MINIREVIEW Malaria ) an overview Renu Tuteja Malaria Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India The term malaria is derived from the Italian ‘mal’aria’, which means ‘bad air’, from the early association of the disease with marshy areas. Towards the end of the 19th century, Charles Louis Alphonse Laveran, a French army surgeon, noticed parasites in the blood of a patient suffering from malaria, and Dr Ronald Ross, a British medical officer in Hyderabad, India, discov- ered that mosquitoes transmitted malaria. The Italian professor Giovanni Battista Grassi subsequently showed that human malaria could only be transmitted by Anopheles mosquitoes. Malaria affects a large num- ber of countries and it has been reported that the inci- dence of the disease in 2004 was between 350 and 500 million cases. Over two billion people, representing more than 40% of the world’s population, are at risk of contracting malaria, and the number of malaria deaths worldwide has been estimated at 1.1–1.3 million per annum in World Health Organization (WHO) reports 1999–2004. Malaria has a broad distribution in both the subtropics and tropics, with many areas of the tropics endemic for the disease. The countries of sub-Saharan Africa account for the majority of all malaria cases, with the remainder mostly clustered in India, Brazil, Afghanistan, Sri Lanka, Thailand, Indo- nesia, Vietnam, Cambodia, and China [1,2]. Malaria is estimated to cost Africa more than $12 billion annu- ally and it accounts for about 25% of all deaths in children under the age of five on that continent [3]. In many temperate areas, such as western Europe and the USA, public health measures and economic develop- ment have been successful in achieving near- or complete elimination of the disease, other than cases imported via international travel. The parasites Malaria is transmitted through the bite of an infected female Anopheles mosquito. Of the approximately Keywords cerebral malaria; erythrocytes; malaria life cycle; malaria parasite; mosquito; parasite genome; parasite transcriptome; pathogenesis; Plasmodium falciparum; red blood cells Correspondence R. Tuteja, Malaria Group, International Centre for Genetic Engineering and Biotechnology, PO Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India Fax: +91 11 26742316 Tel: +91 11 26741358 E-mail: renu@icgeb.res.in (Received 30 April 2007, revised 26 June 2007, accepted 19 July 2007) doi:10.1111/j.1742-4658.2007.05997.x Malaria is caused by protozoan parasites of the genus Plasmodium and is a major cause of mortality and morbidity worldwide. These parasites have a complex life cycle in their mosquito vector and vertebrate hosts. The pri- mary factors contributing to the resurgence of malaria are the appearance of drug-resistant strains of the parasite, the spread of insecticide-resistant strains of the mosquito and the lack of licensed malaria vaccines of proven efficacy. This minireview includes a summary of the disease, the life cycle of the parasite, information relating to the genome and proteome of the species lethal to humans, Plasmodium falciparum, together with other recent developments in the field. Abbreviations CSA, chondroitin sulfate A; IDC, intraerythrocytic developmental cycle; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; RBC, red blood cell. 4670 FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, 30 of which are of major importance. Malaria parasites are eukaryotic single-celled microorganisms that belong to the genus Plasmodium . More than 100 species of Plas- modium can infect numerous animal species such as reptiles, birds and various mammals, but only four species of parasite can infect humans under natural conditions: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. These four species differ morphologically, immunologically, in their geographical distribution, in their relapse pat- terns and in their drug responses. P. falciparum is the agent of severe, potentially fatal malaria and is the principal cause of malaria deaths in young children in Africa [3]. The least common malaria parasite is P. ovale, which is restricted to West Africa, while P. malariae is found worldwide, but also with rela- tively low frequency. The most widespread malaria parasite is P. vivax but infections with this species are rarely fatal. Although P. falciparum and P. vivax can both cause severe blood loss (anemia), mild anemia is more common in P. vivax infections, whereas severe anemia in P. falciparum malaria is a major killer in Africa. In addition, in the case of P. falciparum, the infected erythrocytes can obstruct small blood vessels and if this occurs in the brain, cerebral malaria results, a complication that is often fatal, particularly in Afri- can infants. P. ovale and P. vivax have dormant liver stages named hypnozoites that may remain in this organ for weeks to many years before the onset of a new round of pre-erythrocytic schizogony, resulting in relapses of malaria infection. In some cases P. malariae can produce long-lasting blood-stage infections, which, if left untreated, can persist asymptomatically in the human host for periods extending into several decades. Life cycle of malaria parasites The life cycle of malaria parasites is extremely complex and requires specialized protein expression for survival in both the invertebrate and vertebrate hosts. These proteins are required for both intracellular and extracel- lular survival, for the invasion of a variety of cell