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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cell Host Microbe. Author manuscript; available in PMC Sep 11, 2009.
Published in final edited form as:
PMCID: PMC2610487
NIHMSID: NIHMS69860

Malaria parasite pre-erythrocytic stage infection: Gliding and Hiding

Summary

Malaria is caused by red blood cell-infectious forms of Plasmodium parasites resulting in illness and possible death of infected hosts. The mosquito-borne sporozoite stage of the parasite and the initial infection in the liver, however cause little pathology and no symptoms. Nevertheless, these pre-erythrocytic parasite stages are attracting passionate research efforts not least because they are the most promising targets for malaria vaccine development. Here, we review how the infectious sporozoite makes its way to the liver, subsequently develops in hepatocytes and the factors, both parasite and host, involved in the interactions that occur during this ‘silent’ phase of infection.

Introduction

Malaria is the world’s most deadly parasitic disease and is caused by Plasmodium parasites belonging to the apicomplexan phylum. Over 500 million people suffer clinical malaria episodes annually caused by P. falciparum infection alone resulting in a conservative estimate of 1 million deaths (Guinovart et al., 2006; Snow et al., 2005). However, before a victim ever succumbs to the clinical symptoms of the disease, which present themselves in the erythrocytic stage, the clinically silent pre-erythrocytic life cycle stages, transmitted by Anopheles mosquitoes, invade the body and develop in the liver. The invasive sporozoite stage originates in the mosquito midgut where it develops within a parasite oocyst. Sporozoites are released and invade the mosquito salivary glands. Parasite development in the mosquito and salivary gland infection has been reviewed recently (Matuschewski, 2006) and we will here focus on pre-erythrocytic stage biology in the mammalian host, initiated when sporozoites are deposited in the skin by an infectious mosquito. The sporozoites enter the blood circulation and are next found in the liver. Here, sporozoites leave the circulation through the liver sinusoidal endothelium, migrate through a number of hepatocytes and settle in a final hepatocyte for liver stage development. The liver stage grows and undergoes nuclear replication within a parasitophorous vacuole (PV), culminating in the release of tens of thousands of merozoites into the circulatory system. Once in the blood, merozoites rapidly adhere to and invade erythrocytes, replicate and generate further infectious merozoites (Cowman and Crabb, 2006). This cycle continues, leading to the clinical symptoms of the disease (Greenwood et al., 2005). While in transition between different tissues and cells in their vector and mammalian host, the single-celled malaria parasites adapt effectively to their environment. The sporozoite journey is propelled by a unique actin-myosin system, which allows extracellular migration, cell traversal and cell invasion (Kappe et al., 2004). Sporozoite interactions with host tissues are mediated by proteins expressed on the cell surface and by proteins that are released from a set of secretory organelles called micronemes and rhoptries. Sporozoites undergo extensive developmental regulation of gene expression that underlies their adaptation to the different habitats they encounter in the mosquito vector and the mammalian host (Mikolajczak et al., 2008). During the past decade, an extensive molecular characterization of sporozoites and more recently liver stages have allowed the identification of a number of molecular mechanisms used by the parasite during the pre-erythrocytic life cycle. Reverse genetics tools have enabled functional analysis of parasite proteins in vivo. Furthermore, advances in in vivo imaging techniques have enabled a detailed documentation of pre-erythrocytic stage behavior both in the mosquito and mammalian host (Amino et al., 2005). Most pre-erythrocytic stage research has been conducted using rodent malaria models, but is assumed that similar events govern initial infection by human malaria parasites. Thus it is anticipated that research on rodent malaria will inform intervention strategy development for malaria control and ultimately eradication. This is best exemplified with efforts to develop an anti-infection malaria vaccine. In 1967 a seminal paper was published demonstrating that the inoculation of mice with irradiated P. berghei (a rodent malaria parasite) sporozoites induced protection from a subsequent infection with wildtype sporozoites (Nussenzweig et al., 1967). Thus, the concept of sterile protection against malaria infection was born. This paper was followed with studies in humans using P. falciparum irradiated parasites that gave similar results (Clyde et al., 1973). However, irradiated sporozoites were never considered a practical vaccine and work focused on using the major sporozoite surface protein, CSP as a recombinant vaccine. Unfortunately CSP-based vaccine candidates do not provide sterile protection in malaria-endemic areas (Alonso et al., 2005). Also, recent work using either mice tolerized to CSP (Kumar et al., 2006) or transgenic P. berghei sporozoites expressing P. falciparum CSP (Gruner et al., 2007) showed that sterile protection was still obtained with attenuated sporozoite immunizations, despite the absence of immune responses specific to CSP.

