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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-erythrocytic development and survival. The motivation for the present work is also presented.
TABLE OF CONTENT
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 ofÂ sanitation, 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 preventive 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; Müller & Hyde, 2010; Phyo et al., 2012) and to pyrethroids, the active principle of the insecticides treated nets (ITNs) (Fane et al., 2012; N'Guessan 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 (Nyalwidhe & 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 (Külzer et al., 2010); said to be exported in complex with P. falciparum Hsp70-x (PfHsp70-x) into the J dots (Külzer 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 malaria 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; D'Alessandro, 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; Müller & Hyde, 2010; Phyo et al., 2012) and to pyrethroids, the active principle of the insecticides treated nets (ITNs) (Fane et al., 2012; N'Guessan 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 infection (D'Alessandro, 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 mosquito's 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 & Kumar, 2005; Ferguson & Read, 2004; Hill, 2006).
Figure 1.1: The Life Cycle of Plasmodium falciparum.
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 (Amino et al., 2006; Jones & Good, 2006; Kebaier et al., 2009). Thrombospondin-related anonymous protein (TRAP) family and an actin-myosin motor has been show to help sporozoites in its continuous sequence of stick-and-slip motility (Baum et al., 2006; Münter 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 (Prudêncio 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).Â This is a single cycle phase with no clinical symptoms, unlike the erythrocytic stage, which occurs repeatedly and characterized 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 the 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 receptor-ligand 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 cell. The successful formation of stable parasite-host cell junction is followed by entering into the cells through the erythrocyte bilayer. This entrance is made possible with the aid of the actin-myosin 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, disposal 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; N'Guessan 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.
18.104.22.168 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; D'Alessandro, 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; Müller & Hyde, 2010; Phyo et al., 2012) and to pyrethroids, the active principle of the insecticides treated nets (ITNs) (Fane et al., 2012; N'Guessan 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 parasites (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
MODE OF ACTIONS
MECHANISM OF ACTIONS
Accumulate at high concentrations in the parasite acid food vacuole, binding directly to heme, and inhibition of heme ferriprotoporphyrin-IX polymerase, vacuolar phospholipase and protein synthesis (Bray et al., 1999; Foster, 1994; O'Neill et al., 1998; Pukrittayakamee et al., 2000; Russelltt & Goldberg, 1996)
Possibly by interfering with mitochondrial function (Beaudoin & Aikawa, 1968; Boulard et al., 1983; Lell et al., 2000; Walsh et al., 1999a)
Interacting with heme (Chou et al., 1980), and by inhibition of both heme polymerisation (Chou & Fitch, 1993; Slater & Cerami, 1992) and activity of heme catalase (de Almeida Ribeiro et al., 1997).
May have effect on parasite acid vesicles (Roos & Boron, 1981) similar to that of chloroquine (Krogstad et al., 1985). Mefloquine-binding proteins (Desneves, 1996) and plasmodial P-glycoprotein homolog-1, (Pgh-1) (Cowman et al., 1994) can also be a potential target of mefloquine action and resistance.
Mimick PABA to compete for DHPS active sites, thereby inhibiting the formation of dihydropteroate by dihydropteroate synthetase. (Zhang & Meshnick, 1991)
Mimick dihydrofolate to compete for DHFR active sites, thereby inhibiting the reduction of di- to tetra-hydrofolate which is a cofactor for folic acid biosynthesis (Brown, 1962; Futterman, 1957)
Table 1.1: Classes and Mechanism of Antimalarial drugs (cont.)
CLASSES OF DRUGS
MODE OF ACTIONS
MECHANISM OF ACTIONS
Possibly by acting against 70S ribosomes in the parasite mitochondrion (Divo et al., 1985; Geary & Jensen, 1983).
