Intravital Observation of Plasmodium berghei Sporozoite Infection of the Liver

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.     

Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer

Liver infection is an obligatory step in malarial transmission, but it remains unclear how the sporozoites gain access to the hepatocytes, which are separated from the circulatory system by the liver sinusoidal cell layer. We found that a novel microneme protein, named sporozoite microneme protein essential for cell traversal (SPECT), is produced by the liver-infective sporozoite of the rodent malaria parasite, Plasmodium berghei. Targeted disruption of the spect gene greatly reduced sporozoite infectivity to the liver. In vitro cell invasion assays revealed that these disruptants can infect hepatocytes normally but completely lack their cell passage ability. Their apparent liver infectivity was, however, restored by depletion of Kupffer cells, hepatic macrophages included in the sinusoidal cell layer. These results show that malarial sporozoites access hepatocytes through the liver sinusoidal cell layer by cell traversal motility mediated by SPECT and strongly suggest that Kupffer cells are main routes for this passage. Our findings may open the way for novel malaria transmission-blocking strategies that target molecules involved in sporozoite migration to the hepatocyte.     

Sneaking in through the back entrance: the biology of malaria liver stages

Malaria infection is caused by sporozoites, the life cycle stage of Plasmodium that is transmitted by female anopheline mosquitoes. The inoculated sporozoites migrate in the skin, enter a capillary and use the bloodstream for the long haul to the liver. Here, the parasites invade hepatocytes and differentiate to thousands of merozoites that specifically infect red blood cells. Hepatocytes, however, are not directly accessible to sporozoites entering the liver sinusoid. The liver phase of the malaria life cycle can occur only if the parasites first cross the layer of sinusoidal cells that line the liver capillaries. Experimental observations show that sporozoite entry into the liver parenchyma involves a complex cascade of events, from binding to extracellular matrix proteoglycans via passage through Kupffer cells and transmigration through several hepatocytes, until the final host cell is found. By choosing the liver as their initial site of replication, Plasmodium sporozoites can exploit the tolerogenic properties of this unique immune organ to evade the host's immune response.     

Plasmodium yoelii: Influence of immune modulators on the development of the liver stage

Plasmodium sporozoites suppress the respiratory burst and antigen presentation of Kupffer cells, which are regarded as the portal of invasion into hepatocytes. It is not known whether immune modulation of Kupffer cells can affect the liver stage. In the present study, we found that sporozoites inoculated into Wistar rats could be detected in the liver, spleen, and lung; however, most sporozoites were arrested in the liver. Sporozoites were captured by Kupffer cells lined with endothelial cells in the liver sinusoid before hepatocyte invasion. Pretreatment with TLR3 agonist poly(I:C) and TLR2 agonist BCG primarily activated Kupffer cells, inhibiting the sporozoite development into the exoerythrocytic form, whereas Kupffer cell antagonists dexamethasone and cyclophosphamide promoted development of the liver stage. Our data suggests that sporozoite development into its exoerythrocytic form may be associated with Kupffer cell functional status. Immune modulation of Kupffer cells could be a promising strategy to prevent malaria parasite infection.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

Nomadic or sessile: can Kupffer cells function as portals for malaria sporozoites to the liver?

The initial site of replication for Plasmodium parasites in mammalian hosts are hepatocytes, cells that offer unique advantages for the extensive parasite replication occurring prior to the erythrocytic phase of the life cycle. The liver is the metabolic centre of the body and has an unusual relationship to the immune system. However, to reach hepatocytes, sporozoites must cross the sinusoidal barrier, composed of specialized endothelia and Kupffer cells, the resident macrophages of the liver. Mounting evidence suggests that, instead of taking what would seem a safer route through endothelia, the parasites traverse Kupffer cells yet suffer no harm. Kupffer cells have a broad range of responses towards incoming microorganisms, toxins and antigens which depend on the nature of the intruder, the experimental conditions and the environmental circumstances. Kupffer cells may become activated or remain anergic, produce pro- or anti-inflammatory mediators. Consequently, outcomes are diverse and include development of immunity or tolerance, parenchymal necrosis or regeneration, chronic cirrhotic transformation or acute liver failure. Here we review data concerning the unique structural and functional characteristics of Kupffer cells and their interactions with Plasmodium sporozoites in the context of a model in which these hepatic macrophages function as the sporozoite gate to the liver.     

