Plasmodium Falciparum Life Cycle

Plasmodium Falciparum Life Cycle Malaria is one of the world leading causes of death, especially among people living in sub-Sahara Africa and other tropical regions. Of the five species of the genus Plasmodium, the malaria protozoan parasite, known to infect Man, Plasmodium falciparum is responsible for the most virulent, severe and dangerous form of human malaria. Over the years, chemotherapy has played central role in the strategies towards the eradication of this disease. However, ability of P. falciparum to develop resistance to effective and affordable drugs and to pyrathroids, the active principle of the insecticides treated nets (ITNs) has made constant search for new pharmacotherapy imperative. This review presents an overview of the life cycle of the causative organism (P. falciparum), the efforts at controlling the disease and the molecular and cellular basis of the infection, with special emphasis on molecular chaperones of the heat shock proteins family as critical components of the parasite intra-erythr ocytic development and survival. The motivation for the present work is also presented. 1.1 Introduction Malaria, whose pathogen is transmitted by female Anopheles mosquitoes, is both preventable and curable, but yet still impacting negatively on the health of millions of people and account for high rate of mortality, especially among children in sub-Sahara Africa (Breman, 2001; Greenwood et al., 2008; Hay et al., 2004; Rowe et al., 2006; Snow et al., 2005). Five species of the genus Plasmodium, the protozoan parasites, responsible for malaria infection are known to infect humans. They are P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi. It has been proposed that P. ovale consist of two species (Cox-Singh, 2010) and that zoonosis is the medium through which P. knowlesi infect humans (White, 2008). Of these, P. falciparum and P. vivax are numerically most important, with the former responsible for the most virulent, severe and dangerous form of human malaria (Greenwood et al., 2008). The World Health Organization (WHO) 2011 Malaria reports (WHO | World Malaria Report 2011, 2012) estimated a total of 216 million episodes of malaria in 2010 with at least 655 000 deaths, mostly in Africa and among children under the age of 5 years. Malaria was reported to be prevalent in 99 countries with an estimated 3.3 billion people at risk. Though, supports by international donors has led to rapid decrease in malaria mortality, especially among adults in Africa, Murray et al., (2012) contended that the malaria mortality burden may actually be larger than previously estimated and that for the necessary elimination and eradication to be achieved at larger scale, there is an urgent need for more supports. Factors, such as lack ofsanitation, malnutrition, lack or reduced access to medications, poverty and the location of many of the poor countries affected by malaria in the tropical zones, all combined together to create an enabling environment for the disease to thrive. Though pre ventive approaches such as good sanitation and distribution of insecticide treated nets (ITNs) (Curtis et al., 2006), have been employed as strategy towards the eradication of this disease, chemotherapy remains the most widely used approach. The ability of P. falciparum to develop resistance to effective and affordable drugs (Cheeseman et al., 2012; Jambou et al., 2005; Mller Hyde, 2010; Phyo et al., 2012) and to pyrethroids, the active principle of the insecticides treated nets (ITNs) (Fane et al., 2012; NGuessan et al., 2007) has made constant search for new pharmacotherapy imperative. Malaria parasite life cycle is a complex mechanism involving two hosts, human and female Anopheles mosquitoes. However, the clinical symptoms of the disease are associated with the invasion of the erythrocytes by the parasite, its growth, division inside the host cell and the cyclic cell lysis and reinvasion of new erythrocytes. The intra-erythrocytic survival and development of the parasite as well as the pathology of the infection are linked to structural and functional remodeling of the host cell through the export of parasite-encoded proteins (Botha et al., 2007; Miller et al., 2002; Pesce Blatch, 2009; Przyborski Lanzer, 2005). Meanwhile, attempts have been made to present an extensive description of the protein interaction network for P. falciparum (LaCount et al., 2005) and about 300 parasite-encoded proteins are predicted to be exported (Marti et al., 2004; Sargeant et al., 2006). Among the exported