Act by binds to plasmepsin which is a haemoglobin degrading enzyme and to haematin. (Blauer & Akkawi, 1997; Lelièvre1 et al., 2012)
May be connected with inhibition of the formation of ß-haematin through the formation of complex with haemin. (Lelièvre1 et al., 2012)
Sesquiterpene lactone endoperoxide
Artemisinins and its derivatives
Interacts with reduced iron of heme moiety derived from hemoglobin digestion (Golenser et al., 2006) or free intracellular reduced iron species(O'Neill et al., 2010), leading to bioactivation of ART. They are also involved in heme alkylation (Robert et al., 2005), increased production of ROS (Wang et al., 2010) and oxidative membrane damage by interacting with phospholipids (Hartwig et al., 2009)
Atovaquone acts on the mitochondrial electron transfer chain and interfere with mitochondrial membrane potential (Rottenberg, 1997) possibly by inhibition of dihydroorotate dehydrogenase (DHODase), (Hudson, 1993; Krungkrai, 1995; Vaidya et al., 1993). Also, atovaquone may cause mitochondria depolarization (Fry & Pudney, 1992) or collapse of mitochondria membrane potential by inhibiting electron transfer from 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).
22.214.171.124 Malaria Vaccines
Notwithstanding the centrality of chemoprophylaxis and chemotherapy in efforts at combating the menace of malaria infection (D'Alessandro, 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 (André, 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 offers 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 toÂ P. falciparumÂ antigens 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 parasite.
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 proteinÂ essential forÂ cellÂ traversal 1) and SPECT2 (Ishino et al., 2005, 2004). The absence of SPECT1Â orÂ SPECT2Â in mutant sporozoite does not prevent gliding motility but prevent migration through host cells (Ishino et al., 2004).Â 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 cellÂ traversal protein forÂ ookinete andÂ sporozoite (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 merozoite surface and theÂ proteolytic processing of MSP-1 coincides with merozoite maturation and invasion (Cooper, 1993) and that antibodies against MSP-1 did inhibit invasion (Holder et al., 1994). For the parasite to gain entry into the host erythrocytes, the initial contact is followed by reorientation and erythrocyte deformation.
Figure 1.2: The Merozoites Invasion of Erythrocytes.
The invasion of erythrocytes by the merozoites is a fast process involving attachment, erythrocyte membrane disruption, reorientation, junction formation, PV formation, inward movement and resealing of erythrocyte and PV membranes. The transient nature of this process helps the parasite to evade the host immune system. The invasion is followed by host cell remodelling needed for parasite survival and development. (This image is a modification of QIAGEN's original, copyrighted image by Michael O. Daniyan. The original image may be found at https://www.qiagen.com/geneglobe/pathwayview.aspx?pathwayID=164
Apical membrane antigen-1 (AMA-1), a transmembrane protein localized at the apical end of the merozoite and binds erythrocytes, has been implicated in this reorientation (Mitchell et al., 2004). The inability of the antibodies against AMA-1 to prevent initial contact between merozoite and erythrocyte suggest that it is not involved in merozoite attachment. However, the antibodies prevent reorientation and thus block merozoite invasion (Mitchell et al., 2004). Content of the apical organelles (micronemes, rhoptries, and dense granules) also play important roles in invasion and establishment of the parasite within the host cells. Carruthers & Sibley (1997) showed that the release of the content of micronemes and rhoptries took place during initial contact with the host cell and the formation of the parasitophorous vacuole (PV) respectively. The dense granule contents may be involved in the host cell modification since its release takes place following complete parasite entry.
Merozoite reorientation and microneme release lead to the formation of tight junction between the merozoite apical end and the host cell, thereby providing an avenue for the binding of proteins localized to the micronemes with the receptors on the surface of the erythrocyte. Micromene proteins that have been identified in Plasmodium species include a 175 kDa erythrocyte binding antigen (EBA-175) ofÂ P. falciparum, Duffy-binding protein (DBP) of P. vivaxÂ andÂ P. knowlesi, PlasmodiumÂ sporozoite surface protein-2 (SSP2), also referred to as TRAP (thrombospondin-related adhesive protein) and their homologue, circumsporozoite- and TRAP-related protein.
