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.

Immunohistochemical analyses reveal FoxP3 expressions in spleen and colorectal cancer in mice treated with AOM/DSS, and their suppression by glycyrrhizin.

We previously demonstrated that glycyrrhizin (GL) suppressed inflammation and carcinogenesis in an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced murine model of colorectal cancer (CC). In this study, we found an accumulation of regulatory T cells (Tregs) in the spleen and suppression by GL in model mice. ICR mice were divided into four groups: Control, GL, CC, and GL-treated CC (CC+GL), and were sacrificed 20 weeks after AOM/DSS treatment. We measured spleen weight, areas of white and red pulp, and CD8+ T cells (cytotoxic T lymphocytes, CTL), and CD11c-positive cells (dendritic cells) in splenic tissues and forkhead box protein 3 (FoxP3)-positive cells (Tregs) in colorectal and splenic tissues. In all cases, the CC group showed a significant increase compared with those in Control group, and GL administration significantly attenuated this increase. These results indicate that Tregs accumulated in the spleen may participate in inflammation-related carcinogenesis by suppressing CTL. We also suggest that GL which binds to high-mobility group box 1 (HMGB1), suppresses carcinogenesis with decreasing Tregs in the spleen. Furthermore, there was an expression of FoxP3 in cancer cells, indicating that it may be involved in the malignant transformation of cancer cells.

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent.

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.

MAEBL is essential for malarial sporozoite infection of the mosquito salivary gland.

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.

Malaria-specific and nonspecific activation of CD8+ T cells during blood stage of Plasmodium berghei infection.

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8(+) T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8(+) T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8(+) T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8(+) T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8(+) T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8(+) T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8(+) T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect 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 Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-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.

Identification and characterization of a new kallikrein-kinin system inhibitor from the salivary glands of the malaria vector mosquito Anopheles stephensi.

A new kallikrein-kinin system inhibitor, designated anophensin, was identified in the salivary glands of the malaria vector mosquito, Anopheles stephensi. In vitro reconstitution experiments showed that anophensin inhibits activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII (FXII) and prekallikrein (PK), and subsequent release of bradykinin. Additionally, anophensin inhibits activation of the kallikrein-kinin system on cultured human umbilical vein endothelial cells (HUVECs). Direct binding assays show that this inhibitory effect is due to Zn(2+)-dependent specific binding of anophensin to both FXII and high molecular weight kininogen (HK). Furthermore, anophensin interacts with both the N-terminus of FXII and domain D5 of HK, which are the binding domains for biological activating surfaces. These results suggest that anophensin inhibits activation of the kallikrein-kinin system by interfering with the association of FXII and HK with biological activating surfaces, resulting in the inhibition of bradykinin release in a host animal during insect blood-feeding.

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.

Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans.

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 gamma-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.

Characterization of the Drosophila Methoprene -tolerant gene product. Juvenile hormone binding and ligand-dependent gene regulation.

Juvenile hormones (JHs) of insects are sesquiterpenoids that regulate a great diversity of processes in development and reproduction. As yet the molecular modes of action of JH are poorly understood. The Methoprene-tolerant (Met) gene of Drosophila melanogaster has been found to be responsible for resistance to a JH analogue (JHA) insecticide, methoprene. Previous studies on Met have implicated its involvement in JH signaling, although direct evidence is lacking. We have now examined the product of Met (MET) in terms of its binding to JH and ligand-dependent gene regulation. In vitro synthesized MET directly bound to JH III with high affinity (Kd = 5.3 +/- 1.5 nm, mean +/- SD), consistent with the physiological JH concentration. In transient transfection assays using Drosophila S2 cells the yeast GAL4-DNA binding domain fused to MET exerted JH- or JHA-dependent activation of a reporter gene. Activation of the reporter gene was highly JH- or JHA-specific with the order of effectiveness: JH III >> JH II > JH I > methoprene; compounds which are only structurally related to JH or JHA did not induce any activation. Localization of MET in the S2 cells was nuclear irrespective of the presence or absence of JH. These results suggest that MET may function as a JH-dependent transcription factor.

Identification and characterization of the plasma kallikrein-kinin system inhibitor, haemaphysalin, from hard tick, Haemaphysalis longicornis.

The plasma kallikrein-kinin system inhibitor, haemaphysalin, from the hard tick, Haemaphysalis longicornis, was identified. It was found that haemaphysalin inhibited activation of the plasma kallikrein-kinin system by interfering with reciprocal activation between factor XII and prekallikrein. It did not, however, inhibit amidolytic activities of factor XIIa and kallikrein. Direct binding assay indicated that factor XII/XIIa and high molecular weight kininogen (HK) are the target molecules of haemaphysalin, and that Zn2+ ions are involved in the interactions of haemaphysalin with these target molecules. This suggests that haemaphysalin interacts with target molecules by recognizing their conformational changes induced by Zn2+ ions. Furthermore, haemaphysalin interacted with the fibronectin type II domain and domain D5, the cell binding domains of factor XII and HK, respectively. This finding suggests that haemaphysalin interferes with the association of factor XII and the prekallikrein-HK complex with a biologic activating surface by binding to these cell-binding domains, leading to inhibition of the reciprocal activation between factor XII and prekallikrein.