proteins are the molecular chaperones of the heat shock protein family (Nya lwidhe Lingelbach, 2006). Molecular chaperone are a family of proteins that function to stabilize proteins, facilitate their translocation across intracellular membranes, their degradation, and ensure that proteins in a cell are properly folded and functional (Hartl Hayer-Hartl, 2002; Hartl, 1996). PFA0660w belongs to an extended family of Hsp40 proteins predicted to be transported by the parasite into the host cell (Hiller et al., 2004; Marti et al., 2005; Sargeant et al., 2006). It is a Type II Hsp40 protein, said to be homologous to human DnaJB4, a cytosolic type II Hsp40, known to interact with human Hsp70 to facilitate protein folding, transport and assembly (Botha et al., 2007). Recent studies have localized PFA0660w into structures in the infected erythrocyte, called the J-dots (Kulzer et al., 2010); said to be exported in complex with P. falciparum Hsp70-x (PfHsp70-x) into the J dots (Kulzer et al., 2012) and failure to obtain a viable PFA0660w-knocked-out parasite (Maier et al., 2008), suggests that it may be essential for the survival of the parasite in the infected erythrocytes and therefore a potential target for drug action. 1.2 Malaria Infection 1.2.1 Background information Malaria, though curable and preventable, remains a life-threatening disease that was noted in more than 4,000 years ago and has being responsible for millions of death. World Health Organization (WHO) listed malaria among the most important infectious diseases of the tropics and form part of the sixth millennium development goal (MDG 6) (WHO | MDG 6: combat HIV/AIDS, malaria and other diseases, 2012). The target 6C of MDG 6 is to bring malaria and other major diseases to a halt by 2015 and begin to reverse their incidences. Strategies advocated by WHO to combat malaria includes prevention with the use of long-lasting insecticides treated bed-nets (ITNs) and indoor residual spraying, and rapid treatment with effective anti-malarial medicines with special focus on pregnant women and young children. WHO Roll Back Malaria further recommends that to control Plasmodium falciparum malaria during pregnancy, in addition to individual protection with ITNs and prompt management of anaemia and m alaria using effective anti-malaria drugs, intermittent preventive treatment (IPTp) or chemoprophylaxis should be encouraged (WHO | Malaria in pregnancy, 2012). Though, the World Health Organization (WHO) 2011 Malaria reports (WHO | World Malaria Report 2011, 2012) estimated at least 655 000 deaths as a result of malaria infection, mostly in Africa and among children under the age of 5 years, the mortality burden may actually be larger than previously estimated, thus, the need for improved supports by the funding organization to be able to achieve the much needed malaria elimination and eradication (Murray et al., 2012). Malaria is caused by the transmission of parasites to humans by female Anopheles mosquitoes during a blood meal. Plasmodium falciparum is known to be responsible for high rate of mortality, especially among children in sub-Saharan Africa, mostly under age 5 years (Breman, 2001; Greenwood et al., 2008; Hay et al., 2004; Rowe et al., 2006; Snow et al., 2005). Apart from the fact that many of the countries that are mostly affected are located in the tropical region of the world, increasing level of poverty, with its attendant economic consequences, coupled with lack of or improper sanitation and reduced access to prompt medication are factors that are creating enabling environment for the disease to thrive. Though preventive approaches such as the use of insecticide treated bed nets, IPTp and chemoprophylaxis with good sanitation (Curtis et al., 2006; WHO | Malaria in pregnancy, 2012), have been employed as strategy towards the eradication of this disease, the use of chemotherapeutic drugs remains the most widely used approach (Butler et al., 2010; DAlessandro, 2009). However, the success of this strategy has been hampered by the resilient of the parasite in continually creating resistance to the available drugs. The ability of P. falciparum to develop resistance to effective and affordable drugs (Cheeseman et al., 2012; Jambou et al., 2005; Mller Hyde, 2010; Phyo et al., 2012) and to pyrethroids, the active principle of the insecticides treated nets (ITNs) (Fane et al., 2012; NGuessan et al., 2007), has made constant search for new pharmacotherapy imperative. However, notwithstanding the centrality of chemoprophylaxis and chemotherapy in efforts at combating the menace of malaria infe ction (DAlessandro, 2009), and wide distribution of insecticide-impregnated bed nets, efforts aimed at enhancing long lasting protective immunity through vaccination, of which RTS,S is emerging as most promising vaccine formulation, have also been intensified (Ballou, 2009; Casares et al., 2010). 