types and for the evasion of host immune responses. Once injected into the human host, P. falciparum and P. mal- ariae sporozoites trigger immediate schizogony, whereas P. ovale and P. vivax sporozoites may either trigger immediate schizogony or lead to delayed schizogony as they pass through the hypnozoite stage mentioned above. The life cycle of the malaria parasite is shown in Fig. 1A and can be divided into several stages, starting with sporozoite entry into the bloodstream. Tissue schizogony (pre-erythrocytic schizogony) Infective sporozoites from the salivary gland of the Anopheles mosquito are injected into the human host along with anticoagulant-containing saliva to ensure an even-flowing blood meal. It was thought that spor- ozoites move rapidly away from the site of injection, but a recent study using a rodent parasite species (Plasmodium yoelii) as a model system indicates that, at least in this case, the majority of infective sporo- zoites remain at the injection site for hours, with only slow release into the circulation [4]. Once in the human bloodstream, P. falciparum sporozoites reach the liver and penetrate the liver cells (hepatocytes) where they remain for 9–16 days and undergo asexual replication known as exo-erythrocytic schizogony. The mechanism of targeting and invading the hepatocytes is not yet well understood, but studies have shown that sporozo- ite migration through several hepatocytes in the mam- malian host is essential for completion of the life cycle [5]. The receptors on sporozoites responsible for hepato- cyte invasion are mainly the thrombospondin domains on the circumsporozoite protein and on thrombospon- din-related adhesive protein. These domains specifically bind to heparan sulfate proteoglycans on the hepato- cytes [6]. Each sporozoite gives rise to tens of thousands of merozoites inside the hepatocyte and each merozoite can invade a red blood cell (RBC) on release from the liver. In an interesting study, also using rodent malaria parasites (Plasmodium berghei), it has been shown that liver-stage parasites manipulate their host cells to guarantee the safe delivery of mer- ozoites into the bloodstream [7]. Hepatocyte-derived merosomes appear to act as shuttles that ensure the protection of parasites from the host immune system and the release of viable merozoites directly into the circulation [7]. The time taken to complete the tissue phase varies, depending on the infecting spe- cies; (8–25 days for P. falciparum, 8–27 days for P. vivax, 9–17 days for P. ovale and 15–30 days for P. malariae ), and this interval is called the prepatent period. Erythrocytic schizogony Merozoites enter erythrocytes by a complex invasion process, which can be divided into four phases: (a) ini- tial recognition and reversible attachment of the mero- zoite to the erythrocyte membrane; (b) reorientation and junction formation between the apical end of the merozoite (irreversible attachment) and the release of substances from the rhoptry and microneme organ- elles, leading to formation of the parasitophorous R. Tuteja Malaria ) an overview FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 4671 vacuole; (c) movement of the junction and invagina- tion of the erythrocyte membrane around the merozo- ite accompanied by removal of the merozoite’s surface coat; and (d) resealing of the parasitophorous vacuole and erythrocyte membranes after completion of mero- zoite invasion [8]. Because the invasion of erythrocytes by P. falciparum requires a series of highly specific molecular interactions, it is regarded as an attractive target for the development of interventions to combat malaria [6]. Asexual division starts inside the erythro- cyte and the parasites develop through different stages therein. The early trophozoite is often referred to as the ‘ring form’, because of its characteristic morphol- ogy (Fig. 1). Trophozoite enlargement is accompanied by highly active metabolism, which includes glycolysis of large amounts of imported glucose, the ingestion of host cytoplasm and the proteolysis of hemoglobin into constituent amino acids. Malaria parasites cannot degrade the heme by-product and free heme is poten- tially toxic to the parasite. Therefore, during hemo- globin degradation, most of the liberated heme is polymerized into hemozoin (malaria pigment), a crys- talline substance that is stored within the food vacu- oles [8]. The end of this trophic stage is marked by multiple rounds of nuclear division without cytokinesis resulting in the formation of schizonts (Fig. 1). Each mature schizont contains around 20 merozoites and these are released after lysis of the RBC to invade further un- infected RBCs. This release coincides with the sharp increases in body temperature during the progression of the disease. This repetitive intraerythrocytic cycle of Fuse & make Zyg ote Oocyst Cycle in mo squito Rupturing Oocyst Liver cell Exo-erythrocytic cycle Schizont Ruptured schizont RB C ring stage Trophs Ga me tocytes Male & fe ma le ga me tocytes Ruptured schizont Erythrocytic cycle Mosquito takes a A B blood m eal (injects sporozoites) Tro p hozoite Schizont Rin g Fig. 1. (A) Life cycle of the malaria parasite P. falciparum. The figure has been prepared with the help of the information, artwork and micrographs from the CDC’s websites for parasite identification http://www. dpd.cdc.gov/dpdx and http://www.itg.be. (B) Different intraerythrocytic stages of development of P. falciparum in culture. Malaria ) an overview