All mammalian malaria parasite species have an initial tissue stage in liver cells. The liver stage produces new parasite forms that can enter and live inside red blood cells. Accordingly, the first place of residence provides parasites with a radically different cellular and molecular environment from their subsequent red blood cell home. Liver stages have remained refractory to reveal their secrets, yet the last few years have seen several advances in elucidating their biology. This review looks at the more recent findings concerning the liver stage–host hepatocyte association, some of which may become powerful weapons in the prevention of malaria infection. We also outline areas of liver stage research and technological development that provide promising foci to accelerate a better understanding of this most elusive of the parasites many life cycle stages.
The life of Plasmodium parasites consists of an intricate cycling between mosquito vectors and vertebrate hosts. Within the host the parasite must perpetually infect cells to produce the next generation of invasive stages that can go on and enter new target cells. All of the complex malaria pathologies are associated with blood infection and therefore most malaria research has focused on the blood stages of the parasite's life cycle. However, when the parasite is initially transmitted to a mammalian host by the bite of an infected Anopheline mosquito, the inoculated sporozoite stages are not competent to directly infect red blood cells. Instead they make a beeline to the liver where they leave the blood stream and infect hepatocytes. Within hepatocytes the parasite occupies a unique vacuole called the parasitophorous vacuole (PV) in which the parasite grows rapidly, goes through multiple nuclear divisions and ultimately produces the first generation of red blood cell-infectious merozoite forms. The fact that it took 68 years after the discovery of the parasite blood stages to identify this initial obligatory tissue stage of infection serves as a historic aide memoire about the elusive nature of the malaria liver stages (LS) also referred to as exoeythrocytic forms (EEF). The past 10 years have seen progress in understanding the sporozoite molecular motility and invasion machinery (reviewed in Kappe et al., 2004a) and have revealed new complexities in sporozoite behaviour and host interactions prior to hepatocyte infection (reviewed in Mota and Rodriguez, 2004; Yuda and Ishino, 2004). In this review we will focus on recent advances in elucidating the complex LS–host hepatocyte biology and highlight critical areas of future investigation that may not only contribute to a better understanding of LS but will also provide new impetus to the field of malaria infection intervention.
Sporozoite mode of hepatocyte entry: should I stay or should I go?
The Plasmodium sporozoite has emerged as the most versatile among the parasites invasive stages. The unique molecular motility apparatus, consisting mainly of the transmembrane protein TRAP and a submembraneous actin myosin motor complex (reviewed in Kappe et al., 2004b), drives the three basic types of tissue interaction that sporozoites display in the mammalian host cell environment: gliding motility along cell linings, traversal through cells by membrane rupture wounding (transcellular migration) and invasion into a PV. PV formation leads to intracellular residence. A fourth type of interaction, the transmigration between cells (paracellular migration), is possible but not yet experimentally demonstrated. Intravital microscopy studies revealed sporozoite tissue traversal capacity in the skin subsequent to transmission (Vanderberg and Frevert, 2004). It appears that tissue traversal is an important means to seek out and to breach the vascular lining for entry into the blood stream but it is not clear whether the sporozoite utilizes transcellular migration routes, paracellular migration routes or a combination of both. Once inside the liver sinusoids, sporozoites move along endothelial cells. When they encounter Kupffer cells, resident macrophages that are interspersed in the liver endothelial cell layer, sporozoites enter and traverse them (reviewed in Frevert et al., 2006). This traversal allows sporozoites to gain access to hepatocytes (Frevert et al., 2005). Importantly, gene deletion studies in Plasmodium berghei identified secreted sporozoite proteins that are critical for cell traversal (Ishino et al., 2004; 2005a). Traversal mutants nonetheless retain their capacity to invade with PV formation and show no defect in LS development. The precise function of traversal-associated proteins remains to be elucidated; however, some of them contain perforin-like membrane insertion domains (Kaiser et al., 2004), suggesting a possible role in host cell membrane rupture. Tissue traversal, including direct traversal of cells, therefore enables sporozoites to reach target hepatocytes. Yet, only after traversing and wounding several hepatocytes do sporozoites switch from traversal mode to invasion mode into a PV (Mota et al., 2001). The wounded hepatocytes themselves appear to undergo necrotic cell death (Frevert et al., 2005). Hepatocyte traversal in vivo appears puzzling. Why do sporozoites not invade the first hepatocyte they encounter and transform into LS (Fig. 1)? Based on experimental evidence two models have