1.2.2 Life Cycle of Plasmodium falciparum Malaria parasite life cycle (Figure 1.1) is a complex mechanism involving two hosts, human and female Anopheles mosquitoes. The survival of the parasite during several stages of its development depends on its ability to invade and grow within multiple cell types and to evade host immune responses by using their specialized proteins (Florens et al., 2002; Greenwood et al., 2008). Sporozoites (infective stage), merozoites (erythrocytes invading stage), trophozoites (multiplying form in erythrocytes), and gametocytes (sexual stages) are stages involved in the development of the parasite. These stages are unique in shapes, structures and complementary proteins. The continuous changes in surface proteins and metabolic pathways during these stages help the parasites to survive the host immune response and create challenges for drugs and vaccines development (Florens et al., 2002). The sporogony or sexual phase occurs in mosquitoes, resulting in the development of numerous infective forms of the parasites which when ingested by human host induced disease. During a blood meal by female Anopheles mosquitoes from an individual infected with malaria, the male and female gametocytes of the parasite enter into the gut of the mosquito, adjust itself to the insect host environment and initiate the sporogonic cycle. The fusion of male and female gametes produced zygotes, which subsequently develop into actively moving ookinetes that pierced into the mosquito midgut wall to develop into oocysts. Each oocyst divides to produce numerous active haploid forms called sporozoites which are subsequently released into the mosquitos body cavity following the burst of the oocyst. The released sporozoites travel to and invade the mosquito salivary glands, from where they get injected into the human bloodstream during another blood meal, causing malaria infection (Barillas-Mury Kum ar, 2005; Ferguson Read, 2004; Hill, 2006). The parasite life cycle traverse two hosts (Man and Mosquito) with each stage involving complex cellular and molecular modifications. To prevent blood clots, Sporozoites infected saliva are deposited into Man during blood meal by female Anopheles mosquitoes, make their way to the liver, develop over time into hypnozoites (dormant stage, usually responsible for relapse of infection) or merozoites (that are released into blood stream to invade erythrocytes). The clinical symptoms of the disease are associated with the invasion of the erythrocytes by the parasite, its growth, division inside the host cell and the cyclic cell lysis and reinvasion of new erythrocytes. The schizogony or asexual phase of the life cycle occurs in human host. The cycle is initiated from the liver by the ingested sporozoites and later continues within the red blood cells, resulting in the clinical manifestations of the malaria disease. Following the introduction of invasive sporozoites into the skin after mosquito bite, they are either destroyed by macrophages, enter the lymphatics and drain into the lymph nodes from where they can develop into exoerythrocytic stages (Vaughan et al., 2008) and prime the T cells as a way of mounting protective immune response (Good Doolan, 2007) and/or blood vessel (Silvie et al., 2008b; Vaughan et al., 2008; Yamauchi et al., 2007), from where they made their way into the liver. While in the liver, sporozoites negotiate through the liver sinusoids, entered into hepatocytes, followed by multiplication and growth in parasitophorous vacuoles into schizonts, each of which contains thousands of merozoites, especially with P. falciparum (Ami no et al., 2006; Jones Good, 2006; Kebaier et al., 2009). Thrombospondin-related anonymous protein (TRAP) family and an actinmyosin motor has been show to help sporozoites in its continuous sequence of stick-and-slip motility (Baum et al., 2006; Mnter et al., 2009; Yamauchi et al., 2007) and that it growth and development within the liver cells is facilitated by the circumsporozoite protein of the parasite (Prudncio et al., 2006; Singh et al., 2007). This stick and slip motility prevent the parasite from been washed away by the circulating blood into kidney from where they can be destroyed and removed from the