1.3.4 Host cell remodelling
Host cell remodelling or modification provides an enabling environment for the intra-erythrocytic development and survival of the parasite. Host cell modification, such as cytoadherence of the infected erythrocytes to endothelial cells and subsequent sequestration of the mature parasites in capillaries provides a suitable microaerophilic environment for parasite metabolism, and protection from destruction by the spleen. Another important structural alteration are the knobs formation, which is an electron dense protrusion on the infected erythrocytes and several parasite proteins, including erythrocyte membrane protein-2 (PfEMP2), also called MESA and knob-associated histidine rich protein (KAHRP) are said to be associated with the knobs (Deitsch & Wellems, 1996). Crabb et al. (1997) showed that upon KAHRP disruption, there was a loss of knobs and the ability to cytoadhere. Also localized to the knobs is a polymorphic protein, calledÂ PfEMP1, and it translocation to the erythrocyte surface has been reported to depends in part on PfEMP3 (Waterkeyn et al., 2000) and PfHsp70-x (Külzer et al., 2012).
During the intra-erythrocytic stage of the parasite life cycle, it development and survival in an environment devoid of the necessary cellular machinery for protein trafficking depends on its ability to structurally and functionally remodel the host cell (Botha et al., 2007; Pesce & Blatch, 2009; Przyborski & Lanzer, 2005). This is made possible by exporting parasite proteins, termed exportome, into the host cell (Figure 1.3).
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Figure 1.3: Export of parasite proteins into the cytosol of infected Erythrocytes.
The parasite proteins meant for export are processed in the endoplasmic reticulum (ER) for onward movement into the plasma membrane by vesicular transport. The proteins to be exported are probably unfolded before they can be translocated by the Plasmodium translocon of exported proteins (PTEX) complex across the parasitophorous vacuole membrane (PVM) into the cytosol of the infected red blood cell (RBC). It has been suggested that there may be no need of separate export mechanism for both PEXEL containing and PEXEL negative export proteins (PNEPs) (Spielmann & Gilberger, 2010). (The diagram was adapted from Crabb et al., 2010)
These proteins, especially those with hydrophobic N-terminal sequence, are routed through the endoplasmic reticulum (ER) to the Golgi apparatus, from where they are carried by vesicular transport and release into the lumen of the parasitophorous vacuole (PV) (Adisa et al., 2003). ER plays an important role in protein synthesis, modification and intracellular transport. The ER compartment is where newly-synthesized polypeptides fold, serves as assembly of multimeric proteins, and provide glycoproteins with the needed asparagine-linked glycans. Proteins are retained in ER until they have acquired their correct conformation (Vitale et al., 1993). The transport of the parasite proteins across the parasitophorous vacuolar membrane (PVM) into the host erythrocytes has been proposed to be mediated by a pentameric motif called Plasmodium export element (PEXEL) or host targeting signal (HT) (Hiller et al., 2004; Horrocks & Muhia, 2005; Marti et al., 2004). However, the possibility that the parasite may be using more than one mode of export has been speculated since there are some exported proteins which lack a PEXEL or HT motif (Gormley et al., 1992). The existence of several PEXEL-negative exported proteins (PNEPs) indicates that alternative export pathways might also exist and that an unknown number of proteins might be missing from the currently predicted exportome. Among the PNEPs that has been identified and shown to be localized to Maurer's clefts (Blisnick et al., 2000; Hawthorne et al., 2004; Spielmann et al., 2006; Spycher et al., 2003; Vincensini et al., 2005) are the skeleton binding protein 1 (SBP1) (Blisnick et al., 2000) and the membrane associated histidine-rich protein 1 (MAHRP1) (Spycher et al., 2003) both of which are required for the cytoadherence ligand PfEMP1 to reach the erythrocyte surface (Cooke et al., 2006; Maier et al., 2007; Spycher et al., 2008). Also of importance are the ring-exported protein 1 and 2 (REX1&2) (Hawthorne et al., 2004; Spielmann & Gilberger, 2010; Spielmann et al., 2006). Meanwhile, Bhattacharjee et al. (2008) have reported that HT should be seen to function as a sorting signal that concentrate secretory parasite proteins destined to be exported