been proposed: (i) cell traversal activates the sporozoite secretory capacity needed to release molecules that function during invasion with a PV (Mota et al., 2002) and (ii) hepatocyte growth factor (HGF) released by wounded traversed hepatocytes signals through its receptor called Met on the surface of by-standing hepatocytes (Fig. 1). This renders host hepatocytes more permissive to LS development by inducing important rearrangements in their cytoskeleton and by protecting them from apoptosis (Carrolo et al., 2003; Leiriao et al., 2005a). Both models have been debated in recent reviews (Mota and Rodriguez, 2004; Silvie et al., 2004; Yuda and Ishino, 2004) and it appears that they are not satisfactory. The strongest evidence against the sporozoite activation hypothesis comes from work with sporozoite traversal mutants (Ishino et al., 2004; 2005a). They are completely deficient in transcellular migration using membrane rupture but show no apparent deficiency in invasion with PV formation. Traversal mutants also produce similar numbers of mature LS in hepatoma cells in vitro and in Kupffer cell-depleted rodent livers when compared with wild-type parasites. This indicates that cell wounding may not significantly contribute to enhance hepatocyte permissiveness for LS development.
How do sporozoites commit to PV formation and subsequent LS development? Work by Silvie et al. (2003) sheds some light on the importance of host hepatocyte CD81 in sporozoite invasion and PV formation. They demonstrated that CD81–/– mice and primary hepatocytes prepared from their livers are resistant to Plasmodium yoelii sporozoite infection and LS development (but not P. berghei infection, suggesting important differences in host cell interaction even among closely related malaria species). Antibodies against CD81 also block Plasmodium falciparum infection of human hepatocytes in vitro. Although overexpression of CD81 in normally refractory HepG2 human hepatoma cells renders them permissive to P. yoelii infection, it is not sufficient to support P. falciparum infection (Silvie et al., 2006a). Importantly, P. yoelii sporozoites retain their ability to traverse CD81–/– hepatocytes or HepG2 cells but they cannot form a PV and thus mature LS cannot develop (Fig. 1). However, sporozoites penetrate the nucleus of HepG2 cells and this may result in developmentally arrested LS-like forms. CD81 is a member of the tetraspanin family of proteins expressed on a wide range of cells. Tetraspanins are organized in association with other proteins in membrane microdomains (reviewed in Levy and Shoham, 2005). CD81 has many functions ranging from modulating immune interactions to intracellular trafficking, endocytosis and exocytosis. It is also a receptor for hepatitis C virus (Cocquerel et al., 2006). How CD81 contributes to Plasmodium sporozoite invasion with PV formation is still unknown but it appears not to act as a direct ligand for sporozoite secretory and surface proteins (Silvie et al., 2003). A recent report shows that CD81 contributes to the organization of cholesterol-rich microdomains. Their disruption by cholesterol depletion inhibits sporozoite infection (Silvie et al., 2006b). However, this requirement was again species-specific because cholesterol depletion did not affect P. berghei infection. Future studies need to unravel the mechanisms by which tetraspanins in cholesterol-rich microdomains contribute to PV formation but the current data show that there may be important molecular differences in the ways Plasmodium species engage in allegedly conserved host cell interactions such as PV formation.
LS and hepatocyte death: if I stay there will be trouble
Successful intracellular pathogens are able to safely reside inside a host cell long enough to undergo growth and multiplication, and at the same time exploit the host cell for resources. The intrahepatocytic LS exhibits distinct developmental phases, each possibly interacting with the host hepatocyte in different ways (Figs 1 and 2). First, after productive invasion with PV formation the initial dedifferentiation from invasive sporozoite to liver trophozoite commences. During this phase the parasite likely does not depend on nutrient acquisition from the host cell (Kaiser et al., 2003). In the second intermediate LS growth phase the parasite has to establish an extensive system to remodel and exploit the host hepatocyte. In the third phase the parasite significantly outgrows the ordinary confines of the hepatocyte. This process is perhaps the most unique found among intracellular parasite development strategies because Plasmodium LS increase in cell volume many times that of an uninfected host hepatocyte (Figs 1 and 2), yet apparently intact host cells continue to enclose them. It is evident that the enormous parasite growth must exert significant physical stress on the host cell but somehow the cell stays alive. Recent intravital imaging studies of the final stage of parasite development reveal that LS let loose variably sized portions of their cell body, possibly still surrounded by host hepatocyte membrane, into the sinusoids. These structures, referred to as extrusomes (Tarun et al., 2006) or merosomes (Sturm et al., 2006), may contain fully formed merozoites but it is currently not clear whether merozoites may also differentiate within these structures after their expulsion.