R. Tuteja 4672 FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS invasion–multiplication–release–invasion continues, taking about 48 h in P. falciparum, P. ovale and P. vivax infections and 72 h in P. malariae infection. It occurs quite synchronously and the merozoites are released at approximately the same time of the day. The contents of the infected RBC that are released upon its lysis stimulate the production of tumor necro- sis factor and other cytokines, which are responsible for the characteristic clinical manifestations of the dis- ease. A number of specific ligand–receptor interactions have been identified as involved in invasion and it has been reported that genetic disruption of any one of these results in a shift to using other pathways [9,10]. The P. falciparum genome sequence, completed in 2002, indicates that several of the molecules involved in invasion are members of larger gene families [11,12]. Merozoite surface proteins (MSP)1 to MSP)4) are an important class of integral membrane proteins identi- fied on the surface of developing and free merozoites. These are involved in the initial recognition of the ery- throcytes via interactions with sialic acid residues and are likely to be important for invasion because anti- bodies directed against these proteins can block this process [9]. Erythrocyte binding antigen 175 (EBA- 175) is a P. falciparum protein that binds the major glycoprotein (glycophorin A) found on human erythro- cytes during invasion [8]. The structure of EBA-175 has striking similarities with the Duffy antigen-binding proteins of P. vivax that are essential for successful invasion by this species. After invasion, the principal parasite ligand known as P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is encoded by a multigene family termed var, is expressed at the surface of the infected RBC [13,14]. PfEMP1 has a pivotal role in P. falciparum pathogenesis and several host recep- tors can be concurrently recognized by the numerous adhesion domains located in the extracellular region of PfEMP1 [15,16]. The extensive diversity in the var gene family is mainly responsible for the evasion of specific immune responses and many of these genes are expressed in the parasite population, but at any given time during an infection, parasites within infected cells express only a single var gene [15–17]. In a recent study, a specific epigenetic mark associated with the silenced var genes has been identified and it has been shown that the persistence of this mark provides advantages to the parasite in pathogenesis and immune evasion [18]. A small proportion of the merozoites in the red blood cells eventually differentiate to produce micro- and macrogametocytes (male and female, respectively), which have no further activity within the human host (Fig. 1A). These gametocytes are essential for transmit- ting the infection to new hosts through female Anophe- les mosquitoes. Normally, a variable number of cycles of asexual erythrocytic schizogony occur before any gametocytes are produced. In P. falciparum, erythro- cytic schizogony takes 48 h and gametocytogenesis takes 10–12 days. Gametocytes appear on the fifth day of primary attack in P. vivax and P. ovale infections, and thereafter become more numerous; they appear at anything from 5 to 23 days after a primary attack by P. malariae. Sexual phase in the mosquito (sporogony) A mosquito taking a blood meal on an infected indi- vidual may ingest these gametocytes into its midgut, where macrogametocytes form macrogametes and exflagellation of microgametocytes produces microga- metes. These gametes fuse, undergo fertilization and form a zygote. This transforms into an ookinete, which penetrates the wall of a cell in the midgut and develops into an oocyst (Fig. 1A). In a recent study, it has been shown that gamete surface antigen Pfs230 mediates human RBC binding to exflagellating male parasites to form clusters termed exflagellation centers, from which individual motile microgametes are released. This pro- tein thus plays an important role in subsequent oocyst development, which is a critical step in malaria trans- mission [19]. Sporogony within the oocyst produces many sporozoites and when the oocyst ruptures, they migrate to the salivary glands for onward transmission into another host (Fig. 1A). This form of the parasite is found in the salivary glands after 10–18 days and thereafter the mosquito remains infective for 1–2 months. When an infected mosquito bites a sus- ceptible host, the Plasmodium life cycle begins again. Symptoms, diagnosis and treatment The accumulation and sequestration of parasite- infected RBCs in various organs such as the heart, brain, lung, kidney, subcutaneous tissues and placenta is a characteristic feature of infection with P. falcipa- rum. Sequestration is a result of the interaction between parasite-derived proteins, which are present on the surface of infected RBCs, and a number of host molecules expressed on the surface of uninfected RBCs, endothelial cells and in some cases placental cells [20]. In specific manifestations of malaria, some receptors for parasite adhesion have been implicated, such as hyaluronic acid and chondroitin sulfate A (CSA) in placental infections and intercellular adhesion molecule 1 (ICAM-1) in cerebral malaria [8,13,21]. R. Tuteja Malaria ) an overview FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 4673 Malaria symptoms can develop as soon as 6–8 days after being bitten by an