into the cytosol of the infected erythrocyte into Maurer's clefts, rather than been regarded as mediator of protein translocation across the PVM. Also De Koning-Ward et al. (2009) showed that a translocon of exported proteins, named PTEX, in P. falciparum, is an important requirement for the export of PEXEL-containing proteins. These PTEX proteins were identified on the basis of their restriction within the Plasmodium genus, likelihood of an ATPase powered source, PVM localization, requirement for blood stage growth and binding specifically to their exported proteins. Among the identified proteins that appear to fulfil these criteria are PTEX150 (PF14_0344) and HSP101. These PTEX proteins were shown to bind specifically to PEXEL-motif containing exported proteins such as PF11_0037 and PF08_0137. Also, the inability of the authors to generate gene knockouts of P. falciparum PTEX150 suggests that this protein may be essential for the intra-erythrocytic survival of the parasite. Thus, it was proposed that once the PEXEL-proteins are deposited in the PV, they are recognised by some members of the PTEX translocon and processed for final translocation into cytosol of infected erythrocytes (De Koning-Ward et al., 2009).
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 part of the exportome (Marti et al., 2004; Sargeant et al., 2006). Among the exported proteins are the molecular chaperones of the heat shock protein family which also form a significant part of the PV proteome (Nyalwidhe & Lingelbach, 2006).
1.4 Molecular Chaperones
1.4.1 Background information
To successfully express polypeptide gene to produce functional proteins, it is important that the polypeptide chain is correctly folded into its native three-dimensional conformation, localized appropriately within or secreted from the cell, and properly assembled into multi-component complexes. Although the proper in vitro folding of a small protein into its native conformation can be dictated by the amino acid sequence alone, most polypeptides would fail to fold properly in the highly concentrated, complex, cellular environment without the assistance of yet another type of machinery. This latter type of protein machinery involves the molecular chaperones (Hartl & Hayer-Hartl, 2002; Hartl, 1996; Smith et al., 1998).
Molecular chaperone are family of proteins that function to stabilize proteins, facilitate their translocation across intracellular membranes e.g endoplasmic reticulum, mitochondrion, nucleus, and peroxisome or their degradation, and ensure that proteins in a cell are properly folded and functional (Hartl & Hayer-Hartl, 2002; Hartl, 1996). Purification techniques and the capability of the molecular chaperones to perform their folding and refolding functions has been reviewed (Longshaw et al., 2006). They, however, do not interact with native proteins, nor do they form part of the final folded structures. Some may be non-specific as they tend to interact with a wide variety of polypeptide chains, while others are usually restricted to specific targets (Hayes & Dice, 1996). Heat and other forms of cellular stress can increase the expression of molecular chaperones (Baker et al., 1984).
Though many chaperones function to catalyze the refolding of denatured proteins, some molecular chaperones are constitutively produced, showing that they have important functions even under normal conditions (Craig et al., 1993; Hayes & Dice, 1996; Parsell & Lindquist, 1993). Among the most important classes of molecular chaperones are the heat shock protein family of molecular chaperones of which 40kDa and 70 kDa heat shock proteins (Hsp40s and Hsp70s respectively) constitute and integral components.
1.4.2 Hsp70 Molecular Chaperones
Hsp70 are 70 kDa heat shock protein that constitute an integral components of the network of molecular chaperones and folding catalysts. Hsp70s consist of a 45kDa N-terminal ATP domain and a 25kDa C-terminal substrate binding domain (Figure 1.4A). The functions of Hsp70s can be broadly categorized into two: first as house keeper, in which case they form an integral component of folding and signal transduction pathways and participate in folding and assembling of newly synthesized proteins and their translocation across cell membranes. Second, as quality control manager, in which Hsp70s participate in controlling the activity of regulatory proteins and also screen proteins for any damage and repair the misfolded and aggregated proteins (Bukau et al., 2000; Hartl & Hayer-Hartl, 2002; Ryan & Pfanner, 2001).
