Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis

Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

Sporozoites of mammalian malaria: attachment to, interiorization and fate within macrophages

Sporozoites of Plasmodium berghei and Plasmodium knowlesi, incubated in normal serum readily interact with peritoneal macrophages of mice or rhesus monkeys, respectively. Interiorization of the sporozoite requires that both serum and macrophages be obtained from an animal susceptible to infection by the malaria parasite. Serum requirements for sporozoite attachment to the macrophage are less specific. Phagocytosis is not essential for the parasites to become intracellular. Our findings indicate that active penetration of the sporozites into the macrohages does occur. Antibodies present in the serum of sporozoite-immunized mice are important in determining the fate of both the intracellular sporozoites and the macrophages containing the parasite. Sporozoites coated with antibodies degenerate within vacuoles of the macrophages, which have no morphologic alteration. Sporozoites incubated in normal serum do not degenerate within macrophages, but the parasitized macrophages become morphologically altered and are destroyed. Preliminary experiments indicate that sporozoites appear to interact with rat Kupffer cells in the same way as with the peritoneal mouse macrophages. It is postulated that Kupffer cells play a dual role in sporozoite-host cell interaction. In normal animals these cells might serve to localize the sporozoites in the immediate vicinity of the hepatocytes. In the immunized animals, macrophages would remove and destroy the antibody-coated parasites, thus contributing to sporozoite-induced resistance.     

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.     

To migrate or to invade: those are the options

Plasmodium sporozoites penetrate and migrate through multiple cells in the host before productively invading a hepatocyte and forming a parasitophorous vacuole. In this issue of Cell Host & Microbe, Coppi and colleagues show that sporozoite interaction with the highly sulfated heparan sulfate proteoglycans on liver cells induces proteolytic cleavage of the major sporozoite surface molecule. They conclude that this interaction is the primary trigger that activates sporozoites for productive invasion.     

Molecular mechanisms of malaria sporozoite motility and invasion of host cells.

Malaria sporozoites have the unique capacity to invade two entirely different types of target cell in the mosquito vector and the vertebrate host during the course of the parasite's life cycle. Although little is known about the specific interaction of the sporozoite with its target cells, two sporozoite proteins, circumsporozoite (CS) and thrombospondin-related adhesive protein (TRAP), have been shown to play important roles in the invasion of both cell types. CS protein is a multifunctional protein involved in sporogony, invasion of the salivary glands, the specific arrest of sporozoites in the liver sinusoid, gliding motility of the sporozoite, and hepatocyte recognition and entry. TRAP has been shown to be critical for sporozoite infection of the mosquito salivary glands and liver cells, and is essential for sporozoite gliding motility. This review will focus on the involvement of these molecules in sporozoite motility and the invasion of host cells.     

Plasmodium sporozoites trickle out of the injection site

Plasmodium sporozoites make a remarkable journey from the skin, where they are deposited by an infected Anopheline mosquito, to the liver, where they invade hepatocytes and develop into exoerythrocytic stages. Although much work has been done to elucidate the molecular mechanisms by which sporozoites invade hepatocytes, little is known about the interactions between host and parasite before the sporozoite enters the blood circulation. It has always been assumed that sporozoites rapidly exit the injection site, making their interactions with the host at this site, brief and difficult to study. Using quantitative PCR, we determined the kinetics with which sporozoites leave the injection site and arrive in the liver and found that the majority of infective sporozoites remain in the skin for hours. We then performed sub-inoculation experiments which confirmed these findings and showed that the pattern of sporozoite exit from the injection site resembles a slow trickle. Last, we found that drainage of approximately 20% of the sporozoite inoculum to the lymphatics is associated with a significant enlargement of the draining lymph node, a response not observed after intravenous inoculation. These findings indicate that there is ample time for host and parasite to interact at the inoculation site and are of relevance to the pre-erythrocytic stage malaria vaccine effort.     

Sporozoites of Mammalian Malaria: Attachment To, Interiorization and Fate Within Macrophages

Sporozoites of Plasmodium berghei and Plasmodium knowlesi, incubated in normal serum readily interact with peritoneal macrophages of mice or rhesus monkeys, respectively. Interiorization of the sporozoite requires that both serum and macrophages be obtained from an animal susceptible to infection by the malaria parasite. Serum requirements for sporozoite attachment to the macrophage are less specific. Phagocytosis is not essential for the parasites to become intracellular. Our findings indicate that active penetration of the sporozoites into the macrophages does occur.     