Liver invasion by malarial parasites – how do malarial parasites break through the host barrier?

Malarial transmission to the human host is established by sporozoite infection of the liver. Sporozoites are released from the mosquito salivary glands and carried by the blood flow to the liver sinusoid. In the sinusoid, sporozoites leave the blood circulation by crossing the sinusoidal cell layer to infect hepatocytes, the site for their development into the erythrocyte-invasive forms. Traversal of the sinusoidal cell layer and subsequent hepatocyte infection are the most important events in sporozoite liver invasion, but the molecular basis of both events remains to be elucidated. The present review of sporozoite liver invasion focuses on recent advances in this topic obtained by application of reverse genetics. Sporozoites traverse host cells, rupturing the host cell membrane in the process. Three microneme proteins have important roles in this motility. Disruption of one of these genes abolishes or severely impairs cell traversal without affecting other types of invasive motility. Studies using these disruptant parasites indicate that cell-traversal ability is required for crossing the sinusoidal cell layer and accessing the hepatocytes for infection. This process is homologous to midgut epithelium penetration by the malarial ookinete, because identical or paralogous genes are critically involved in both processes. After arrival at the hepatocyte, the invasion mode of the sporozoites switches from cell traversal to hepatocyte infection.     

CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts

The malarial parasite has two hosts in its life cycle, a vertebrate and a mosquito. We report here that malarial invasion into these hosts is mediated by a protein, designated cell-traversal protein for ookinetes and sporozoites (CelTOS), which is localized to micronemes that are organelles for parasite invasive motility. Targeted disruption of the CelTOS gene in Plasmodium berghei reduced parasite infectivity in the mosquito host approximately 200-fold. The disruption also reduced the sporozoite infectivity in the liver and almost abolished its cell-passage ability. Liver infectivity was restored in Kupffer cell-depleted rats, indicating that CelTOS is necessary for sporozoite passage from the circulatory system to hepatocytes through the liver sinusoidal cell layer. Electron microscopic analysis revealed that celtos-disrupted ookinetes invade the midgut epithelial cell by rupturing the cell membrane, but then fail to cross the cell, indicating that CelTOS is necessary for migration through the cytoplasm. These results suggest that conserved cell-passage mechanisms are used by both sporozoites and ookinetes to breach host cellular barriers. Elucidation of these mechanisms might lead to novel antimalarial strategies to block parasite's transmission.     

A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection

Plasmodium sporozoites are injected into the mammalian host during mosquito blood feeding and carried by the blood stream to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. To reach the hepatocytes, sporozoites must cross the liver sinusoidal cell layer, which separates the hepatocytes from the circulatory system. Little is known about the molecular mechanisms by which sporozoites breach this cellular barrier. Here we report that a protein with a membrane attack complex/perforin (MACPF)-related domain is involved in this step. This molecule is specifically expressed in liver-infective sporozoites and localized in micronemes, organelles engaged in host cell invasion. Gene disruption experiments revealed that this protein is essential for the membrane-wounding activity of the sporozoite and is involved in its traversal of the sinusoidal cell layer prior to hepatocyte-infection. Disruptants failed to leave the circulation, and most of them were eliminated from the blood by liver perfusion. Our results suggest that rupture of the host plasma membrane by the pore-forming activity of this molecule is essential for cell passage of the sporozoite. This report is the first to demonstrate an important role of a MACPF-related protein in host cell invasion by a pathogenic microorganism.     

Migration through host cells activates Plasmodium sporozoites for infection

Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, migrate through several hepatocytes before infecting a final one. Migration through hepatocytes occurs by breaching their plasma membranes, and final infection takes place with the formation of a vacuole around the sporozoite. Once in the liver, sporozoites have already reached their target cells, making migration through hepatocytes prior to infection seem unnecessary. Here we show that this migration is required for infection of hepatocytes. Migration through host cells, but not passive contact with hepatocytes, induces the exocytosis of sporozoite apical organelles, a prerequisite for infection with formation of a vacuole. Sporozoite activation induced by migration through host cells is an essential step of Plasmodium life cycle.     