infected mosquito, or as late as several months after departure from a malarious area. People infected with malaria parasites typically experience fever, shivering, cough, respiratory distress, pain in the joints, headache, watery diarrhea, vomiting and convulsions [8]. Severe malaria is usually complex and several key pathogenic processes such as jaundice, kidney failure and severe anemia can combine to cause serious and often fatal disease [8]. There are no life-threatening complications in most cases of malaria, but what triggers the transition from an uncomplicated to a serious infection is not well understood [22]. Malaria is especially dangerous to pregnant women and small children and in endemic countries it is an important determinant of perinatal mortality [23]. Parasite sequestration in the placenta is a key feature of infection by P. falciparum during preg- nancy and is associated with severe adverse outcomes for both mother and baby, such as premature delivery, low birthweight and increased mortality in the new- born [24]. PfEMP1, a ligand for CSA, is a major target of antibodies associated with protective immunity and P. falciparum isolates that sequester in the placenta primarily bind CSA [25]. After repeated exposure to malaria during pregnancy, women in areas of endemic- ity slowly develop immunity; thus multigravid women are comparatively less susceptible to pregnancy-associ- ated malaria than primagravid women. Malaria is diagnosed using a combination of clinical observations, case history and diagnostic tests, princi- pally microscopic examination of blood [26]. Ideally, blood should be collected when the patient’s tempera- ture is rising, as that is when the greatest number of parasites is likely to be found. Thick blood films are used in routine diagnosis and as few as one parasite per 200 lL blood can be detected. Rapid diagnostic ‘dipstick’ tests, which facilitate the detection of malaria antigens in a finger-prick of blood in a few minutes are easy to perform and do not require trained person- nel or a special equipment [26]. However, they are relatively expensive and although P. falciparum can be diagnosed, P. ovale, P. malariae and P. vivax cannot be distinguished from one another using this method. Three consecutive days of tests that do not indicate the presence of the parasite can rule out malaria. Malaria is a curable disease if treated adequately and promptly. Quinine from the bark of the Andean Cinchona tree was the first widely used antimalarial treatment and was discovered long before the causes of malaria were known. However, the parasite can rap- idly develop resistance to common antimalarial drugs. In many parts of the world P. falciparum has become resistant to Fansidar and chloroquine, which are the two most commonly used and most affordable antima- larial drugs [27,28]. To overcome this problem and to prolong the useful life of current drugs, combination therapy is being increasingly employed. Artemisinin, which is obtained from the plant Artemisia annua,is an extremely effective antimalarial, and this drug, or its derivatives such as artesunate or artemether, are being used in mainly pairwise combinations with sev- eral other drugs such as Fansidar [29] and mefloquine [30], the latter an important and still highly efficacious drug against which resistance, especially in southeast Asia is, however, of increasing concern. The inexorable spread of drug resistance is a major problem in malaria control, especially as there are no clinically approved malaria vaccines available to date, even though a number are in development and testing. Recent reports have described state-of-the-art malaria vaccine development and selected malaria vaccines in current clinical development [31,32]. Several major international initiatives have been launched to tackle malaria (Table 1) [33]. These include the WHO’s Roll Back Malaria program, the Multilateral Initiative in Malaria [34], the Medicines for Malaria Venture , the Malaria Vaccine Initiative, and the Global Fund to Fight AIDS, TB and Malaria, which supports the implementation of prevention and treatment programs. There are a number of ways to decrease malaria transmission but none currently offers a complete block, therefore new methods are urgently required [35]. The three combined strategies of drug treatment, vaccination and vector control will ulti- mately be required to significantly reduce malaria transmission [29,36]. With respect to the last of these, another potential option for reducing malaria is by the use of genetically modified mosquitoes that are refractory to transmis- sion of the pathogen [37]. Recently, important techni- cal advances, which include germ-line transformation of mosquitoes, the characterization of tissue-specific promoters and the identification of effector molecules that interfere with parasite development, have resulted in the production of transgenic mosquitoes incapable of spreading the malaria parasite [37]. However, in order for Plasmodium-refractory mosquitoes to be effective, they need to be able to thrive in the wild and compete successfully with their wild-type counterparts. One major concern about the use of these engineered mosquitoes is whether the modification would be sta- ble long-term [37]. Even though the possibility of genetically modifying mosquito vector competence has been well studied in the laboratory, much work is still needed to develop strategies for the release and Malaria ) an overview