont-infected erythrocytes from the vascular endothelium and release them into the general circulation, where they are removed by the spleen. This mechanism of parasite destruction is probably more efficient than any other and may be the most important means of controlling infections in clinically immune adults. RESEARCH FOCUS: The identification and characterization of parasite cytoadherence antigens, with the aim of understanding their role in parasite development and pathogenesis. Parasite Genes The DNA of malaria parasites is very difficult to clone into stable vector systems, making construction of an ordered parasite gene library in Escherichia

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MALARIA: Obstacles and Opportunities coli virtually impossible. Knowing the number and location of genes within the parasite DNA, and the proteins produced by each, would greatly aid efforts at understanding parasite function. RESEARCH FOCUS: A complete genomic library for P. falciparum and P. vivax and an in vitro system for translating genes into their protein products. RESEARCH FOCUS: The construction of complementary DNA libraries specific for the many stages of parasite development. Such a library would be a powerful tool for unfolding the mysteries of specific parasite developmental processes such as pre-erythrocytic and erythrocytic schizogony, gametogeny, and sporogeny. RESEARCH FOCUS: The identification of a system for the transformation of P. falciparum that would be of tremendous benefit in defining the roles of parasite genes. In Vitro Cultivation Perhaps no research tool has promoted research in malaria more thoroughly during the past 50 years than the continuous cultivation of P. falciparum. Although other Plasmodium species have been successfully propagated in vitro, no other species that infects humans can be cultured. RESEARCH FOCUS: The development of an in vitro system for the continuous cultivation of P. vivax and the other species that cause disease in humans. REFERENCES Adams, J. H., D. E. Hudson, M. Torii, G. E. Ward, T. E. Wellems, M. Aikawa, and L. H. Miller. 1990. The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites. Cell 63:141-153. Aikawa, M., L. H. Miller, J. Johnson, and J. Rabbege. 1978. Erythrocyte entry bymalaria parasites: a moving junction between erythrocyte and parasite. Journal of Cell Biology 77:72-82. Atkinson, C. T., and M. Aikawa. 1990. Ultrastructure of malaria-infected erythrocytes. Blood Cells 16:351-368. Bannister, L. H., and A. R. Dluzewski. 1990. The ultrastructure of red cell invasion in malaria infections: a review. Blood Cells 16:257-292. Barnwell, J. W. 1990. Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes. Blood Cells 16:379-395. Bernard, F., and J. Schrevel. 1987. Purification of a Plasmodium berghei neutral endopeptidase and its localization in merozoites.

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