body. Motility is driven by an actin-myosin motor located underneath the plasma membrane. The sporozoite journey is propelled by a unique actin-myosin system, which allows extracellular migration, cell traversal and cell invasion (Kappe et al., 2004).This is a single cycle phase with no clinical symptoms, unlike the erythrocytic stage, which occurs repeatedly and characterize d with clinical manifestation. The hepatocytic merozoites are stored in vesicles called merosomes where they are protected from the phagocytotic action of Kupffer cells. The release of these merozoites into the blood stream via the lung capillaries initiates the blood stage of the infection (Silvie et al., 2008b). In some cases (as it can be found with P. vivax and P. ovale malaria) dormant sporozoites, called hypnozoites, are formed and remain in the liver for a long time. These hypnozoites are usually responsible for the development of relapse of clinical malaria infection and has been reported to be genotypically different from the infective sporozoites ingested after a mosquito bite (Cogswell, 1992; Collins, 2007). The development of the parasite within the red blood cells occur with precise cyclic accuracy with each repeated cycles producing hundreds of daughter cells that subsequently invades more red blood cells. The clinical symptoms of the disease are associated with the invasion of the erythrocytes by th e parasite, its growth, division inside the host cell and the cyclic cell lysis and reinvasion of new erythrocytes. The invasion of RBCs by the merozoites takes place within seconds and made possible by series of receptorligand interactions. The ability of the merozoites to quickly disappear from circulation into the RBCs protect its surface antigens from exposure to the host immune response (Cowman Crabb, 2006; Greenwood et al., 2008; Silvie et al., 2008b). Unlike P. Vivax, which invade the RBCs by binding to Duffy blood group, the more virulent P. falciparum possess varieties of Duffy binding-like (DBL) homologous proteins and the reticulocyte binding-like homologous proteins that allows it to recognize and bind effectively to different RBC receptors (Mayer et al., 2009; Weatherall et al., 2002). Micronemes, rhoptries, and dense granules are the specialized apical secretory organelles of the merozoite that help the merozoites to attach, invade, and establish itself in the red cel l. The successful formation of stable parasitehost cell junction is followed by entering into the cells through the erythrocyte bilayer. This entrance is made possible with the aid of the actinmyosin motor, proteins of the thrombospondin-related anonymous protein family (TRAP) and aldolase, leading to the creation of a parasitophorous vacuole, that isolate the intracellular ring parasite from the host-cell cytoplasm, thereby creating a conducive environment for its development (Bosch et al., 2007; Cowman Crabb, 2006; Haldar Mohandas, 2007). The intra-erythrocytic parasite is faced with the challenge of surviving in an environment devoid of standard biosynthetic pathways and intracellular organelles in the red cells. This challenge is overcome by the ability of the parasite to adjust its nutritional requirement to haemoglobin only, formation of a tubovesicular network, thereby expanding it surface area and by export of a range of remodeling and virulence factors into the red cell (Silvie et al., 2008b). Following the ingestion of the hemoglobin into the food vacuole, it is degraded to make available the amino acids for protein biosynthesis. Heme is a toxic free radical capable of destroying the parasite within the red blood cells. Heme polymerase is used by the parasite for the detoxification of heme and the resulting hemozoin is sequestrated as hemozoin. As the parasite grows and multiplies, new permeation pathways are created in the host cell membrane to help in the uptake of solutes from the extracellular medium, disp osal of metabolic wastes, and in initiating and sustaining electrochemical ion gradients, thereby preserving the osmotic stability of the infected red cells and thus, premature hemolysis (Kirk, 2001; Lew et al., 2003). 