In the last few years, genetically attenuated parasites (GAPs) have been created that circumvent the need for irradiation. Mining of the sporozoite transcriptome has enabled the discovery of genes whose protein products are essential for liver stage development. In every case, deletion of these genes caused the early cessation of liver stage development and prevented the onset of blood stage infection (Mikolajczak et al., 2007a). When mice were inoculated with these knockout sporozoites, they were subsequently completely protected against wildtype sporozoite infection. Therefore, the possibility arises that P. falciparum GAPs could be used as a whole-organism malaria vaccine.

Here, we discuss the most recent data that elucidate the cell biology and molecular biology of the parasites on the way from inoculation at the site of a mosquito bite, through their development in the liver and into the blood stream. We will also highlight areas where our current understanding of this complex journey is inadequate.

The sporozoite journey from the skin to the liver

When an Anopheles female mosquito takes a blood meal its proboscis probes into the host’s skin. In doing so, saliva is deposited to prevent the blood from coagulating (Beier, 1998). Sporozoites move with gliding motility from the salivary gland cavities and are ejected within the skin of the host (Frischknecht et al., 2004; Vanderberg and Frevert, 2004). A few hundred sporozoites can be deposited under the skin of a mouse by a single mosquito during a blood meal (Jin et al., 2007). Shortly after their intradermal deposition, sporozoites start to glide in a comparable fashion and speed to that seen in vitro (Amino et al., 2007). The in vivo gliding motility of sporozoites appears random and follows a corkscrew movement pattern. These quantitative real-time imaging studies have shown that most sporozoites move by continuous gliding in the skin in order to reach a blood vessel where they then breach the endothelial barrier to enter the blood circulation (Amino et al., 2007, Vanderberg, 2004 #325). The sporozoites can also breach a lymphatic vessel to reach the draining lymph node of the injection site, where some of the sporozoites can partially develop into exoerythrocytic stages (Amino et al., 2006). The ability of sporozoites to glide, as well as to migrate through and invade cells has been attributed to a number membrane anchored and secreted proteins. One of these, the thrombospondin-related anonymous protein (TRAP), a micronemal protein powers the gliding motility and invasion both in the mosquito vector salivary gland and in the mammalian host (Kappe et al., 1999). However, as well as invading cells, the sporozoite has the ability to migrate through host cells by membrane disruption, allowing for movement in and out of cells (Mota et al., 2002; Mota et al., 2001; Vanderberg and Stewart, 1990). Traversal, unlike invasion, does not lead to the production of a PV, within which the developing parasite grows. Initially, two molecules were identified that have a role in host cell traversal, named SPECT1 (sporozoite microneme protein essential for cell traversal 1) and SPECT2 (Ishino et al., 2005a; Ishino et al., 2004). The SPECT proteins are secreted by micronemes. SPECT1 has no similarity to any known proteins whilst SPECT2 (also called PPLP1) has a membrane attack complex/perforin related domain (Kaiser et al., 2004), suggesting that it might insert into membranes. Mutant sporozoites lacking either SPECT1 or SPECT2 show in vitro gliding motility but are unable to migrate through host cells in vitro. Liver infection rates in vivo were greatly reduced suggesting that host cell traversal is needed so sporozoites can cross the sinusoidal cell layer, likely through the resident macrophage, known as the Kupffer cell (Ishino et al., 2004). Since the mutant sporozoites showed in vitro gliding motility but no cell traversal activity, it was not clear if in vivo gliding motility or host cell traversal were necessary for sporozoite traversal to the blood stream from the skin. Recently though, it has been shown that in vivo host cell traversal is important for sporozoite progression from the skin to the circulatory system, and then to the liver (Amino et al., 2008). In this study, in vivo imaging of P. berghei spect1 and spect2 parasites expressing green fluorescent protein (GFP) was accomplished in the skin of a mouse ear. More than 90% of the mutant sporozoites were immotile in the skin after mosquito deposition. Furthermore, mutant sporozoites were found associated with phagocytes and dermal fibroblasts in the skin post mosquito injection. Despite the apparent lack of cell traversal, a number of spect mutant parasites were able to breach the endothelial barrier to the blood stream and initiate liver stage development. The authors suggest that traversal is important for the passage of the sporozoite through the dermis to the liver but not for hepatocyte traversal of the capillary endothelium. In addition to SPECT1 and SPECT2, a number of other proteins have a role in sporozoite cell traversal capacity prior to hepatocyte infection, including a second TRAP family member, TLP (TRAP-Like Protein) (Moreira et al., 2008), a sporozoite secreted phospholipase (Bhanot et al., 2005), and CelTOS (cell traversal protein for ookinete and sporozoite) (Kariu et al., 2006). It is important to note that most of the studies conducted on sporozoite motility, host cell traversal and host cell invasion have been carried out with P. berghei. P. berghei is able to invade and grow in a number of types of host cell and it is currently not known if this phenomenon exists in all Plasmodium species. Therefore, since the body of evidence for sporozoite traversal and invasion has been conducted on P. berghei, inter-species in vivo imaging studies are needed to reveal possible differences in the behavior of malaria parasite sporozoites and their interaction in the skin prior to their one-way journey to the liver.

