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.

SummaryPlasmodium 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 liverinfective 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.

SummaryThe 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.

SummaryPlasmodium 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.

Malarial sporozoites mature in the oocysts formed in the mosquito midgut wall and then selectively invade the salivary glands, where they wait to be transmitted to the vertebrate host via mosquito bite. Invasion into the salivary gland has been thought to be mediated by specific ligand–receptor interactions, but the molecules involved in these interactions remain unknown. MAEBL is a single transmembrane-like protein that is structurally related to merozoite adhesive proteins. We found MAEBL of the rodent malaria parasite, Plasmodium berghei, to be specifically produced by the sporozoites in the oocyst and localized in their micronemes, which are secretory organelles involved in malarial parasite invasion into the host cell. A targeted disruption experiment of the P. berghei MAEBL gene revealed that it was essential for sporozoite infection of the salivary gland and was involved in the attachment to the salivary gland surface. In contrast, the disruption of the MAEBL gene did not affect sporozoite motility in vitro nor infectivity to the vertebrate host. These results suggest that P. berghei MAEBL is a sporozoite attachment protein that participates in specific binding to and infection of the mosquito salivary gland.

SummaryMany 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.

CTRP (circumsporozoite protein and thrombospondin-related adhesive protein [TRAP]-related protein) of the rodent malaria parasite Plasmodium berghei (PbCTRP) makes up a protein family together with other apicomplexan proteins that are specifically expressed in the host-invasive stage 1. PbCTRP is produced in the mosquito-invasive, or ookinete, stage and is a protein candidate for a role in ookinete adhesion and invasion of the mosquito midgut epithelium. To demonstrate involvement of PbCTRP in the infection of the vector, we performed targeting disruption experiments with this gene. PbCTRP disruptants showed normal exflagellation rates and development into ookinetes. However, no oocyst formation was observed in the midgut after ingestion of these parasites, suggesting complete loss of their invasion ability. On the other hand, when ingested together with wild-type parasites, disruptants were able to infect mosquitoes, indicating that the PbCTRP gene of the wild-type parasite rescued infectivity of disruptants when they heterologously mated in the mosquito midgut lumen. Our results show that PbCTRP plays a crucial role in malaria infection of the mosquito midgut and suggest that similar molecular mechanisms are used by malaria parasites to invade cells in the insect vector and the mammalian host.

SummaryThe 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 liverstage 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 midto-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.