1.2.3 Control of Malaria infection Preventive measures are a critical step towards the control and eradication of malaria. Preventive approach can broadly be divided into two Infection control and Vector control. Infection control focuses on preventing the development of the disease as a result of occasional mosquito bite or relapse of previous infection (Lell et al., 2000; Walsh et al., 1999b). This involves the use of chemoprophylaxis. Travellers to malaria endemic countries are expected to start prophylaxis at least two weeks before and to continue up to two weeks after. One important target group in the infection control using chemoprophylaxis are the pregnant women. Intermittent preventive treatment for pregnant women (IPTp) is the globally acknowledge approach for prevention of malaria in pregnancy (Vallely et al., 2007; WHO | Malaria in pregnancy, 2012). Sulphadoxin-pyrimethamine (SP) has been used for this purpose and there are compelling arguments for the use of artesunate-SP (Jansen, 2011). To ensure long lasting prevention, this approach should be combined with vector control. Vector control focuses on protecting against mosquitoes bite, thereby preventing the transmission of the parasite to Man. Strategies for vector control include the use of residual spraying of insecticides, insect repellent cream or spray, sleeping under bed nets, especially, the insecticide impregnated bed nets (ITNs) and proper sanitation (Curtis et al., 2006; Lavialle-Defaix et al., 2011; WHO | Insecticide-treated materials,). WHO provides guideline for the production, preparation, distribution and the use of the ITNs (WHO | Insecticide-treated materials,). With the reported resistance to pyrathroids, an active principle of the insecticides treated bed nets (Fane et al., 2012; NGuessan et al., 2007), all strategies involving the use of chemical agents, also faces the global challenge of developing resistance. Training in proper sanitation and its sustainability from generation to generation is most probably the best approach in controlling the malaria disease. Personal and general hygiene which involve in-door and out-door cleaning, good refuse disposal practices, eradication of stagnant water, proper sewage disposal and clean, dry and uninterrupted drainages are examples of good sanitation practices that will not only prevent malaria infection, but also other killer diseases of the tropics. Sanitation is not only cheap and affordable; it is within the reach of everybody. 1.2.3.2 Malaria Chemotherapy Despite the use of preventive approaches outlined above (Curtis et al., 2006; WHO | Malaria in pregnancy, 2012), as strategy towards the eradication of this malaria, the use of chemotherapeutic drugs remains the most widely used approach (Butler et al., 2010; DAlessandro, 2009). They are widely employed as prophylaxis, suppressive and curative. However, the success of this strategy has been hampered by the resilient of the parasite in continually creating resistance to the available drugs. The ability of P. falciparum to develop resistance to effective and affordable drugs (Cheeseman et al., 2012; Jambou et al., 2005; Mller Hyde, 2010; Phyo et al., 2012) and to pyrethroids, the active principle of the insecticides treated nets (ITNs) (Fane et al., 2012; NGuessan et al., 2007), has made constant search for new pharmacotherapy imperative. Various approaches have been employed to identify new antimalaria agents with a view to reducing cost, ensuring availability and reducing the incidences of resistance (Rosenthal, 2003). Chemical modification of the existing antimalarial is a simple approach and required no extensive knowledge of the mechanism of drug action and the biology of the infection. Many drugs in use today have been produced using this approach, including chloroquine, primaquine and mefloquine from quinine (Stocks et al., 2001), 8-aminoquinoline, tafenoquine, from primaquine (Walsh et al., 1999a) and lumefantrine from halofantrine (van Vugt et al., 2000). Another approach is the use of plant derived compound with little or no chemical modification has led to the discovery of potent antimalarial such as artemisinins (Meshnick, 2001). Also, the use of other agents not originally designed for malaria such as folate antagonists, tetracyclines and antibiotics that were reported to be active against malaria parasit es (Clough Wilson, 2001) is another viable approach to drug discovery. Resistance reversals such verapamil, desipramine and trifluoperazine (van Schalkwyk et al., 2001) have also been used in combination with antimalaria drugs to improve therapy. Optimization of therapy with existing antimalaria agents is widely used as a productive approach towards improving therapy. Optimization of therapy underscore the need for combination therapy with newer and older drugs and with agents that are not original designed as antimalaria but which can potentiate the antimalaria property and/or block resistance to antimalaria agents. Thus for