Sporozoite infection of the liver: traversal versus invasion

Once the infectious sporozoite enters the bloodstream it homes to the liver. How does the sporozoite sense where it is in the host and what mechanisms enable it to recognize when it has entered the liver sinusoid - the route it must take to ultimately gain access to its place of further development – a single hepatocyte? The liver sinusoid is a unique type of blood vessel with a fenestrated, discontinuous endothelium through which the oxygen-rich blood from the hepatic artery and the nutrient-rich blood from the portal vein can flow. A family of liver-specific highly sulfated heparan sulfate proteoglycans (HSPGs) protrudes into the sinusoid from the extracellular matrix in the space of Disse (which separates the endothelial cells of the sinusoid and the hepatocytes of the liver) through the fenestration. The HSPGs are produced by stellate cells within the space of Disse. As well as endothelial cells, the liver sinusoids are lined with Kupffer cells, resident liver macrophages that can take up and destroy foreign material such as bacteria, thereby preventing such material from entering the liver. An elegant study using intravital imaging has shown the movement of sporozoites in the liver (Frevert et al., 2005). The sporozoites used in the study expressed fluorescent proteins under the control of the circumsporozoite protein (CSP), which is highly expressed at this stage of the parasite life cycle. In combination with mice expressing fluorescent proteins in sinusoidal endothelial cells and Kupffer cells, the authors were able to use fluorescent microscopy to visualize sporozoite behavior in the liver sinusoid. Once they have reached the liver, the sporozoite glides freely for a number of minutes along the sinusoidal epithelium, a process which can occur both with and against the flow of blood. It appears that sporozoites then invade Kupffer cells traverse them and cross into the space of Disse. Once inside the liver parenchyma, sporozoites continue to migrate through several hepatocytes. The sporozoite eventually invades a final hepatocyte, with formation of a PV and begins liver stage (LS) growth.

To assess the requirement for Kupffer cells in sporozoite liver infection, the liver infection rate of P. yoelii in homozygous op/op mice, which have 77% fewer Kupffer cells than their wildtype littermates, was studied. Liver infection rates were decreased by 84% in the op/op mice, which indicates the importance of the Kupffer cell as the portal through which the sporozoite travels on its way to the liver (Baer et al., 2007b). Surprisingly, treatment of mice with liposome-encapsulated clodronate, which destroys Kupffer cells (and other phagocytic cells) increased sporozoite liver infection up to 15-fold. However, further analysis using electron microscopy showed that clodronate treatment had caused the formation of gaps in the liver sinusoid, possibly allowing sporozoites direct access to the hepatocytes (Baer et al., 2007b). The fact that sporozoites enter the liver through a resident macrophage raises an obvious question. Why is the sporozoite not phagocytozed by the liver’s primary defense mechanism to foreign attack? It turns out that the ubiquitous CSP plays a role in preventing the respiratory burst necessary for Kupffer cells to destroy sporozoites (Usynin et al., 2007). CSP is highly expressed by sporozoites and both sporozoites and CSP alone were able to induce the generation of cyclic AMP (cAMP) in Kupffer cells. cAMP stimulates adenyl cyclase activity which then inhibits the generation of reactive oxygen species – a potent macrophage defense mechanism. The ability of sporozoites to induce cAMP induction was mediated by both HSPGs and the low-density lipoprotein receptor-related protein LRP-1, both of which are found to be highly expressed by Kupffer cells (Usynin et al., 2007).