Direct Infection of Hepatocytes by Sporozoites of Plasmodium berghei1

ABSTRACT. To identify the unknown liver cell type initially invaded by sporozoiles of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

Direct infection of hepatocytes by sporozoites of Plasmodium berghei

To identify the unknown liver cell type initially invaded by sporozoites of mammalian malaria, young rats were inoculated intravenously with large numbers of Plasmodium berghei sporozoites obtained from infected Anopheles stephensi mosquitoes. Fine structural studies of liver specimens obtained from the rats within 2 min after inoculation demonstrated the presence of morphologically unaltered sporozoites in the cytoplasm of hepatocytes. Many sporozoites were also observed undergoing cytolysis within the lysophagosomes of Kupffer cells, as well as other phagocytic cells. These observations strongly suggest direct infection of the hepatocyte by the sporozoite.     

Invasion of mammalian host cells by Plasmodium sporozoites

Malaria is transmitted through the bite of an infected mosquito, which introduces Plasmodium sporozoites into the mammalian host. Sporozoites rapidly reach the liver of the host where they are sequestered, a process probably mediated by circumsporozoite (CS) protein. Once in the liver, sporozoites migrate through several hepatocytes by breaching their plasma membranes before infecting a final hepatocyte with formation of a vacuole around the sporozoite, where development occurs into blood stage parasites. We propose that migration through several host cells activates sporozoites for ultimate productive invasion. This migration triggers sporozoite exocytosis, which is necessary for hepatocyte invasion, probably because it provides molecules, such as thrombospondin-related anonymous protein (TRAP), likely required for sporozoite invasion with the formation of a vacuole. How sporozoites migrate from the skin to the liver and invade hepatocytes remains unclear. Understanding this initial stage of malaria is crucial for the development of new approaches against the disease.     

Interactions of Plasmodium berghei sporozoites and murine Kupffer cells in vitro

Malaria sporozoites must leave the bloodstream and cross a layer of sinusoidal lining cells in order to infect hepatocytes and undergo exoerythrocytic schizogony. To determine whether Kupffer cells (KC) derived from this layer interact with sporozoites, murine KC were isolated from perfused livers of BALB/cJ mice and incubated in vitro with Plasmodium berghei sporozoites. Isolated KC had characteristic macrophage surface Ag and were phagocytic, ingesting both latex particles and Leishmania major amastigotes. In the absence of immune serum, sporozoites associated with fewer than 10% of these KC. By 30 min, 10% of the cell-associated sporozoites were completely ingested, 30% were in the process of being ingested, and 60% were attached to the surface of the cells. Opsonization of sporozoites with monoclonal or polyclonal antibodies directed against P. berghei circumsporozoite protein markedly enhanced sporozoite association with KC. Up to 40% of cells exposed to opsonized sporozoites had parasites inside or attached to their surfaces. Sporozoites attached to or ingested by KC were uniformly destroyed within 240 min in all cultures; there was no evidence of conversion of sporozoites to the exoerythrocytic stage within KC by light microscopy, and there was no evidence of residual sporozoites, either inside or outside of cells, by either light or electron microscopy. These data suggest that under nonimmune conditions, KC play a minor role in resistance to infection by malaria sporozoites. However, when sporozoites are opsonized by circumsporozoite antibodies, phagocytosis by KC may be an important immune mechanism that prevents parasitization of hepatocytes.     

The silent path to thousands of merozoites: the Plasmodium liver stage

Plasmodium sporozoites are deposited in the skin of their vertebrate hosts through the bite of an infected female Anopheles mosquito. Most of these parasites find a blood vessel and travel in the peripheral blood circulation until they reach the liver sinusoids. Once there, the sporozoites cross the sinusoidal wall and migrate through several hepatocytes before they infect a final hepatocyte, with the formation of a parasitophorous vacuole, in which the intrahepatic form of the parasite grows and multiplies. During this period, each sporozoite generates thousands of merozoites. As the development of Plasmodium sporozoites inside hepatocytes is an obligatory step before the onset of disease, understanding the parasite's requirements during this period is crucial for the development of any form of early intervention. This Review summarizes our current knowledge on this stage of the Plasmodium life cycle.     