the combination to be ideal, it should improve antimalarial efficacy, providing additive or synergistic antiparasitic activity and slow the progression of parasite resistance to the antimalaria agents. For example, combination of artesunate with sulfadoxine/pyrimethamine (von Seidlein et al., 2000) or with amodiaquine (Adjuik et al., 2002), if devoid of underlying resistance to the artesunate partners which can lead to high rates recrudescence (Dorsey et al., 2002), may prove to be optimal antimalarial agents. Other combinations that have been effectively used include artesunate and mefloquine (Price et al., 1997) and artemether and lumefantrine (Lefevre et al., 2001). The combination of analog of proguanil (chlorproguanil) with dihydropteroate synthase (DHPS) inhibitor (dapsone), originally produced to treat leprosy (Mutabingwa et al., 2001) has open up a new and effective approach in antimalaria drug therapy. The use of dapsone and other drug resistance reversers such as verapamil, desipramine, trifluoperazine (van Schalkwyk et al., 2001) and Chlorpheniramine (Sowunmi et al., 1997) has shown potential for reducing the rate of drug resistance. Table 1.1: Classes and Mechanism of Antimalarial drugs CLASSES OF DRUGS Gametocidal Tafenoquine Gamatocidal Gametocidal biguanides (proguanil, cycloguanil), Trimethoprim CLASSES OF DRUGS Clindamycin, Spiramycin m ubiquinol to cytochrome C (Vaidya, 2001). Meanwhile, one important and innovative approach towards drug discovery in malaria chemotherapy is the search for new antimalaria drug target. Such targets include parasite membrane (Vial Calas, 2001), food vacuole (Banerjee et al., 2002), mitochondrial and apicoplast (Ralph et al., 2001; Vaidya, 2001). The cytosol, which is the centre of metabolic activities (e.g. folate metabolism and glycolysis) and enzymes activities have proven to be valuable as potential target for drug action (Plowe, 2001; Razakantoanina et al., 2000). To survive and develop within the erythrocytes, Plasmodium falciparum export most of its virulent factors into the cytosol of the infected erythrocytes. Among these are the molecular chaperones of the heat shock proteins which are focus of many researches and are increasingly gaining ground as potential target of drug action (Behr et al., 1992; Kumar et al., 1990). 1.2.3.3 Malaria Vaccines Notwithstanding the centrality of chemoprophylaxis and chemotherapy in efforts at combating the menace of malaria infection (DAlessandro, 2009), and wide distribution of insecticide-impregnated bed nets, efforts aimed at enhancing long lasting protective immunity through vaccination, of which RTS,S is emerging as most promising vaccine formulation, has been intensified (Ballou, 2009; Casares et al., 2010). These attempts at producing an effective vaccine against malaria infection has, however, for many years proved unsuccessful (Andr, 2003; Artavanis-Tsakonas et al., 2003). Having a vaccine that can completely block transmission from human to mosquito host can be a major limp towards global eradication of malaria. But, the absences of such immunity may explain the possible partnership between the parasite and the host, developed over a long time of co-habitation (Evans Wellems, 2002). On the other hand, a vaccine developed in line with the model of naturally acquired immunity that o ffers protections against morbidity and mortality, offers more encouragement. Such a vaccine will be a major step in the right direction and may not require regular booster vaccination like it would with vaccine that target infection transmission blockage (Struik Riley, 2004). Meanwhile, the development of natural immunity, after a long term exposure to the infection, especially with people living in the endemic areas has been reported (Baird, 1995; Hoffman et al., 1987; Rogier et al., 1996). The rate of acquired immunity in infants is faster than older children, but they also stand the chance of higher risk of developing severe malaria infection and anaemia (Aponte et al., 2007). Though, adults who, having obtained naturally acquired immunity, migrated to malaria-free zones, stands the risk of contacting the diseases upon return to their endemic region, documentary evidences however revealed that their responses to such re-infection are very rapid and tend to respond to treatment and recover faster than those who have not been previously xposed. (Di Perri G et al., 1994; Jelinek et al., 2002; Lepers et al., 1988). While this naturally acquired immunity is beneficial, it leaves the most vulnerable population (children and pregnant