Once the sporozoite has migrated through the Kupffer cell, it then passes through a number of hepatocytes before finally taking up residence. Traversal damage and subsequent necrosis in hepatocytes has been confirmed in liver sections which revealed clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes (Frevert et al., 2005). It is not currently understood exactly why sporozoites migrate through a number of hepatocytes before finally invading and residing in the hepatocyte in which they will replicate. The traversal of sporozoites through hepatocytes occurs by rupturing of their plasma membrane followed by repair (Mota et al., 2001), It appears that traversal through hepatocytes is essential for the induction of apical-regulated exocytosis (Mota et al., 2002); a process which is necessary for cell invasion (Bannister and Mitchell, 1989) but the exact mechanism by which this occurs is not known. Apical-regulated exocytosis has been shown in sporozoites by measuring release of TRAP at the apical end of the parasite and its subsequent release into the medium. Increasing the cytosolic cAMP levels in Plasmodium sporozoites with the addition of the cell permeable cAMP analogue 8 bromo (8Br)-cAMP induces exocytosis in vitro, as measured by an increase in the accumulation of extracellular TRAP at the apical end of sporozoites (Ono et al., 2008). cAMP is synthesized by adenylyl cyclase (AC) and the incubation of sporozoites with the AC inhibitor MDL-12.330A prevented sporozoite exocytosis. Thus, it is presumed that the synthesis of cAMP by AC increases sporozoite exocytosis. Indeed, the treatment of P. yoelii sporozoites with 8Br-cAMP decreased their ability to migrate through monolayers of the hepatoma cell line Hepa 1–6 and increased their ability to invade the cells (Ono et al., 2008). The major downstream target of cAMP is protein kinase A (PKA) and inhibition of PKA activity by H89 was also able to reduce sporozoite exocytosis. Searching the malaria genome for AC genes revealed the presence of two different genes – ACalpha and ACbeta. Based on microarray analysis, P. falciparum ACα is expressed in sporozoites (Le Roch et al., 2003) and the deletion of P. berghei ACα did not alter parasite growth during blood stages or in the mosquito (Ono et al., 2008). However, activation of apical exocytosis in the acα sporozoite was greatly reduced and resulted in a defective invasion of Hepa1–6 cells in vitro and a decrease in liver parasite load forty hours after sporozoite infection of mice in vivo.

Sporozoite traversal through hepatocytes induces the secretion of host hepatocyte growth factor (HGF), which renders hepatocytes susceptible to infection (Carrolo et al., 2003). Studies with P. berghei showed that the removal of HGF with antibodies decreased the number of liver stages formed in vitro after sporozoite infection of HepG2 cells. The receptor for HGF is the tyrosine kinase MET and the incubation of Hepa1–6 cells with P. berghei sporozoites activated MET and HepG2 cells expressing a constitutively active MET were more susceptible to sporozoite infection, suggesting that the effect of HGF on sporozoite infection was mediated through MET. The HGF/MET signaling induced host cell actin reorganization and this was shown to be necessary for early liver stage development. In addition, HGF/MET signaling prevents the apoptosis of parasite infected cells, thus ensuring a successful parasite infection (Leiriao et al., 2005). Thus, the host protein HGF, released upon sporozoite traversal appears to be important for the downstream invasion and early liver stage development of the parasite.

Sporozoite entry into cells (both traversal and invasion) is dependant on organelles termed micronemes, which secrete proteins necessary for the parasite’s ability to cross the plasma membrane of host cells but also to form the moving junction when invasion accompanied with PV membrane (PVM) formation occurs. As has already been mentioned, SPECT1 and SPECT2 are intimately involved in host cell traversal. What about sporozoite invasion of hepatocytes with the formation of a PVM? Two parasite molecules are involved in this process - P36 and P52/P36p (Ishino et al., 2005b), the genes for which are arranged in tandem within the Plasmodium genome. These two proteins are members of the 6-cys protein family (Templeton and Kaslow, 1999) and P52/P36p has a putative glycophosphatidylinositol (GPI)-anchoring domain which allows the attachment of the protein to the sporozoite membrane via a GPI-anchor. Disruption of either of the genes in P. berghei gave rise to normal numbers of sporozoites but the sporozoites, although able to traverse cells, were defective in the final invasion of hepatocytes. Instead, the sporozoites continued to migrate through cells (Ishino et al., 2005b) with the result that liver stage infection was severely reduced. However, rodents still developed blood stage infection when inoculated with sporozoites of either knockout. In P. yoelii, the simultaneous disruption of both P36 and P52/P36p gave rise to sporozoites that were completely unable to form a PVM (Labaied et al., 2007). The result of this dual gene deletion was that the liver stage of the parasite did not develop and a blood stage infection did not occur indicating that P52/P36p and P36 might have a partially redundant function. Together, it appears that they have a critical role in a process that leads to formation of a PVM. However, further studies are needed to elucidate the molecular mechanisms of PVM induction. The identification of host cell receptors that bind P36 and P52/P36p is an important step in this direction but has yet to be accomplished. Interestingly, the tetraspanin ‘cluster of differentiation 81’ (CD81) is required on hepatocytes for P. yoelii sporozoite invasion with PVM formation (Silvie et al., 2003). P. yoelii sporozoites were unable to infect CD81-deficient mouse hepatocytes, in vivo and in vitro and antibodies against mouse and human CD81 inhibited the in vitro hepatic development of P. yoelii and P. falciparum, respectively. Further study has revealed that cholesterol is involved in the assembly of CD81 microdomains on the cell surface and is necessary for sporozoite infection (Silvie et al., 2006). Using CD9/CD81 chimeras, it has been shown that a 21 amino acid stretch of CD81 located in a domain structurally conserved in the large extracellular loop of all tetraspanins is sufficient in an otherwise CD9 background to confer susceptibility to in vitro P. yoelii infection (Yalaoui et al., 2008). Interestingly, P. berghei does not depend on CD81 for invasion of human hepatoma cell lines and can invade mouse hepatoma cell lines in a CD81 dependent and independent manner (Silvie et al., 2007), further evidence for the promiscuous nature of this rodent parasite.

