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.

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.

The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their bloodfeeding behavior. Here, we report a salivary protein of the malarial vector mosquito, Anopheles stephensi, that inhibits activation of the plasma contact system. This factor, named hamadarin, is a 16-kDa protein and a major component of the saliva of this mosquito. Assays using human plasma showed that hamadarin dose-dependently inhibits activation of the plasma contact system and subsequent release of bradykinin, a primary mediator of inflammatory reactions. Reconstitution experiments showed that hamadarin inhibits activation of the plasma contact system by inhibition of the reciprocal activation of factor XII and kallikrein. Direct binding assays demonstrated that this inhibitory effect is due to hamadarin binding to both factor XII and high molecular weight kininogen and interference in their association with the activating surface. The assays also showed that hamadarin binding to these proteins depends on Zn 2؉ ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn 2؉ binding. We propose that hamadarin may attenuate the host's acute inflammatory responses to the mosquito's bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely.

We previously found that a Salmonella typhimurium vector engineered to secrete soluble tumor antigen induces CD4 +

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.