Contribution of the N-terminal and C-terminal domains of haemaphysalin to inhibition of activation of plasma kallikrein-kinin system.

Haemaphysalin is a kallikrein-kinin system inhibitor from hard tick Haemaphysalis longicornis, and consists of two Kunitz type protease inhibitor domains. Each domain as well as haemaphysalin inhibited intrinsic coagulation by inhibiting activation of the kallikrein-kinin system without affecting the amidolytic activities of intrinsic coagulation factors, indicating that both domains were involved in the inhibition through a similar mechanism to that for haemaphysalin. Reconstitution experiments showed that the C-terminal domain contributed more predominantly to this inhibition. Direct binding assaying showed that the C-terminal domain could bind to the cell-binding region of high molecular weight kininogen (HK), suggesting that it also binds to the cell-binding region of factor XII. Judging from these findings, the C-terminal domain may more effectively inhibit the association of factor XII and HK with the cell surface by binding to cell-binding regions, and hence would predominantly contribute to the inhibition of activation of the kallikrein-kinin system.

Immuno-electron microscopic observation of Plasmodium berghei CTRP localization in the midgut of the vector mosquito Anopheles stephensi.

The subcellular localization of Plasmodium berghei circumsporozoite protein and thrombospondin-related adhesive protein (PbCTRP) in the invasive stage ookinete of P. berghei was studied in the midgut of Anopheles stephensi by immuno-electron microscopic observations using polyclonal antibodies and immuno-gold labeling. PbCTRP was found to be associated with the micronemes of a mature ookinete throughout the movement from the endoperitrophic space to the basal lamina of the midgut epithelium. PbCTRP was also observed in the electron-dense area outside the ookinete, which might have been secreted from the apical pore. PbCTRP is found most abundantly at the site of contact between the apical end of an ookinete and the basal lamina of an epithelial cell. These results suggest that PbCTRP functions as an adhesion molecule for ookinete movement into the midgut lumen and epithelial cell and for ookinete association with the midgut basal lamina and transformation into an oocyst.

Effect of corosolic acid on dietary hypercholesterolemia and hepatic steatosis in KK-Ay diabetic mice.

Corosolic acid (CA), contained in the leaves of the banaba plant (Lagerstroemia speciosa L.), is a pentacyclic triterpene, and has hypoglycemic effects. The effects of CA on dietary hypercholesterolemia and hepatic steatosis were assessed in KK-Ay mice, an animal model of type 2 diabetes. Two kinds of high cholesterol diet with or without 0.023% CA, were prepared for the study. KK-Ay mice were fed a normal diet (controls), the high cholesterol diet with CA (CA-mice) or that without CA (HC-mice) for 10 weeks. CA inhibited the mean blood cholesterol level by 32% (P<0.05) and the liver cholesterol content by 46% (P<0.05) compared with those of HC-mice 10 weeks after the start of dietary intake. Acutely, CA inhibited the mean blood cholesterol level 4 h after the administration of a high-cholesterol cocktail in an oral cholesterol-loading test, compared with that of control mice (P<0.05). These results suggest that CA has some direct effects on the cholesterol absorption process in the small intestine. CA may inhibit the activity of cholesterol acyltransferase, which acts in the re-esterification of cholesterol in the small intestine, in type 2 diabetes.

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.

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 parasite's transmission.

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.

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.

Analysis of involvement of the 3'-untranslated regions in regulating mRNA stability for vitellogenin, cyanoprotein alpha, and cyanoprotein beta from the bean bug, Riptortus clavatus.

The degradation of the 3'-untranslated regions (UTRs) of vitellogenin, cyanoprotein alpha, and cyanoprotein beta from the bean bug, Riptortus clavatus, was analyzed in vitro. The degradation pattern was similar for all three RNAs, with a high degradation rate in non-diapausing adult insects and no degradation in the fifth instar nymphs and in diapausing adults, and was not correlated with the expression levels of these three proteins. Proteins binding to the 3'-UTRs were detected in polysomal and cytosolic extracts. These factors, however, were present in all developmental stages. The abundance of the polysomal factor showed little variation, but the cytosolic factor was enriched in adult insects. Cross-competition experiments demonstrated that the same factors bound to all three RNAs with similar affinity. The pattern of degradation, presence of the binding factors in all stages, and their inability to distinguish between the target sequences indicate that the 3'-UTRs do not participate in controlling the expression of these three proteins.

A mosquito salivary protein inhibits activation of the plasma contact system by binding to factor XII and high molecular weight kininogen.

The salivary glands of female mosquitoes contain a variety of bioactive substances that assist their blood-feeding 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.