R. Tuteja 4674 FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS survival of these engineered mosquito populations in the field. In a recent study, it was reported that when fed on Plasmodium -infected blood, transgenic malaria- resistant mosquitoes had a significant fitness advantage over wild-type mosquitoes [38]. The genome, proteome and transcriptome The genome of P. falciparum clone 3D7 was the first to be sequenced and annotation of the predicted genes is at an advanced stage [12]. The availability of the P. falciparum genome sequence has the potential to reveal a large number of possible new drug targets and genes important for parasite biology and pathogenesis. Genome information for P. falciparum and other species of Plasmodium is freely available at http:// www.plasmodb.org, and it has been shown that the P. falciparum genome covers  23 megabase pairs of DNA, split into 14 chromosomes. P. falciparum also has a circular plastid-like genome and a linear mito- chondrial genome [39]. The nuclear genome is the most (A+T)-rich genome sequenced to date, with an overall (A+T) composition of  81%, which increases to  90% in intergenic regions and introns [12]. About 5300 genes have been predicted from the genome sequence, of which only a few have been identified to date as encoding enzymes. The regions near the ends of each chromosome are interesting; the genes residing here encode surface proteins or antigens that are some- times recognized by the human immune system to stimulate immune responses. However, exchange of material between chromosome ends gives the parasite a considerable capacity for changes in antigen expres- sion and thereby immune evasion. The genome sequence of P. falciparum has also revealed new gene families encoding proteins responsible for mediating erythrocyte invasion [9]. It is interesting to note that, although the homologs of genes involved in basic path- ways such as translation initiation, DNA replication, repair and recombination are present in the genome of the parasite [12,40], it appears to lack some key meta- bolic pathways; for example, the synthesis of a major- ity of the 20 amino acids, synthesis of purines and the salvage of pyrimidines, as well as two protein compo- nents of ATP synthase (a mitochondrial ATP-pro- ducing enzyme) and components of a conventional NADH dehydrogenase complex [12]. It has also been proposed that the regulation of protein levels is con- trolled through mRNA processing and translation, in addition to the level of gene transcription [12]. Molec- ular transfection technology, together with the ability to introduce fluorescent reporter proteins, is a rela- tively recent development that is facilitating a greater understanding of many other aspects of the parasite’s cell biology [41]. It is noteworthy that components of some anabolic pathways for the synthesis of fatty acids, isoprenoid precursors, heme and iron sulfur complexes seem to be localized in the apicoplast, a structure within the cell related to the plastids of plant species that has its own genome [12,42–46], as mentioned above. Studies have shown that the apicoplast is essential for survival of the parasite [47,48]. Its genome is 35 kb and encodes only 57 proteins but it is estimated that around 10% of the proteins encoded by the nucleus may be des- tined for this structure [49]. Such proteins are targeted into the organelle by the use of a bipartite-targeting signal [49]. One protein in this class is encoded by an unusual gene on chromosome 14 specifying contiguous DNA polymerase, DNA primase and DNA helicase activities and thought to play a key role in the replica- tion of the apicoplast genome [12,50]. The organellar genome sequence also identified molecules within the Table 1. Important websites. No. Description Website 1 WHO Roll Back Malaria program http://www.rbm.who.int 2 Multilateral Initiative in Malaria http://www.mim.su.se 3 Medicines for Malaria Venture http://www.mmv.org 4 Malaria Vaccine Initiative http://www.malariavaccine.org 5 Global Fund to Fight AIDS, TB and Malaria http://www.theglobal fund.org 6 Plasmodium genome database PlasmoDB http://www.plasmodb.org/ 7 Plasmodium falciparum Gene database http://www genedb.org/genedb/malaria/ 8 Malaria Parasite Metabolic Pathways http://sites.huji.ac.il/malaria/ 9 Malaria Transcriptome database http://malaria.ucsf.edu/comparison 10 Plasmodium falciparum genome ⁄ pathway database http://plasmocyc.stanford.edu/ 11 Malaria Research and Reference Reagent Resource Center http://www.mr4.org/ 12 Understanding higher-order function from genome information http://www.genome.ad.jp/kegg/ 13 Detection of enzyme-encoding genes in P. falciparum genome http://bioinformatics.leeds.ac.uk/shark/ R. Tuteja Malaria ) an overview FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 4675 apicoplast that, in other systems, are the targets of sev- eral existing drugs, such as antibiotics, and there are now experimental data showing that such compounds can also inhibit the growth of P. falciparum by target- ing this bacterium-derived endosymbiotic organelle [51,52]. At the proteomics level, the proteins from four stages of the life cycle of P. falciparum (clone 3D7), i.e. sporozoites, merozoites, trophozoites and gameto- cytes, have been profiled using multidimensional pro- tein identification technology and MS analysis [53]. It has been reported that the sporozoite proteome is markedly different from the other stages and about half of the sporozoite