Antisporozoite Antibodies with Protective and Nonprotective Activities: In Vitro and In Vivo Correlations Using Plasmodium gallinaceum, an Avian Model

ABSTRACT. A correlation was observed between in vivo and in vitro activity of six monoclonal antibodies (mAb) against the major circumsporozoite protein of the avian malaria Plasmodium gallinaceum as follows. (1) Two mAb were protective, totally abrogating sporozoite infectivity to chicks, its natural host, in vivo; they caused 100% inhibition of sporozoite invasion (ISI) in vitro to SL-29 chicken fibroblasts and intense ISI to cultured chicken macrophages, as well as inhibited the exoerythrocytic development of sporozoites taken up by macrophages, the initial cell host of P. gallinaceum sporozoites. (2) Two mAb were partially protective in that they reduced sporozoite infectivity to chicks, caused partial ISI to SL-29 and macrophage cells and partial inhibition to the exoerythrocytic development of sporozoites in macrophages in vitro. (3) Two mAb were totally inactive in vivo although they both bound to the sporozoite antigens as detected by indirect immunofluorescence, western blot, and ELISA; they both failed to induce ISI or inhibit the exoerythrocytic development in macrophages. The possible participation of macrophages as the initial cell type involved in sporozoite destruction in the presence of anti-circumsporozoite antibodies is discussed.     

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.     

Early transcriptional responses of HepG2-A16 liver cells to infection by Plasmodium falciparum sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.     

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.     

The Journey of Malaria Sporozoites in the Mosquito Salivary Gland

The life cycle of malaria parasites in the mosquito vector is completed when the sporozoites infect the salivary gland and are ready to be injected into the vertebrate host. This paper describes the fine structure of the invasive process of mosquito salivary glands by malaria parasites. Plasmodium gallinaceum sporozoites start the invasion process by attaching to and crossing the basal lamina and then penetrating the host plasma membrane of the salivary cells. The penetration process appears to involve the formation of membrane junctions. Once inside the host cells, the sporozoites are seen within vacuoles attached by their anterior end to the vacuolar membrane. Mitochondria surround, and are closely associated with, the invading sporozoites. After the disruption of the membrane vacuole, the parasites traverse the cytoplasm, attach to, and invade the secretory cavity through the apical plasma membrane of the cells. Inside the secretory cavity, sporozoites are seen again inside vacuoles. Upon escaping from these vacuoles, sporozoites are positioned in parallel arrays forming large bundles attached by multilammelar membrane junctions. Several sporozoites are seen around and inside the secretory duct. Except for the penetration of the chitinous salivary duct, our observations have morphologically characterized the entire process of sporozoite passage through the salivary gland.     

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.     

Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha), a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.     

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.     

Ultrastructure of the invasion of Eimeria magna sporozoites into cultured cells.

Monolayers of bovine kidney cells were overlaid with Eimeria magna sporozoites and observed with phase-contrast optics until penetration of the cells by the parasites had begun. Cells and penetrating parasites were fixed with glutaraldehyde and OsO4-containing ruthenium red, dehydrated, and embedded in situ. Cells being penetrated were selected for study in the electron microscope. The lack of intracellular staining with ruthenium red and intact plasmalemmas of cells being penetrated, was accepted as evidence that the sporozoites did not disrupt the plasma membranes. The sporozoite caused invagination of the host cell plasmalemma until the parasite was entirely within the cell, after which the invagination was sealed off by short pseudopodia enclosing the sporozoite within a membrane=lined vacuole inside the cell. Often myelin-forms, apparently of host cell origin, were seen in the space between the sporozoite and the cell.     

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 sporozoite invasion into insect and mammalian cells is directed by the same dual binding system

Plasmodium sporozoites, the transmission form of the malaria parasite, successively invade salivary glands in the mosquito vector and the liver in the mammalian host. Sporozoite capacity to invade host cells is mechanistically related to their ability to glide on solid substrates, both activities depending on the transmembrane protein TRAP. Here, we show that loss-of- function mutations in two adhesive modules of the TRAP ectodomain, an integrin-like A-domain and a thrombospondin type I repeat, specifically decrease sporozoite invasion of host cells but do not affect sporozoite gliding and adhesion to cells. Irrespective of the target cell, i.e. in mosquitoes, rodents and cultured human or hamster cells, sporozoites bearing mutations in one module are less invasive, while those bearing mutations in both modules are non-invasive. In Chinese hamster ovary cells, the TRAP modules interact with distinct cell receptors during sporozoite invasion, and thus act as independently active pass keys. As these modules are also present in other members of the TRAP family of proteins in Apicomplexa, they may account for the capacity of these parasites to enter many cell types of phylogenetically distant origins.