To successfully grow inside hepatocytes malaria parasites have to devise ways to keep the host cell alive because unlike anucleate red blood cells, hepatocytes have ways to interrupt parasite development by triggering their own death. The host cell itself may be alerted to the invader and terminate its own existence by activating pro-apoptotic signalling. The host cell may also expose its parasitic inhabitant by antigen presentation and rely on help from, for example, cytotoxic T cells (CTL), which would kill the infected cell. Little is known about the LS's ability to avoid host cell death but some evidence points to active interference with the host cell apoptotic programme. A recent report shows that infection of hepatoma cells by P. berghei renders them more resistant to external pro-apoptotic stimuli (van de Sand et al., 2005). Infected hepatocytes have an increased ability to withstand starvation, TNFα and d-galactosamine treatment (potent agents of hepatocyte pro-apoptotic stimulation) as well as peroxide-induced apoptosis (Fig. 1). It is unknown, however, how the parasite accomplishes this. As alluded to earlier, Leiriao and co-workers proposed that by wounding neighbouring hepatocytes, which causes HGF to be secreted, sporozoites are able to prepare their future host cell through Met receptor signalling (Carrolo et al., 2003; Leiriao et al., 2005a). Activation of a receptor tyrosine kinase such as Met results in recruitment of adaptor proteins to the receptor's intracellular domain. This in turn activates signalling pathways leading to cellular proliferation, adhesion or increased cell survival (Hammond et al., 2004). One of the most prominent signalling pathways induced by HGF is phosphoinositide 3-kinase (PI3K) implicated to have pro-survival effects on cells (Rosario and Birchmeier, 2003). Interestingly, PI3K inhibition reduces LS numbers grown in hepatoma cells if applied before invasion (Leiriao et al., 2005a) but has no effect on the parasite if administered at 24 h after infection (van de Sand et al., 2005). It is thus not clear whether PI3K pathways are important for invasion with PV formation or whether they act as early pro-survival signals for the infected hepatocyte, which are not utilized in later stages of infection. It has also been observed that resistance to apoptosis increases with progression of LS development (van de Sand et al., 2005). However, it was recently suggested that in the final stage of LS development, the parasite allows the host hepatocyte to commit an unusual form of suicide that facilitates parasite evasion of host defences and deposition of merozoites into the blood stream (Sturm et al., 2006). Sturm et al. observed that in late in vitro LS-hepatoma cell infections, the host cells lose their mitochondrial membrane potential and release mitochondrial cytochrome c. Interestingly, the released cytochrome c was detected in LS but not in the host cell cytoplasm. However, infected hepatoma cells do not exhibit a typical apoptotic phenotype including DNA fragmentation, caspase activation and phosphatidylserine (PS) redistribution to the outer membrane leaflet. Nevertheless, infected cells lose their adhesive properties but are not phagocytosed by macrophages in an in vitro assay, presumably because they lack PS on the surface. The authors concluded that these in vitro observed events possibly aid merozoite release in vivo into the sinusoids and help the parasite avoid phagocytosis by Kupffer cells (Sturm et al., 2006). This conclusion, however, remains speculative.