Hsp70s are known to be involved in protein folding by ATP-controlled cycles of substrate binding and release (Cheetham et al., 1994; Suh et al., 1998; Young et al., 2003). The ATPase cycle of Hsp70s is characterized by alternating between a low affinity ATP bound state and a high affinity ADP bound state. The low affinity ATP bound state speeds up the rate of substrate exchange while the high affinity ADP bound state lowers the rate of substrate exchange (Mayer & Bukau, 2005). Also, genetic and biochemical evidence have shown that ATP hydrolysis is essential for the Hsp70s chaperone activity. (Ha et al., 1999; Mayer & Bukau, 2005). It appears that the interaction of Hsp70s with hydrophobic peptide portion of proteins in an ATP-controlled manner is responsible for its activities (Mayer & Bukau, 2005).
However, in a thermodynamically coupled process, the rate of substrate stimulated ATP hydrolysis is very low for any functional Hsp70 cycle to take place. Thus, for functional Hsp70 cycle to take place, co-chaperones are required to couple with substrate, thereby increasing the rate of ATP hydrolysis (Mayer & Bukau, 2005). Among the co-chaperones that have been implicated for this function are those belonging to the J domain containing proteins of which Hsp40 family of heat shock proteins are one (Laufen et al., 1999). PfHsp70s and PfHsp40s have been identified in the parasitophorous vacuole and Maurer's clefts, thus implicating them in possible erythrocyte proteins quality control and export of parasitic proteins into the host erythrocyte (Lanzer et al., 2006; Nyalwidhe & Lingelbach, 2006; Sargeant et al., 2006).
Recent studies have reported the identification and potential pharmacological uses of small molecules that specifically interact with and modulate the activities of Hsp70 (Chang et al., 2008; Cockburn et al., 2011; Fewell et al., 2004). Among identified molecules is polyamine 15-deoxyspergualin which was reported to binds and enhances the steady state ATPase activity of Hsp70 by 20-40% (Smith et al., 1998). Also reported was NSC 630668-R/1 (R/1), which inhibits ATPase activity and blocks Hsp70-mediated trafficking of polypeptides (Fewell et al., 2001). Another resent discovery is the class of dihydropyrimidines (Fewell et al., 2004). Dihydropyrimidines includes unique classes of chaperone modulators, in which some directly inhibited ATPase activity, while others such as MAL3-101, selectively blocked the ATPase enhancing ability of specific J domain proteins (Fewell et al., 2004). It is important to note that despite their diverse activities, many of the Hsp70-binding compounds share a central dihydropyrimidine core and vary only in their pendant functionality. High-throughput screening for small molecules that modulate the ATPase activity of the molecular chaperone DnaK has been reported (Chang et al., 2008).
1.4.3 Hsp40 Molecular co-Chaperones
Hsp40 proteins are defined by the presence of the J domain, an approximately 70 amino acid domain with similarity to the initial 73 amino acids of the E. coli Hsp40, DnaJ (Cyr et al., 1994; Ohki et al., 1986). They mainly function as co-chaperones of Hsp70s (Cheetham et al., 1994; Kelley, 1998; Suh et al., 1998; Young et al., 2003). The J domain was first discovered to be present in E. coli DnaJ with conserved HPD tripeptide that represents the signature motif of the Hsp40 protein family (Fan et al., 2003; Yochem et al., 1978). Therefore they facilitate the folding of nascent polypeptides through their interaction and regulation of partner Hsp70 proteins (Feldheim et al., 1992). In addition, Hsp40 proteins have been implicated in protein translocation (Jubete et al., 1996), protein degradation (Jiang et al., 1997), clathrin uncoating (Campell et al., 1997; Ma et al., 2002), and viral infection (al-Herran & Ashraf, 1998).
