Tomoko Ishino

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.

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.

A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell.

Plasmodium parasites are fertilized in the mosquito midgut and develop into motile zygotes, called ookinetes, which invade the midgut epithelium. Here we show that a calcium‐dependent protein kinase, CDPK3, of the rodent malarial parasite (Plasmodium berghei) is produced in the ookinete stage and has a critical role in parasite transmission to the mosquito vector. Targeted disruption of the CDPK3 gene decreased ookinete ability to infect the mosquito midgut by nearly two orders of magnitude. Electron microscopic analyses demonstrated that the disruptant ookinetes could not access midgut epithelial cells by traversing the layer covering the cell surface. An in vitro migration assay showed that these ookinetes lack the ability to migrate through an artificial gel, suggesting that this defect caused their failure to access the epithelium. In vitro migration assays also suggested that this motility is induced in the wild type by mobilization of intracellular stored calcium. These results indicate that a signalling pathway involving calcium and CDPK3 regulates ookinete penetration of the layer covering the midgut epithelium. Because humans do not possess CDPK family proteins, CDPK3 is a good target for blocking malarial transmission to the mosquito vector.

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.

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 parasites transmission.

Two proteins with 6-cys motifs are required for malarial parasites to commit to infection of the hepatocyte

Many intracellular pathogens have host cells suitable for their proliferation, and selectively invade them using specific host–parasite interactions. Malarial sporozoites, the liver‐invasive forms, are effectively targeted to hepatocytes and proliferate in them. So far, however, sporozoite molecules that mediate the specific infection of hepatocytes remain unknown. Here we report that two proteins, Pbs36p and Pbs36, belonging to the plasmodium 6‐cys domain protein family, carry out this function. We found that these molecules are specifically produced in liver‐infective sporozoites. Target disruption of the respective genes nearly abolished sporozoite infectivity in the mammalian host. Invasion assays revealed that the mutant parasites could not commit to infection, even when they encounter with hepatocytes, resulting in continuous traversal of hepatocytes. These results suggest that these proteins are necessary for sporozoites to recognize hepatocytes and commit to infection. This finding might lead to novel anti‐malarial strategies that prevent sporozoite infection of the hepatocyte.

Development of the malaria parasite in the skin of the mammalian host

The first step of Plasmodium development in vertebrates is the transformation of the sporozoite, the parasite stage injected by the mosquito in the skin, into merozoites, the stage that invades erythrocytes and initiates the disease. The current view is that, in mammals, this stage conversion occurs only inside hepatocytes. Here, we document the transformation of sporozoites of rodent-infecting Plasmodium into merozoites in the skin of mice. After mosquito bite, ∼50% of the parasites remain in the skin, and at 24 h ∼10% are developing in the epidermis and the dermis, as well as in the immunoprivileged hair follicles where they can survive for weeks. The parasite developmental pathway in skin cells, although frequently abortive, leads to the generation of merozoites that are infective to erythrocytes and are released via merosomes, as typically observed in the liver. Therefore, during malaria in rodents, the skin is not just the route to the liver but is also the final destination for many inoculated parasites, where they can differentiate into merozoites and possibly persist.

LISP1 is important for the egress of Plasmodium berghei parasites from liver cells.

Most Apicomplexa are obligatory intracellular parasites that multiply inside a so‐called parasitophorous vacuole (PV) formed upon parasite entry into the host cell. Plasmodium, the agent of malaria and the Apicomplexa most deadly to humans, multiplies in both hepatocytes and erythrocytes in the mammalian host. Although much has been learned on how Apicomplexa parasites invade host cells inside a PV, little is known of how they rupture the PV membrane and egress host cells. Here, we characterize a Plasmodium protein, called LISP1 (liver‐specific protein 1), which is specifically involved in parasite egress from hepatocytes. LISP1 is expressed late during parasite development inside hepatocytes and locates at the PV membrane. Intracellular parasites deficient in LISP1 develop into hepatic merozoites, which display normal infectivity to erythrocytes. However, LISP1‐deficient liver‐stage parasites do not rupture the membrane of the PV and remain trapped inside hepatocytes. LISP1 is the first Plasmodium protein shown by gene targeting to be involved in the lysis of the PV membrane.

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.

Liver-specific protein 2: a Plasmodium protein exported to the hepatocyte cytoplasm and required for merozoite formation.

The liver stage is the first stage of the malaria parasite that replicates in the vertebrate host. However, little is known about the interplay between the parasite liver stage and its host cell, the hepatocyte. In this study, we identified an exported protein that has a critical role in parasite development in host hepatocytes. Expressed sequence tag analysis of Plasmodium berghei liver‐stage parasites indicated that transcripts encoding a protein with an N‐terminal signal peptide, designated liver‐specific protein 2 (LISP2), are highly expressed in this stage. Expression of LISP2 was first observed 24 h after infection and rapidly increased during the liver‐stage schizogony. Immunofluorescent staining with anti‐LSP2 antibodies showed that LISP2 was carried to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Gene targeting experiments demonstrated that majority of the LISP2‐mutant liver‐stage parasites arrested their development during formation of merozoites. These results indicate that exported LISP2 is involved in parasite–host interactions required for the development of liver‐stage parasites inside hepatocytes. This study demonstrated that mid‐to‐late liver‐stage malarial parasites have a system for exporting proteins to the host cell as intraerythrocytic stages do and presumably to use the proteins to modify the host cell and improve the environment.