LS growth deficiencies: if I go it will be double
The development and replication capacity of LS is remarkable, achieving one of the fastest growth rates among eukaryotic cells. After hepatocyte invasion the parasite spends a significant period of its total intracellular residence of 2–7 days (2–3 days in rodent malaria parasites, 5–7 days in human malaria parasites) dedifferentiating into a trophozoite (Figs 1 and 2). Interestingly, sporozoites can undergo this transformation without host cells. Solely exposing sporozoites to 37°C and serum triggers a transformation process that appears indistinguishable from that occurring inside hepatocytes (Kaiser et al., 2003). Some of the profound cellular and molecular changes occurring in parasite-stage transition are therefore independent of host cell contact, interaction and residence. However, upon completion of transformation, LS initiate growth only when inside a host cell. For complete development the PV appears critical. Recently, two parasite proteins, UIS3 and UIS4, were shown to be essential for LS growth in P. berghei (Mueller et al., 2005a,b). UIS4 is a small integral membrane protein expressed in the sporozoites secretory organelles. It is released after invasion and localizes to the PV membrane (PVM) throughout LS development (Figs 1 and 2). UIS4 knockout sporozoites invade hepatocytes, form a PV and transform into liver trophozoites but subsequently they show a severe growth defect in cultured hepatoma cells and in mouse livers. UIS3 knockout parasites suffer a similar growth deficiency. UIS3, which is also a predicted integral membrane protein but exhibits no sequence similarity to UIS4, has yet to be localized but it may also be positioned in the PVM. Therefore, UIS-PVM proteins fulfil a critical function during parasite growth. Whether these functions lie in essential pathways of LS interactions with the host hepatocyte such as uptake of host molecules and inhibition of apoptosis, or whether UIS-PVM proteins have a structural function for the PVM remains to be investigated.
A remarkable application of growth-deficient parasites is their use as live attenuated vaccines. Mice immunized with UIS3 or UIS4 knockout sporozoites are completely protected against infection with wild-type sporozoites (Mueller et al., 2005a,b). Intriguingly, this protection lasts for at least 9 month but the mutant parasites appear undetectable in the liver 40 h after inoculation (A. Tarun and S. Kappe, unpubl. obs.). The immunological mechanisms of protection by mutant parasites, their potential as live vaccines in humans and their similarities and potential differences with irradiation-attenuated parasites have been the subject of discussion elsewhere and we refer the reader to some current articles (Frevert and Nardin, 2005; Good, 2005; Waters et al., 2005; Hill, 2006). Another P. berghei sporozoite protein first described as P52 in P. yoelii and P. falciparum (Kappe et al., 2001) was recently implicated in LS growth of P. berghei (van Dijk et al., 2005). P52 (named P36p in P. berghei) is a putative GPI-anchored protein and belongs to the Plasmodium 6-cysteine protein family. Other members of this family are expressed on gametocytes and play a critical role in fertilization and, thus, are likely to be involved in cell–cell interaction (van Dijk et al., 2001). Some members are also expressed in asexual blood stages where they localize to the surface and the invasive organelles of merozoites but their function for merozoites has not been elucidated (Gilson et al., 2006). van Dijk et al. (2005) showed that P52/P36p knockout sporozoites retain their ability to invade hepatocytes but unlike the UIS3 and UIS4 mutants do not form a PVM and suffer an in vivo growth arrest similar to but more severe than radiation attenuated parasites. As in the case of UIS3 and UIS4 knockout sporozoites, P52/P36p knockout sporozoites also confer protection against infectious sporozoite challenge (van Dijk et al., 2005). Interestingly, the P52/P36p knockout parasites appear unable to inhibit host hepatocyte apoptosis, which as discussed earlier is blocked in hepatocytes infected with wild-type sporozoites. Another study using P. berghei P52/P36p knockout parasites, however, observed a severe deficiency in hepatocyte invasion (Ishino et al., 2005b). Cell traversal was not negatively affected in this mutant but rather increased. Accordingly, data on the role of P52/P36p in hepatocyte infection seem incongruous. However, because both studies found that P52/P36p mutants cannot form a PVM, their results point to some function for P52/P36p in the pathways that lead to induction of this essential compartment.
The discussed studies raise intriguing questions with important implications for vaccine design: do sporozoites have to enter hepatocytes with PV formation and initiate intrahepatocytic development to induce protective immune responses? Is necrotic cell death in traversed and wounded cells combined with the inability of mutant parasites to inhibit apoptosis of the invaded host cell ideal for stimulation of protective immune responses? Irradiation-attenuated sporozoite-infected hepatocytes have been shown to undergo apoptosis. This could be an effective means to provide antigen to dendritic cells and may be important for the initiation of a protective immune response (Leiriao et al., 2005b). Earlier studies indicated that dead sporozoites are not protective, but what about live sporozoites that are unable to productively invade cells with PV formation? Recent intravital studies using P. berghei show that live sporozoites engage in complex tissue interactions in skin and lymph nodes, including interactions with antigen presenting cells (Amino et al., 2006). Furthermore, P. berghei sporozoites form early LS-like structures in the lymph nodes but their development is aborted. Although these observations have yet to be made with other Plasmodium species, it is possible that induction of protective immune responses takes place in organs other than the liver.