Hsp40 possesses four canonical domains (Figure 1.4B): a J domain, with a highly conserved HPD (His-Pro-Asp) motif that is needed for the stimulation of ATPase activity of Hsp70s (Cheetham & Caplan, 1998), a Gly/Phe-rich region (GF-domain), which is said to regulate and ensure stabilization of Hsp70-substrate binding (Hennessy et al., 2000; Tsai & Douglas, 1996); a cysteine-rich zinc binding domain, which possibly ensure stability of the Hsp40 tertiary structure (Martinez-Yamout et al., 2000); and a C-terminal domain, which has been implicated in the capturing of protein substrates and dimerization (Borges et al., 2005; Wu et al., 2005). On the basis of these domains, Hsp40 proteins were classified by Cheetham & Caplan, 1998 as follows: Type I Hsp40 proteins possess all the four canonical domains; Type II Hsp40 proteins possess all but lack zinc binding domain; while Type III Hsp40 proteins only have the J domain. Also, a Type IV Hsp40 protein, having a J-like domain, but with corrupted tripeptide HPD has been described (Botha et al., 2007).
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Figure 1.4: Schematic representation of Hsp70 and Hsp40 domains.
(A) Hsp70 domains organization showing ATPase and substrate binding domains joined together by a liker region. ATPase domain is important for it chaperone activity and interaction with Hsp40 co-chaperones. (B) Hsp40 type I to IV. With J domain, it could interact with and stimulate the ATPase activity of Hsp70. Other domains in Hsp40 regulate and ensure stabilization of Hsp70-substrate binding (GF region); stability of the Hsp40 tertiary structure (Zn-binding domain) and capturing of protein substrates and dimerization (C-terminal domain). GF region = Gly/Phe-rich region (GF-domain), Zn-binding domain = Cysteine-rich zinc binding domain.
1.4.4 Hsp70-Hsp40 Interactions
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Figure 1.5: Model of Hsp70-Hsp40 Interaction
Chaperone co-chaperone interactions constitute and important mechanism in biochemical processes needed for the survival and development of the parasite. The ATPase domain of Hsp70 and the J-domain of Hsp40 are essential components of these interactions. The interaction consist of the binding of substrate to Hsp40 (1 and 2), followed by interaction of substrate bound Hsp40 with the ATPase domain of Hsp70 (3), leading to the conversion of ATP to ADP. At ADP bound state, the rate of Hsp70-substrate interaction is high and very strong compared to it ATP bound state. Following the conversion to ADP bound state, Hsp40 dissociate and release substrate to Hsp70 for refolding to take place (4 and 5). The introduction of Pi through the activity of nucleotide exchange factor (NEF) convert Hsp70 back to ATP bound state, leading to the release of the folded or partially folded substrate (6). This process is repeated until the substrate is well folded. (Adapted from Pesce & Blatch, 2009)
The ability of the J domain proteins to mediate ATP hydrolysis dependent locking of substrates into the cavity of Hsp70 proteins is crucial for almost all the chaperone functions of Hsp70s. Hsp40s are a large family of chaperones with ability to specify the actions of Hsp70 proteins (Cheetham & Caplan, 1998). By their interactions with Hsp70s, Hsp40s often perform specialized functions in which a single Hsp70 can interact with more than one Hsp40s to generate unique Hsp70-Hsp40 pairs that can facilitate specific processes at distinct location within the cell (Horton et al., 2001). It has been shown that the regulation of ATP dependent polypeptide binding of Hsp70 is the major function of Hsp40s (Langer et al., 1992; Palleros et al., 1993; Szabo et al., 1994). This is made possible through their J domain.