In the process of plasma membrane rupture during cell traversal, cytosolic factors are released into the microenvironment which activate NF-kappaB, the main regulator of host inflammatory responses (Torgler et al., 2008). This activation of NF-kappaB occurred shortly after cell rupture and led to a reduction of infection load in a time-dependent manner both in vitro and in vivo. NF-kappaB activation was not observed when SPECT knockout parasites were used since they are unable to traverse cells. Infection rates were increased by the addition of an NF-kappaB inhibitor demonstrating the importance of its induction in the process of the host inflammatory response. Furthermore, primary hepatocytes from mice lacking myeloid differentiation primary response 88 (MyD88), which is an adapter protein used by all Toll-like receptors to activate NF-kappaB, showed no NF-kappaB activation upon rupture suggesting a role of the Toll-like receptor family sensing cytosolic factors. In fact, the lack of MyD88 significantly increased infection in vitro and in vivo. Thus, host cell wounding, although apparently beneficial for the sporozoite to adopt an invasive state, is also likely to limit host cell infection through activation of the inflammatory response. Intriguingly, pre-erythrocytic stages also appear to counter inflammation by inducing the release of host anti-inflammatory factors. The anti-inflammatory enzyme heme oxygenase-1 (HO-1) has recently been shown to be upregulated in the liver following both P. berghei and P. yoelii infection (Epiphanio et al., 2008). Mice expressing an increased amount of HO-1 by infection with an HO-1-expressing adenovirus developed a more severe parasite liver load after P. berghei sporozoite infection. Conversely, mice lacking the HO-1 gene (Hmox1) did not develop blood stage parasitemia after infection with P. berghei sporozoites. The induction of HO-1 during P. berghei liver infection protects the infected hepatocytes by controlling the inflammatory response, as seen by a dramatic reduction in inflammatory foci and a decrease in the number of inflammatory cells and cytokines when comparing mice treated with the HO-1 adenovirus with the Hmox1−/− mice (Epiphanio et al., 2008). At present, it is not known how sporozoite infection increases HO-1 activity and it will of interest to determine which parasite factors play a role in this protective mechanism.

As has already been discussed, sporozoites traverse cells but for replication to take place, the sporozoite must invade a cell and form a PV, which protects it from the host cell and allows its development into infectious merozoites. What parasite and host factors induce the switch form traversal to productive invasion? Recent data indicate that the sulfation levels on HSPGs appear to be a cue that guides the sporozoite to choose its infection mode. In 1993 it was discovered that CSP recognizes and binds HSPG expressed on the surface of the hepatoma cell line HepG2 (Frevert et al., 1993). The binding was abolished by heparitinase treatment – an enzyme that removes the glycosaminoglycan chains from HSPGs, indicating that the recognition was via the glycosaminoglycan chains of the HSPGs. It was later discovered that the degree of sulfation of the glycosaminoglycan chains at both the N- and O-positions of the HSPGs were important for the binding of CSP (Pinzon-Ortiz et al., 2001) and that highly sulfated heparins (naturally occurring HSPGs) had an enhanced ability to competitively inhibit the attachment of sporozoites to HepG2 cells. Further studies analyzing the overall extent of sulfation of heparan sulfate on a variety of cell types demonstrated that the hepatoma cell line Hepa 1–6 had the highest levels, when compared to a mouse dermal fibroblast cell line, an endothelial cell line, adding further evidence that the increased HSPG content of the hepatocyte triggers the attachment of the migrating sporozoite (Coppi et al., 2007). Interestingly, the change in sporozoite behavior – from traversal to invasion was directly related to the cleavage of CSP (Coppi et al., 2007). This cleavage is mediated by a member of the papain family of cysteine proteases and is of sporozoite origin (Coppi et al., 2005). The treatment of sporozoites with E-64, a specific inhibitor of cysteine proteases prior to an in vitro infection of hepatoma cells or the treatment of mice with E-64 prior to an in vivo sporozoite infections was able to completely inhibit sporozoite infectivity (Coppi et al., 2005) demonstrating the requirement for CSP processing for active sporozoite invasion. The incubation of sporozoites with soluble heparin also triggered the invasion response, allowing them to invade typically non-permissive cells such as dermal fibroblasts and endothelial cells as well as Hepa 1–6 cells that had been treated with the sulfation inhibitor chlorate (Coppi et al., 2007). Undoubtedly, the triggering of CSP cleavage is associated with signaling events that enable the onset of sporozoite invasion. Such cascades are typically mediated by protein kinases and the broad range protein kinase inhibitor, staurosporine, is able to inhibit sporozoite invasion (Mota et al., 2002). Plasmodium has a family of calcium-dependent proteins kinases (CDPKs) (Ward et al., 2004), and calcium signaling plays a central role in the regulation of sporozoite cell traversal and invasion (Mota et al., 2002). Although no selective Plasmodium CDPK inhibitors have been discovered, the antagonist W-7 is known to inhibit plant CDPKs and KN-93 inhibits structurally similar animal calmodulin-dependent protein kinases. Like staurosporine, these inhibitors were also able to decrease sporozoite invasion and CSP processing (Coppi et al., 2007), suggesting a role for CDPKs in the signaling cascade involved in sporozoite invasion. The transcription level of a novel member of the Plasmodium CDPK-family, CDPK-6 was found to be significantly higher in P. falciparum sporozoites (Le Roch et al., 2003) and was subsequently knocked-out in P. berghei (Coppi et al., 2007). Sporozoites from CDPK-6 parasites showed a marked decrease in invasive capabilities when compared to wild type sporozoites and a severe decrease in CSP cleavage. Furthermore, incubation of the CDPK-6 parasites with heparin did not increase their ability to invade Heap 1–6 cells (Coppi et al., 2007). Taken together, this data suggests that CDPK-6 is involved in the signaling cascade brought about by the binding of CSP to the highly sulfated hepatocyte HSPGs that leads to the induction of the invasive phenotype of sporozoites that leads to establishment of the PV. It is tempting to speculate that CDPK-6 signaling leads to release of P36 and P52/P36p, the aforementioned proteins that are necessary for PV formation.