Perspectives and thoughts for the future
Recent work by many laboratories has breached the experimental intractability of Plasmodium pre-erythrocytic stages and LS have finally entered the mainstream of malaria research. It is sensible to emphasize that this work has built on earlier pioneering work that could not be discussed here, which provided a solid observational framework for the combined application of powerful tools such as differential gene expression screens, reverse genetics, state of the art cell biology and in vivo imaging. As in the past most recent progress has come from work with rodent model malaria pre-erythrocytic stages. It is generally accepted that rodent parasites are good surrogates for human malaria parasites but, like any model, they have limitations. Key findings that are built on rodent malaria species need confirmatory studies in human malaria parasites. Unfortunately the lack of tractable in vivo models for P. falciparum LS has made this difficult at best and in vitro studies of P. falciparum LS required growth in primary hepatocytes until recently. A new continuous hepatocyte cell line that supports both P. falciparum and Plasmodium vivax LS (Sattabongkot et al., 2006) allows better and easier in vitro access and mouse models with humanized livers are a promising new avenue for in vivo studies (Morosan et al., 2006; Sacci et al., 2006). Also, P. falciparum parasite strains that have adapted to monkey livers are a tool that deserves attention (Collins et al., 2006).
Although increasingly fruitful LS research is now being conducted, it is still hampered by the lack of comprehensive transcriptome and proteome data and as such these areas should be a major focus of enabling research. Published expressed sequence tag data from developmentally late LS are encouraging (Sacci et al., 2005) but genome-wide expression data for all developmental forms of LS are needed and will reveal the sets of genes unique to LS or shared with other life cycle stages. Recently reported techniques to efficiently isolate early and late LS are the first step to achieve this goal (Tarun et al., 2006). Furthermore, isolation of LS in combination with nanoscale proteomic sample preparation (Wang et al., 2005) and high-sensitivity Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) (Page et al., 2004) may for the first time allow efficient proteomic analysis of LS. Uniquely LS-expressed genes are easy targets for the limited set of genetic tools that are available for malaria parasites because their deletion or modification will not interfere with blood stage replication, transmission to mosquitoes and parasite development in the mosquito stages. Some of the work discussed herein has proven the power of this approach. This will give important insights into how malaria parasites occupy their initial niche in the mammalian host. In conjunction with expression profiling of the infected host hepatocyte the mentioned methods will uncover co-ordinated changes in gene expression in parasite and host. Host genes that respond to parasite infection by increased or decreased expression are amenable to study by siRNA approaches in vitro, and transgenic mouse models may exist for a number of these genes that allow their rapid evaluation in in vivo infections with rodent malaria parasites. The potential interaction network of LS and hepatocyte proteomes can be explored using global yeast two-hybrid screens. This may unravel candidate interactions underlying remodelling of the infected hepatocyte, inhibition of host cell apoptosis, antigen presentation and acquisition of host components by the parasite. Essential host–parasite interactions will provide targets for novel drug intervention strategies because it may be possible to specifically inhibit the interacting host factor without interfering with its normal cellular function. This strategy will avoid the ever-looming specter of parasite drug resistance.
In closing, the authors hope that this review helps foster an increasing appreciation of the fascinating and sophisticated cell biology of malaria parasite LS. Furthermore, the fact that immunization with live-attenuated parasites renders this stage completely vulnerable to sterilizing immune responses should also serve as a call to arms for vaccine developers and funding agencies to facilitate development of a pre-erythrocytic malaria vaccine.
We thank our colleagues for their exciting recent findings in malaria LS biology. We thank Lawrence Bergman for kindly providing us with the MSP-1 antibody. This work is supported by the Ellison Medical Foundation, the National Institutes of Health, and the Foundation for the National Institutes of Health through the Grand Challenges in Global Health Initiative.