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.

No abstract

Injury to the blood vessel wall exposes the subendothelial extracellular matrix, which is rich in collagens, providing a substrate for platelet adhesion and aggregation. This is the first step of hemostasis ending in formation of the thrombus. Several proteins on the platelet membrane participate in collagen-platelet interactions in a direct or indirect manner [1]. Glycoprotein (GP)VI [2-5] and a 2 b 1 integrin [6] bind to collagen directly, and GPIb-V-IX [7] and a IIb b 3 integrin [6,8] bind via von Willebrand factor (vWF). In the current 'two-site, two-step' model of platelet-collagen interaction [9][10][11][12], platelet aggregation proceeds as follows: first, GPIb-IX-V rapidly binds to vWF immobilized on collagen so that passing platelets are tethered to the latter. Following this event, GPVI, a surface signaling receptor, binds to collagen with low affinity, which triggers the signaling cascade for platelet activation. This leads to 'inside-out' activation of a 2 b 1 and a IIb b 3 integrins and secretion of platelet agonists such as ADP and thromboxane A 2 , accelerating platelet aggregation and thrombus formation. Therefore, GPVI has a central role in the initial phase of thrombus formation as the major signaling receptor for collagen. GPVI is composed of two extracellular immunoglobulin domains, a mucin-rich stalk, a single transmembrane domain, and a short cytoplasmic tail [2][3][4]. GPVI is coupled to the Fc receptor (FcR) c-chain homodimer in the transmembrane domain via a salt bridge [5,9,10,13,14]. The binding of collagen to GPVI leads to cross-linking of GPVI molecules [15], inducing tyrosine phosphorylation of the cytoplasmic tail of FcR c-chains. This phosphorylation leads to binding To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor c-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates.

The plasma kallikrein (EC 3.4.21.34)-kinin system plays an important role in the initiation and amplification of surface-mediated, acute inflammatory responses following tissue injury [1][2][3][4]. This system is composed of three serine protease zymogens [prekallikrein (PK), factor XII (FXII) (EC 3.4.21.38) and factor XI] and the nonenzymatic procofactor, high molecular weight kininogen (HK). Kallikrein-kinin system activation is initiated by binding of FXII and a PK-HK complex to a biological activating surface, such as an endothelial cell surface, and is then accelerated by the reciprocal activation of FXII and PK on the surface. Zn 2+ is essential for binding of FXII and HK to a biological activating surface, and induces their conformational changes [5][6][7][8][9][10][11]. Activation of the kallikrein-kinin system results in the release of bradykinin, a primary mediator of acute inflammatory responses [3,4,12]. Bradykinin causes vasodilation, increases microvascular permeability, and enhances pain sensitivity, resulting in inflammatory symptoms such as redness, edema and pain around the injured site. Activated FXII (FXIIa) Keywords factor XII; high molecular weight kininogen; kallikrein-kinin system; salivary gland; Triatoma infestans Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn 2+ -dependent manner, suggesting that they specifically recognize Zn 2+ -induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.Abbreviations APTT, activated partial thromboplastin time; DS 500, dextran sulfate of M r 500