women though the mother may be immune, the foetus is not) at risk, as they are yet to gain enough exposure for such immunity to take place. Aponte et al., (2007) also showed that a reduced exposure toP. falciparumantigens through chemoprophylaxis early in life have the potential to delay immunity acquisition. Furthermore, it does not appear that naturally acquired immunity have any effect on transmission of malaria. This further explained the possibility of an evolving host-parasite relationship (Evans Wellems, 2002), which might have been developed over a long time host-parasite co-evolution. Therefore, understanding the compromises that may have developed over time between the parasite and the host may be an important approach towards developing a much needed vaccine. 1.3 Molecular and Cellular Basis of Malaria Infection Following blood meal by an Anopheles female mosquito accompany with the release of saliva to prevent blood coagulation (Beier, 1998), malaria parasites are deposited or ejection of into the skin (Frischknecht et al., 2004; Vanderberg Frevert, 2004). By continuous gliding in the skin, the sporozoite reach a blood vessel, breach the endothelial barrier and enter the blood circulation (Amino et al., 2007; Vanderberg Frevert, 2004) and/or breach a lymphatic vessel to enter the draining lymph node, where exoerythrocytic stages of sporozoites development may take place (Amino et al., 2006). A micronemal protein, called thrombospondin-related anonymous protein (TRAP), has been shown to be responsible for the gliding motility and invasion mosquito vector salivary gland and in mammalian host (Kappe et al., 1999). The sporozoite transversal to the liver and the merozoites invasion and remodeling of the host cells are complex but necessary processes for the survival and development of the par asite. 1.3.1 Cell Transversal Sporozoite possesses the ability to transverse cells i.e move in and out of the host cells by membrane disruption (Mota et al., 2002, 2001; Vanderberg Stewart, 1990). Among the proteins secreted by the micronemes that have been implicated in host cell traversal are SPECT1 (sporozoite microneme proteinessential forcelltraversal 1) and SPECT2 (Ishino et al., 2005, 2004). The absence of SPECT1orSPECT2in mutant sporozoite does not prevent gliding motility but prevent migration through host cells (Ishino et al., 2004).Other proteins of importance to sporozoite cell traversal prior to hepatocyte infection, includes TRAP-Like Protein (Moreira et al., 2008), a sporozoite secreted phospholipase (Bhanot et al., 2005), and celltraversal protein forookinete andsporozoite (Kariu et al., 2006). Similarly, the circumsporozoite protein (CSP) probably plays a role in targeting sporozoites to hepatocytes by interacting with heparin sulfate proteoglycans (Sinnis Sim, 1997). 1.3.2 Liver stage development Upon entering the bloodstream, infectious sporozoite makes it way to the liver. Circumsporozoite protein (CSP) is highly expressed at this stage of the parasite life cycle. Using the sporozoites that expresses fluorescent proteins under the control of CSP and intravital imaging, Frevert and colleagues were able to show the movement of sporozoites in the liver (Frevert et al., 2005). The study showed that sporozoite migrates through several hepatocytes before finally settling in one, form PV and begin the liver stage development. CSP, mediated by low-density lipoprotein receptor-related protein LRP-1, and other highly expressed proteins by Kupffer cells, play an important role in inhibiting the generation of reactive oxygen species via the generation of cyclic AMP (cAMP) which stimulates adenyl cyclase activity (Usynin et al., 2007). Ishino and co-workers reported that two parasite molecules P36 and P52/P36p are involved in sporozoite invasion of hepatocytes with the formation of a PV membrane (PVM) (Ishino et al., 2005). Apart from CSP, other gene product that has been implicated in liver stage development of the parasite includes sporozoite low complexity asparagine-rich protein (SAP1) (Aly et al., 2008) and sporozoite and liver stage asparagine-rich protein (SLARP) (Silvie et al., 2008a) 1.3.3 Erythrocyte Invasion Erythrocyte invasion involves four steps, namely, initial merozoites binding, reorientation and erythrocyte deformation, specific interaction and junction formation and parasite entry (Figure 1.2). Merozoite surface protein-1 (MSP-1) is a well characterized merozoite surface proteins implicated in initial merozoite binding. It has been reported to be uniformly distributed over the merozo