proteins are unique to this stage. In contrast, trophozoites, merozoites and gametocytes have fewer unique proteins, sharing a greater propor- tion of the total. Of the proteins found in multiple stages, the most common were mainly housekeeping proteins such as ribosomal proteins, transcription fac- tors, histones and cytoskeletal proteins [53]. The results also suggested that the P. falciparum genome encodes a large number of unique proteins, many of which might be required for specific host–parasite interac- tions. These interesting proteins with no homology to sequences in other organisms represent potential Plas- modium-specific molecules that might provide targets for new drug and vaccine development [53]. In a simi- lar study the proteomic analysis of selected stages of P. falciparum (NF54 isolate) by high-accuracy MS revealed 1289 proteins, of which 645 were identified in gametes, 931 in gametocytes and 714 in asexual blood stages, respectively [54]. Previous studies have shown that in many cases, the proteins from P. falciparum are consistently bigger than their homologous counterparts from other species, but the role of these parasite-spe- cific inserts in the sequences of P. falciparum proteins is uncertain [55]. Using ORF-specific DNA microarrays, the expres- sion profile across 48 individual 1-h time points from the complete asexual intraerythrocytic developmental cycle (IDC) of the HB3 clone of P. falciparum has been examined [39,56]. This transcriptome analysis revealed that at least 60% of the genome is transcrip- tionally active during this stage and that > 75% of these expressed genes are activated only once during the IDC [39]. These interesting data demonstrate that P. falciparum exhibits an unusual and quite specialized mode of transcriptional regulation, which produces a continuous cascade of gene expression, starting with genes corresponding to general cellular processes, such as protein synthesis, and ending with Plasmodium- specific functionalities, such as genes involved in erythrocyte invasion [39]. Recently, the same group determined the transcriptome of the IDC for two more clones of P. falciparum, 3D7 and Dd2, with different geographical origins from HB3 [57]. Their results revealed that the transcriptome is remarkably well con- served among all three clones but there are some dif- ferences in the expression of genes coding for surface antigens involved in host–parasite interactions [57]. All of these strain-specific data are publicly available at both http://malaria.ucsf.edu/comparison/ and http:// www.plasmoDB.org. Table 1 is a compilation of important websites that have been created to organize and exploit data arising from postgenomic studies of P. falciparum and its related species. For a better understanding of the biolog- ical, physiological and biochemical roles of a particular gene, a website summarizing malaria parasite metabolic pathways as maps has been constructed and is continu- ously being expanded [58] (http://sites.huji.ac.il/malaria/). In addition to classical biochemical pathways, this website contains maps dealing with biological processes such as cell–cell interactions, protein trafficking and transport, and fundamental pathways including replica- tion, transcription and translation [58]. PlasmoCyc is another genome ⁄ pathway database that specifically developed for P. falciparum (http://plasmocyc.stanford. edu/). In this database, the metabolic pathways are displayed with detailed information about individual enzymatic reactions with the chemical structures of the substrates and reactants. The database also contains information about antimalarial drugs and their targets, as well as an overview of all the metabolic pathways and tools for comparing pathways between organisms. Another important website, Kyoto Encyclopedia of Genes and Genomics (KEGG) at (http://www.genome. ad.jp/kegg/), can also be used for exploring higher-order functional aspects of parasite biology from its genome information [59]. A new fully automated software pack- age, the metashark can be used for the detection of enzyme-encoding genes within unannotated genome data from organisms such as P. falciparum and their visualization in the context of the relevant metabolic network(s) [60]. The sharkhunt package can be downloaded from the metashark website at (http:// bioinformatics.leeds.ac.uk/shark/). This search method was successfully used to detect the experimentally demo- nstrated but unannotated pantothenate to coenzyme A pathway encoded in the P. falciparum genome [60]. Conclusions Malaria caused by the mosquito-transmitted parasite P. falciparum is the cause of an enormous number of deaths every year in the tropical and subtropical areas Malaria ) an overview R. Tuteja 4676 FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS of the world. There is an urgent need to design new drugs and⁄ or vaccines that can substantially and con- sistently interrupt the life cycle of this complex para- site. A wealth of information has been generated from genome-wide studies of the transcriptome and prote- ome of the parasite and now it is a real challenge to use this information efficiently to determine the appro- priate therapeutic targets for developing and testing new formulations. Malaria vaccine development is cur- rently at an encouraging stage and it is critical that the momentum achieved to date be maintained in the future. A combination of new antimalarial drugs and vaccines with efficient vector