Liver stage growth and merozoite egress

Due to their low number and inaccessibility, Plasmodium liver stages (LS) are easily the most difficult life stage of the parasite to study. Thus, little is known about the intracellular existence of this rare and elusive stage of the parasite. The liver stage exhibits discrete developmental stages. Initially, after invasion with the consequence of PV formation and establishment of the parasite inside a PV membrane (PVM) the invasive sporozoite dedifferentiates and develops into a liver trophozoite. Surprisingly, this transformation can take place outside of a host cell hepatocyte and only requires serum and a temperature increase to 37°C, suggesting that the host cell factors are not required for this transformation (Kaiser et al., 2003). Beyond the trophozoite stage, liver stage growth and replication through schizogony is rapid and intense and undoubtedly requires nutrients from the host cell and also an extensive manipulation of the hepatocyte since the parasite, although still within the hepatocyte is able grow and increase cell volume dramatically without annihilating the host cell – an exceptional achievement. Cell stress is typically known to trigger programmed cell death (apoptosis) and examination of the hepatoma cell line HepG2 infected with P. berghei showed a lack of apoptotic signaling (van de Sand et al., 2005). Indeed, parasite infection confers resistance to apoptosis of the host cell and to show the physiological relevance of this, mice were infected with high numbers of P. berghei sporozoites and then treated with tumor necrosis factor (TNF)-α and d-glucosamine to induce liver apoptosis. Liver sections of these mice, stained for degraded DNA, confirmed that infected cells containing viable parasites were protected from programmed cell death.

In addition to the activities already mentioned for CSP, this multifunctional protein also promotes liver stage development. CSP contains PEXEL (Plasmodium export element) motifs which export parasite proteins to the host cell cytoplasm. These motifs are functional in CSP and their deletion from P. berghei CSP leads to a defective transport of the protein to the hepatocyte cytoplasm and a decrease in liver stage development (Singh et al., 2007). Interestingly, CSP also contains a functional nuclear localization signal (NLS) which binds primarily to host importinα3 and deletion of the NLS diminishes liver stage growth. Furthermore, the NLS domain of CSP competes with NF-kappaB for binding to importinα3 and thus is believed to play a role in inhibiting the host inflammatory responses necessary to prevent liver stage development (Singh et al., 2007). It is thus obvious that CSP plays a major role in ensuring the sporozoite reaches the liver and initiates liver stage development.