Electron microscopic studies on the interaction of rat Kupffer cells and Plasmodium berghei sporozoites

The interactions between Plasmodium berghei sporozoites and Kupffer cells in rat liver were studied by transmission electron microscopy. Between 10 and 45 min after inoculation, sporozoites were found in the process of entering Kupffer cells and inside phagolysosomes. The sporozoites entered the Kupffer cells by phagocytosis as determined by the presence of pseudopods and local accumulations of aggregated microfilaments and the resulting exclusion of other organelles in the phagocyte cytoplasm beneath the attached parasite. Sporozoites were taken up either with their anterior end first, or backwards. Scanning electron microscopy of in vitro sporozoite Kupffer cell interaction confirmed these observations. It was concluded that sporozoites are taken up in a normal phagocytic way by the Kupffer cells, regardless of their initial place of contact or position. Thirty min after inoculation sporozoites found in phagolysosomes were still morphologically intact but after 45 min we could encounter completely digested sporozoites.     

Plasmodium 6-cysteine proteins determine the commitment of sporozoites to liver-infection

Plasmodium sporozoites travel a long way from the site where they are released by a mosquito bite to the liver, where they infect hepatocytes and develop into erythrocyte-invasive forms. The success of this infection depends on the ability of the sporozoites to correctly recognize the hepatocyte as a target and change their behavior from migration to infection. However, how this change is accomplished remains incompletely understood. In this paper, we report that 6-cysteine protein family members expressed in sporozoites including B9 are responsible for this ability. Experiments on parasites using double knockouts of B9 and SPECT2, which is essential for sporozoite to migrate through the hepatocyte, showed that the parasites lacked the capacity to stop migration. This finding suggests that interactions between these parasite proteins and hepatocyte-specific cell surface ligands mediate correct recognition of hepatocytes by sporozoites, which is an essential step in malaria transmission to humans.     

Host cell traversal is important for progression of the malaria parasite through the dermis to the liver

The malaria sporozoite, the parasite stage transmitted by the mosquito, is delivered into the dermis and differentiates in the liver. Motile sporozoites can invade host cells by disrupting their plasma membrane and migrating through them (termed cell traversal), or by forming a parasite-cell junction and settling inside an intracellular vacuole (termed cell infection). Traversal of liver cells, observed for sporozoites in vivo, is thought to activate the sporozoite for infection of a final hepatocyte. Here, using Plasmodium berghei, we show that cell traversal is important in the host dermis for preventing sporozoite destruction by phagocytes and arrest by nonphagocytic cells. We also show that cell infection is a pathway that is masked, rather than activated, by cell traversal. We propose that the cell traversal activity of the sporozoite must be turned on for progression to the liver parenchyma, where it must be switched off for infection of a final hepatocyte.     

AMA1 and MAEBL are important for Plasmodium falciparum sporozoite infection of the liver

The malaria sporozoite injected by a mosquito migrates to the liver by traversing host cells. The sporozoite also traverses hepatocytes before invading a terminal hepatocyte and developing into exoerythrocytic forms. Hepatocyte infection is critical for parasite development into merozoites that infect erythrocytes, and the sporozoite is thus an important target for antimalarial intervention. Here, we investigated two abundant sporozoite proteins of the most virulent malaria parasite Plasmodium falciparum and show that they play important roles during cell traversal and invasion of human hepatocytes. Incubation of P. falciparum sporozoites with R1 peptide, an inhibitor of apical merozoite antigen 1 (AMA1) that blocks merozoite invasion of erythrocytes, strongly reduced cell traversal activity. Consistent with its inhibitory effect on merozoites, R1 peptide also reduced sporozoite entry into human hepatocytes. The strong but incomplete inhibition prompted us to study the AMA‐like protein, merozoite apical erythrocyte‐binding ligand (MAEBL). MAEBL‐deficient P. falciparum sporozoites were severely attenuated for cell traversal activity and hepatocyte entry in vitro and for liver infection in humanized chimeric liver mice. This study shows that AMA1 and MAEBL are important for P. falciparum sporozoites to perform typical functions necessary for infection of human hepatocytes. These two proteins therefore have important roles during infection at distinct points in the life cycle, including the blood, mosquito, and liver stages.