A model of Hsp70-Hsp40 interaction has been proposed (Figure 1.5; Pesce & Blatch, 2009). Its involve the coupling of substrate with Hsp40s followed by subsequent attachment to the N-terminal ATP domain of Hsp70s. The coupling of Hsp40s with substrates drives the ATP hydrolysis thereby increasing the functional activity and affinity of Hsp70s for the substrates. With successful hydrolysis of ATP leading to a high substrate affinity ADP state, Hsp40 releases the substrate to Hsp70 and disengaged. The process of refolding by Hsp70 continues until the substrate bound Hsp70 attain another ATP bound state, a low affinity state. The cycle is repeated for as long as it is necessary for the substrate to be correctly folded (Pesce & Blatch, 2009). Furthermore, the functions of Hsp70s within a cellular compartment are multidimensional. It is therefore important to have substrate specific targeting of essential proteins for the survival of the cell. This regulatory activity is performed by the Hsp40s co-chaperones by connecting Hsp70s with their specific proteins, including substrates and other chaperones, by their interactions with both the chaperones and the substrate (Fan et al., 2003).
1.4.5 PfHsp70 and PfHsp40
At least six genes for Hsp70 proteins have been identified in P. falciparum with features spanning across the cytosolic, endoplasmic reticulum (ER), and the mitochondrial forms (Sargeant et al., 2006; Shonhai et al., 2007). These genes include PfHsp70-1, PfHsp70-2, PfHsp70-3, PfHsp70-x, PfHsp70-y and PfHsp70-z.
PfHsp70-2 is a homolog of the mammalian ER 78-kDa glucose-regulated protein or immunoglobulin-binding protein (Grp78/ BiP) (Kappes et al., 1993), reported to be confined to ER-like structures in P. falciparum (Kumar et al., 1991) and also found in the Maurer's clefts (Vincensini et al., 2005). However, unlike Grp78/BiP, which contains a typical eukaryotic C-terminal ER retention signal, PfHsp70-2 possesses the ER N-terminal leader sequence and a possible C-terminal ER retention signal (Pelham, 1989). PfHsp70-3 is the mitochondrial Hsp70 homolog with weakly conserved mitochondrial pre-sequence and a distinct phylogenetic features, suggesting a distinct role for this protein (Sargeant et al., 2006; Slapeta & Keithly, 2004). The detection of PfHsp70-3 in the Maurer's clefts (Lanzer et al., 2006; Vincensini et al., 2005) and its reported association with a malaria antigen (MAL13P.304) and two asparagines rich antigen proteins (PF08_0060 and PF11_0111) that are exported into the erythrocyte (Barale et al., 1997; Weber et al., 1988), suggests a possible role of PfHsp70-3 in facilitating the export of proteins of parasitic origin into the erythrocyte. PfHsp70-x is a 76kDa PfHsp70 with close identity to PfHsp70-1, except for the replacement of the C-terminal EEVD motif with EEVN motif (Sargeant et al., 2006; Shonhai et al., 2007). However, they both share high sequence identity, and possesses highly conserved bipartite nuclear localization signals (Robbins et al., 1991), suggesting that PfHsp70-x is also likely to be found in the nucleus (Shonhai et al., 2007). Recent report however localized PfHsp70-x to the cytosol and PV and showed that it is exported into the cytosol of infected erythrocyte in complex with other PfHsp40s in J-dot (Külzer et al., 2012). PfHsp70-z and PfHsp70-y, though, possess relatively conserved ATPase domains, they display very low conservation in the peptide-binding domains (Shonhai et al., 2007), lack conserved nuclear localization signal (Robbins et al., 1991) and no threonine residue that is said to be a crucial phosphorylation site of DnaK (McCarty & Walker, 1991). These distinct features suggest a different regulatory control and distinct functions for PfHsp70-z and PfHsp70-y when compared with the typical Hsp70 chaperones (Shonhai et al., 2007).