control measures will be required to halt the transmission of malaria in the affected areas of the world. Acknowledgements The author is grateful to Professor John Hyde (Uni- versity of Manchester, UK) and Dr C. Chitnis (IC- GEB, New Delhi) for critical reading and corrections on the manuscript and the referees for constructive suggestions. The author thanks Arun Pradhan for help in the preparation of figure. The work in author’s lab- oratory is supported by grants from Department of Biotechnology, Defence Research and Development Organization and Department of Science and Technol- ogy. Infrastructural support from the Department of Biotechnology, Government of India is gratefully acknowledged. References 1 Snow RW, Craig M, Deichmann U & Marsh K (1999) Estimating mortality, morbidity and disability due to malaria among Africa’s non-pregnant population. Bull WHO 77, 624–640. 2 Breman JG, Egan A & Keusch GT (2001) The intolera- ble burden of malaria: a new look at the numbers. Am J Trop Med Hyg 64 (Suppl. 1–2), iv–vii. 3 Snow RW, Korenkromp EL & Gouws E (2004) Pediat- ric mortality in Africa: Plasmodium falciparum malaria as a cause or risk. Am J Trop Med Hyg 71 (Suppl. 2), 16–24. 4 Yamauchi LM, Coppi A, Snounou G & Sinnis P (2007) Plasmodium sporozoites trickle out of the injection site. Cell Microbiol [Epub ahead of print]. 5 Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Fre- vert U, Nussenzweig RS, Nussenzweig V & Rodriguez A (2001) Migration of Plasmodium sporozoites through cells before infection. Science 291, 141–144. 6 Frevert U, Sinnis P, Cerami C, Shreffler W, Takacs B & Nussenzweig V (1993) Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes. J Exp Med 177, 1287–1298. 7 Sturm A, Amino R, van de Sand C, Regen T, Retzlaff S, Rennenberg A, Krueger A, Pollok JM, Menard R & Heussler VT (2006) Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science 313, 1287–1290. 8 Miller LH, Baruch DI, Marsh K & Doumbo OK (2002) The pathogenic basis of malaria. Nature 415, 673–679. 9 Cowman AF & Crabb BS (2002) The Plasmodium falci- parum genome – a blueprint for erythrocyte invasion. Science 298, 126–128. 10 Tolia NH, Enemark EJB, Sim KL & Joshua-Tor L (2005) Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum. Cell 122, 183–193. 11 Bowman S, Lawson D, Basham D, Brown D, Chilling- worth T, Churcher CM, Craig A, Davies RM, Devlin K, Feltwell T et al. (1999) The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum. Nature 400, 532–538. 12 Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511. 13 Newbold C, Craig A, Kyes S, Rowe A, Fernandez- Reyes D & Fagan T (1999) Cytoadherence, pathogene- sis and the infected red cell surface in Plasmodium falci- parum. Int J Parasitol 29, 927–937. 14 Chen Q, Schlichtherle M & Wahlgren M (2000) Molecu- lar aspects of severe malaria. Clin Microbiol Rev 13 , 439–450. 15 Su XZ, Heatwole VM, Wertheimer SP, Guinet F, Herr- feldt JA, Peterson DS, Ravetch JA & Wellems TE (1995) The large diverse gene family var encodes pro- teins involved in cytoadherence and antigenic variation of Plasmodium falciparum-infected erythrocytes. Cell 82, 89–100. 16 Baruch DI, Pasloske BL, Singh HB, Bi X, Ma XC, Feldman M, Taraschi TF & Howard RJ (1995) Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes. Cell 82, 77–87. 17 Beeson JG & Brown GV (2002) Pathogenesis of Plasmo- dium falciparum malaria: the roles of parasite adhesion and antigenic variation. Cell Mol Life Sci 59, 258–271. 18 Chookajorn T, Dzikowski R, Frank M, Li F, Jiwani AZ, Hartl DL & Deitsch KW (2007) Epigenetic mem- ory at malaria virulence genes. Proc Natl Acad Sci USA 104, 899–902. 19 Eksi S, Czesny B, van Gemert GJ, Sauerwein RW, Eling W & Williamson KC (2006) Malaria transmis- sion-blocking antigen, Pfs230, mediates human red R. Tuteja Malaria ) an overview FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 4677 blood cell binding to exflagellating male parasites and oocyst production. Mol Microbiol 61, 991–998. 20 Baruch DI (1999) Adhesive receptors on malaria-para- sitized red cells. Baillieres Best Pract Res Clin Haematol 12, 747–761. 21 Ockenhouse CF, Ho M, Tandon NN, Van Seventer GA, Shaw S, White NJ, Jamieson GA, Chulay JD & Webster HK (1991) Molecular basis of sequestration in severe and uncomplicated Plasmodium falciparum malaria: differential adhesion of infected erythrocytes to CD36 and ICAM-1. J Infect Dis 164, 163–169. 22 Snow RW & Marsh K (1998) New insights into the epidemiology of malaria relevant for disease control. Br Med Bull 54, 293–309. 23 Van Geertruyden JP, Thomas F, Erhart A & D’Aless- andro U (2004) The contribution of malaria in preg- nancy to perinatal mortality. Am J Trop Med Hyg 71 (Suppl. 2), 35–40. 24 Beeson JG, Reeder JC, Rogerson SJ & Brown GV (2001) Parasite adhesion and immune evasion in placen- tal malaria. Trends Parasitol 17, 331–337. 25 Beeson JG, Brown GV, Molyneux ME, Mhango C, Dzinjalamala F & Rogerson SJ (1999) Plasmodium falci- parum isolates from infected pregnant women and chil- dren are associated with distinct adhesive and antigenic properties. J Infect Dis 180, 464–472. 26 Bell D, Wongsrichanalai C & Barnwell JW (2006) Ensuring quality and access for malaria diagnosis: how can it be achieved? Nat Rev Microbiol 4, 682–695. 27 Ridley RG (2002) Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 415, 686–693. 28 Rosenthal P (2001) Antimalarial Chemotherapy and Mechanisms of Action. Resistance and New Directions in Drug Discovery. Humana Press, Totowa, NJ. 29 Miller LH & Greenwood B (2002) Malaria – a shadow over Africa. Science 298, 121–122. 