It is likely that the hepatocyte, due to its unique metabolic activity – a major synthesizer of lipids and purines as well as a store for glycogen, is able to provide the developing liver stage with the nutrients it requires for the generation of the thousands of red blood cell-infectious merozoites that are released into the circulation. Recent studies of the liver stage PVM have shown that it is associated with the host endoplasmic reticulum but not with host mitochondria or lysosomes and the PV contains pores that restrict the passage of solutes to less than 855 Daltons (Bano et al., 2007). Nevertheless, the liver stage PV appears to be an active and highly permeable compartment that ensures the continued supply of nutrients to support parasite growth. It is currently not clear how larger nutrients cross the PVM but parasite proteins expressed on the surface of the PVM are definitely involved in liver stage growth progression. Advances in understanding the liver stage PVM came initially from studies that aimed to elucidate why sporozoites from the mosquito salivary glands are highly infectious for the liver (Matuschewski et al., 2002). Using suppression subtractive hybridization, a number of genes that were upregulated in infectious sporozoites (UIS genes) were isolated. Two of these genes, UIS3 and UIS4, when independently deleted from P. berghei or P. yoelii caused liver stage growth arrest and the subsequent prevention of blood stage infection (Mueller et al., 2005a; Mueller et al., 2005b; Tarun et al., 2007). UIS4 is expressed on the PVM and presumably plays an essential role in either maintaining the integrity of the PVM or is able to interact with host cell factors for nutrient acquisition. Studies on UIS3 are more advanced. This protein is also expressed on the PVM and it has recently been shown, using a yeast two-hybrid screen that it is able to interact with hepatocyte liver fatty acid binding protein (L-FABP) (Mikolajczak et al., 2007b). As its name suggests, L-FABP is a hepatocyte specific fatty acid carrier which enables fatty acids to be moved through the cytoplasm. Using L-FABP specific siRNA to down regulate its expression in the hepatoma cell line HUH7, significantly decreased the growth of P. berghei liver stages when compared to cells transfected with a control siRNA. Conversely, when L-FABP expression was increased with transfection of a plasmid carrying the L-FABP cDNA into HUH7 cells, P. berghei growth was increased. It is tempting to speculate that this interaction fuels the transfer of fatty acids from L-FABP to UIS3 and subsequently to the parasite for lipid synthesis, although there is currently no evidence for this. Importantly, P. falciparum UIS3 has recently been crystallized in association with the lipid phosphatidylethanolamine and has also been shown to directly interact with L-FABP (Sharma et al., 2008), adding fuel to the hypothesis that UIS3 has an important function for transporting lipids to the developing liver stage.

A further gene that is expressed exclusively in sporozoites and liver stages has also been shown to be essential for liver stage development. The gene product has been named both sporozoite low complexity asparagine-rich protein (SAP1) (Aly et al., 2008) and sporozoite and liver stage asparagine-rich protein (SLARP) (Silvie et al., 2008). Targeted deletion of SAP1 in P. yoelii and of SLARP in P. berghei did not have any effect on parasite blood stage replication or mosquito development. However, even though both sap1 and slarp sporozoites were able to invade hepatocytes, they were unable to initiate liver stage development and consequently were unable to generate a blood stage infection. Strikingly, the absence of SAP1/SLARP in P. yoelii/P. berghei abolished the expression of a number of essential UIS genes, including UIS3 and UIS4 but genes encoding proteins such as CSP and TRAP mostly unaffected. It is tempting to hypothesize that the protein is either acting on transcription and turns on UIS genes or it might stabilize UIS gene mRNA in the salivary gland sporozoites and early liver stages.

Although liver stages are difficult to study, the generation of recombinant P. berghei (Franke-Fayard et al., 2004) and P. yoelii (Tarun et al., 2006) parasites which actively express green fluorescent protein (GFP) have enabled intravital imaging of liver stages and also the isolation of liver stages by fluorescence activated cell sorting (FACS) (Tarun et al., 2006). Using FACS to isolate liver stages from GFP-expressing P. yoelii parasites has allowed a detailed analysis of both the transcriptome and proteome of this previously intractable life stage (Tarun et al., 2008). This effort has finally enabled a detailed analysis of the genes that are upregulated in liver stage development when compared to other life cycle stages. One of the findings from this work was that genes encoding enzymes of the type II fatty acid synthesis (FAS II) pathway were highly upregulated in late liver stage development as well as being well represented in the proteome. Furthermore, inhibitors of this pathway, which is expressed in the parasite apicoplast (Ralph et al., 2004), were able to reduce liver stage development in vitro. This is exciting, as it raises the possibility that inhibitors of the FAS II pathway, which is not present in the human host could prevent liver stage development. Clearly, our ability to mine the liver stage gene transcription and protein expression has great potential for the discovery of the molecules involved in liver stage development.