However, most research into this group of chaperones have focused only on investigating the chaperone properties of PfHsp70-1 (Matambo et al., 2004; Ramya et al., 2006; Sharma, 1992) and its potential as vaccine candidate (Behr et al., 1992; Kumar et al., 1990). PfHsp70-1 is a 70kDa cytosolic/nuclear-localized P. falciparum Hsp70 and its nuclear localization is enhanced in response to heat stress (Kappes et al., 1993). PfHsp70-1 has features to justify its candidature as co-expression partner, including successful overexpression in E. coli (Matambo et al., 2004). Its expression during the blood stages of the parasite (Joshi et al., 1992), solubility (Kappes et al., 1993) and the detection of antibodies to PfHsp70-1 in malaria patients (Kumar et al., 1990) may explain its importance in intra-erythrocytic survival and development of the parasite and its candidature for vaccine development (Behr et al., 1992; Kumar et al., 1990). Other studies have reported that it is present in the PV (Nyalwidhe & Lingelbach, 2006) and Maurer's clefts (Vincensini et al., 2005), raising the possibility of being exported into the erythrocyte. However, all the PfHsp70s lack export signal motifs (Marti et al., 2004; Sargeant et al., 2006) and are therefore not predicted to be exported into the erythrocyte (Botha et al., 2007). Also, like other eukaryotic cytosolic Hsp70s, PfHsp70-1 have been shown to possesses a characteristic C-terminal EEVD motif which binds to co-chaperones to facilitate its interaction with other partner proteins (Demand et al., 1998).
Of the 43 P. falciparum Hsp40s that have been identified, a total of 19, consisting of four each of Types II and III and eleven Type IV, belong to the malaria exportome (Sargeant et al., 2006). PFA0660w, a Hsp40 protein, regarded to be essential for the intra-erythrocytic development and survival of the parasite, together with PFB0090c and PFE0055c are Type II Hsp40 proteins exported into the cytosol of infected erythrocytes (Sargeant et al., 2006) and are said to be homologous to human DnaJB4, a cytosolic type II Hsp40, known to interacts with human Hsp70 to facilitate protein folding, transport and assembly (Botha et al., 2007). A recent study has shown that the J domains of PfHsp40 proteins (PfJ4 and PfJ1) were able to exert their functionality through a specific interaction with E. coli Hsp70, DnaK in vivo and replace the J-domain of the prokaryotic A. Tumefaciens DnaJ J-domain (Nicoll et al., 2007; Pesce et al., 2008). However, a mutation at position 26 of the helix II compromised the J domains functionality, indicating that this basic residue in association with the HPD motif may constitute important elements of a fundamental binding surface required for J domain-based Hsp40-Hsp70 interaction (Nicoll et al., 2007). Also, further investigation into the activity of Pfj4 revealed that it full length expression in OD259 bacteria disallow cells growth at non-permissive temperatures, suggesting a distinct roles from the type I Hsp40s and that the information for the specificity of substrate and the partner Hsp70 may be retained in G/F-rich and C-terminal regions of Pfj4 (Pesce et al., 2008). The expression of Pfj4 appeared to increase upon heat shock and localise in the cytoplasm and nucleus of trophozoites and schizonts (Pesce et al., 2008).
Meanwhile, the first functional characterization of the interactions between Pfj1 and PfHsp70-1 was reported by Misra & Ramachandran, (2009). The biophysical characterization of the C-terminus of PfHsp70-1 provided evidence for its involvement in stabilizing the otherwise unstable nucleotide-binding domain (NBD). This may explain how the parasite survives in the face of drastic change in temperature during its lifecycle that involve two hosts. Thus, Pfj1 function as a co-chaperone, enhancing the chaperone activity of PfHsp70-1 (Misra & Ramachandran, 2009).
1.4.5 The J dots and PFA0660w
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). Interestingly, in a study that used a loss of function approach to identify proteins that are involved in parasite virulence and rigidity, an attempt to produce a viable PFA0660w-knocked out parasite failed (Maier et al., 2008), suggesting that it may be essential for the survival and development of the parasite in the infected erythrocytes (Pesce & Blatch, 2009). In a study con