30 Wiseman V, Kim M, Mutabingwa TK & Whitty CJ (2006) Cost-effectiveness study of three antimalarial drug combinations in Tanzania. PLoS Medicine 3, e373. 31 Todryk SM & Hill AV (2007) Malaria vaccines: the stage we are at. Nat Rev Microbiol 5, 487–489. 32 Girard MP, Reed ZH, Friede M & Kieny MP (2007) A review of human vaccine research and development: malaria. Vaccine 25, 1567–1580. 33 Sachs JD (2002) A new global effort to control malaria. Science 298, 122–124. 34 Heddini A, Keusch GT & Davies CS (2004) The multi- lateral initiative on malaria: past, present and future. Am J Trop Med Hyg 71 (Suppl. 2), 279–282. 35 Greenwood B & Mutabingwa T (2002) Malaria in 2002. Nature 415, 670–672. 36 Ballou WR, Herrera MA, Carucci D, Richie TL, Corradin G, Diggs C, Druilhe P, Giersing BK, Saul A, Heppner DG et al. (2004) Update on the clinical development of candidate malaria vaccines. Am J Trop Med Hyg 71 (Suppl. 2), 239–247. 37 Christophides GK (2005) Transgenic mosquitoes and malaria transmission. Cell Microbiol 7, 325–333. 38 Marrelli MT, Li C, Rasgon JL & Jacobs-Lorena M (2007) Transgenic malaria-resistant mosquitoes have a fitness advantage when feeding on Plasmodium -infected blood. Proc Natl Acad Sci USA 104, 5580–5583. 39 Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J & DeRisis JL (2003) The transcriptome of the intraerythr- ocytic developmental cycle of Plasmodium falciparum. PLoS Biology 1, E5. 40 Tuteja R & Pradhan A (2006) Unraveling the ‘DEAD- box’ helicases of Plasmodium falciparum. Gene 376, 1–12. 41 Tilley L, McFadden G, Cowman A & Klonis N (2007) Illuminating Plasmodium falciparum-infected red blood cells. Trends Parasitol [Epub ahead of print]. 42 Surolia N & Surolia A (2001) Triclosan offers protec- tion against blood stages of malaria by inhibiting enoyl- ACP reductase of Plasmodium falciparum. Nature Med 7, 167–173. 43 Gornicki P (2003) Apicoplast fatty acid biosynthesis as a target for medical intervention in apicomplexan para- sites. Int J Parasitol 33, 885–896. 44 Jomaa H, Wiesner J, Sanderbrand S, Altincicek B, Weidemeyer C, Hintz M, Turbachova I, Eberl M, Zeidler J, Lichtenthaler HK et al. (1999) Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285, 1573–1576. 45 Wiesner J & Jomaa H (2007) Isoprenoid biosynthesis of the apicoplast as drug target. Curr Drug Targets 8, 3–13. 46 Sato S & Wilson RJ (2002) The genome of Plasmodium falciparum encodes an active delta-aminolevulinic acid dehydratase. Curr Genet 40, 391–398. 47 Fichera ME & Roos DS (1997) A plastid organelle as a drug target in apicomplexan parasites. Nature 390, 407–409. 48 He CY, Striepen B, Pletcher CH, Murray JM & Roos DS (2001) Targeting and processing of nuclear-encoded apicoplast proteins in plastid segregation mutants of Toxoplasma gondii. J Biol Chem 276, 28436–28442. 49 Waller RE, Reed MB, Cowman AF & McFadden GI (2000) Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. EMBO J 19, 1974–1802. 50 Seow F, Sato S, Janssen CS, Riehle MO, Mukhopadhy- ay A, Phillips RS, Wilson RJ & Barrett MP (2005) The plastidic DNA replication enzyme complex of Plasmo- dium falciparum. Mol Biochem Parasitol 141, 145–153. 51 McConkey GA, Rogers MJ & McCutchan TF (1997) Inhibition of Plasmodium falciparum protein synthesis. Targeting the plastid-like organelle with thiostrepton. J Biol Chem 272, 2046–2049. Malaria ) an overview R. Tuteja 4678 FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 52 Goodman CD, Su V & McFadden GI (2007) The effects of anti-bacterials on the malaria parasite Plasmo- dium falciparum. Mol Biochem Parasitol 152, 181–191. 53 Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL et al. (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520–526. 54 Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, Eling WM, Hall N, Waters AP, Stunnenberg HG et al. (2002) Analysis of the Plasmo- dium falciparum proteome by high-accuracy mass spec- trometry. Nature 419, 537–542. 55 Pizzi E & Frontali C (2001) Low-complexity regions in Plasmodium falciparum proteins. Genome Res 11, 218–229. 56 Bozdech Z, Zhu J, Joachimiak MP, Cohen FE, Pulliam B & DeRisi JL (2003) Expression profiling of the schiz- ont and trophozoite stages of Plasmodium falciparum with a long-oligonucleotide microarray. Genome Biol 4 (2), R9. 57 Llina ´ s M, Bozdech Z, Wong ED, Adai AT & DeRisi JL (2006) Comparative whole genome transcriptome analy- sis of three Plasmodium falciparum strains. Nucleic Acids Res 34, 1166–1173. 58 Ginsburg H (2006) Progress in in silico functional ge- nomics: the malaria metabolic pathways database. Trends Parasitol 22, 238–240. 59 Kanehisa M, Goto S, Kawashima S, Okuno Y & Hat- tori M (2004) The KEGG resource for deciphering the genome. Nucleic Acids Res 32, D277–D280. 60 Pinney JW, Shirley MW, McConkey GA & Westhead DR (2005) metaSHARK: software for automated meta- bolic network prediction from DNA sequence and its application to the genomes of Plasmodium falciparum and Eimeria tenella. Nucleic Acids Res 33, 1399–1409. R. Tuteja Malaria ) an overview FEBS Journal 274 (2007) 4670–4679 ª 2007 The Author Journal compilation ª 2007 FEBS 4679 . attachment) and the release of substances from the rhoptry and microneme organ- elles, leading to formation of the parasitophorous R. Tuteja Malaria ) an overview FEBS. mosquitoes transmitted malaria. The Italian professor Giovanni Battista Grassi subsequently showed that human malaria could only be transmitted by Anopheles