Intravital microscopy of GFP-expressing P. yoelii and P. berghei parasites has finally allowed for a detailed analysis of how exo-erythrocytic merozoites egress from their host hepatocyte and enter the bloodstream. The parasites induce the death and detachment of their host hepatocyte and this is followed by the budding of merozoite-filled vesicles (extrusomes/merosomes) into the sinusoidal lumen (Sturm et al., 2006; Tarun et al., 2006). The extrusomes/merosomes are surrounded by host cell membrane and do not expose the classical apoptotic signal – phosphatidylserine – at their surface, suggesting that the infected hepatocyte did not undergo apoptosis before extrusome/merosome release (Baer et al., 2007a; Sturm et al., 2006). In vivo studies on GFP expressing P. yoelii liver stages have shown that he majority of extrusomes/merosomes exit the liver intact and adapt to a relatively uniform size, in which 100–200 merozoites reside (Baer et al., 2007a). Extrusomes/merosomes then survive the subsequent passage through the right heart and accumulate in the lungs. Ex vivo analysis showed that extrusomes/merosomes break up inside pulmonary capillaries with the subsequent liberation of merozoites into the bloodstream.

Conclusions

This review has concentrated on the recent advances made to elucidate the parasite and host molecules involved in ensuring that the Plasmodium sporozoite reaches the liver and subsequently develops into blood stage-infectious merozoites. However, current experimental data provide incomplete information and sometimes differences in data on a number of key issues and these have yet to be resolved. For one, little is known about how the proteins that are all released from the micronemes can have such distinct functions in motility, traversal and invasion. Are their subpopulations of micronemes that are released at different times? In addition– although involved in cell traversal and invasion –what are SPECT1, SPECT2, CelTOS, P36 and P52/P36p actually doing? No one knows. Still up for great debate - does the sporozoite, as has been suggested (Mota et al., 2002; Mota et al., 2001), have an absolute need to traverse through cells in order for its ultimate invasion of a hepatocyte? Deletion of spect genes give rise to sporozoites that are unable to traverse through cells in vitro but can cause an in vivo infection (Amino et al., 2008; Ishino et al., 2005a; Ishino et al., 2004). How is this possible? Perhaps, sporozoites are able to glide between cells and finally reach the hepatocyte but this seems unlikely. It has also been suggested that the highly sulfated HSPGs found in the liver promote a switch from traversal to invasion and yet, the sporozoite continues to migrate through hepatocytes before reaching its place of further development (Coppi et al., 2007). Further evidence suggests that traversal is ‘turned on’ for the journey to the liver and then ‘turned off’ before invasion of the final hepatocyte (Amino et al., 2008). Gene deletions of molecules involved in these processes only add to the confusion as in vivo, they all appear to diminish passage to the liver but not prevent it – suggesting a degree of redundancy in sporozoite infection. The passage of sporozoites through the Kupffer cell is also contentious. Removal of Kupffer cells still allows liver stage infection, yet this could be due to ‘holes’ appearing in the endothelial layer of the sinusoid (Baer et al., 2007b), thus allowing a direct passage for the sporozoite to a hepatocyte. Experimental evidence suggests a direct role of CSP in preventing the Kupffer cell from phagocytozing infectious sporozoites (Usynin et al., 2007), but does this mean that the sporozoite must enter this cell in order to reach the liver? Could the sporozoite simply pass through an endothelial cell as it appears to do to enter skin capillaries? P. yoelii and P. falciparum sporozoites are known to interact with CD81 to enter hepatocytes (Silvie et al., 2003) but the receptor on the parasite remains elusive.

In contrast to the apparent redundancy of molecules involved in the sporozoite invasion of the liver, a number of gene deletions completely prevent early liver stage development and these knock out sporozoites are also excellent GAPs. Nevertheless, UIS3 is the only liver stage protein currently known to interact directly with the host (Mikolajczak et al., 2007b; Sharma et al., 2008), presumably to funnel nutrients into the developing liver stage. What other proteins are involved in assuring the liver stage grows and evades the host cell – what is the function of UIS4 and what other proteins are expressed on the liver stage PVM? Late liver stage development is still a black box – the parasite is undergoing colossal growth and replication and yet we know almost nothing about the processes involved. Certainly, recent transcriptome and proteome data on late liver stages have unearthed candidate metabolic pathways, including fatty acid synthesis, that are up-regulated in this major expansion but the necessity of these pathways has yet to be determined.

The availability of new tools to study liver stage development (both in vitro and in vivo) will hopefully soon herald an enhanced understanding of this elusive life cycle stage. Ultimately preventing liver stage development will prevent malaria and new methodologies for liver stage intervention (both drugs and vaccines) will emanate from novel research in this field.

Figure 1
The sporozoite journey to the hepatocyte and subsequent liver stage development: parasite and host interactions

Acknowledgements

Work by the authors on this subject was funded by the NIH and a Grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health initiative.

Footnotes

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