Masao Yuda

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 Zn2+ ions, suggesting that hamadarin binds to these contact factors by recognizing their conformational change induced by Zn2+ binding. We propose that hamadarin may attenuate the hosts acute inflammatory responses to the mosquitos bites by inhibition of bradykinin release and thus enable mosquitoes to take a blood meal efficiently and safely.

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.

von Willebrand Factor A domain-related protein, a novel microneme protein of the malaria ookinete highly conserved throughout Plasmodium parasites.

The mosquito-invasive form of the malarial parasite, the ookinete, develops numerous secretory organelles, called micronemes, in the apical cytoplasm. Micronemal proteins are thought to be secreted during midgut invasion and to play a crucial role in attachment and motility of the ookinete. We found a novel ookinete micronemal protein of rodent malarial parasite Plasmodium berghei, named P. berghei von Willebrand factor A domain-related protein (PbWARP), and report it here as a putative soluble adhesive protein of the ookinete. The PbWARP gene contained a single open reading frame encoding a putative secretory protein of 303 amino acids, with a von Willebrand factor type A module-like domain as a main component. Western blot analysis demonstrated that PbWARP was firstly produced 12 h after fertilization by maturing ookinetes as SDS-resistant complexes. Recombinant PbWARP produced with a baculovirus system also formed SDS-resistant high-order oligomers. Immuno-electron microscopic studies showed that PbWARP was randomly distributed in the micronemes. PbWARP homologues also exist in human malarial parasites, Plasmodium falciparum and Plasmodium vivax. Highly conserved primary structures of PbWARP homologues among these phylogenetically distant Plasmodium species suggest their functional significance and the presence of a common invasion mechanism widely utilized throughout Plasmodium parasites.

Identification of an AP2-family Protein That Is Critical for Malaria Liver Stage Development

Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite Plasmodium berghei and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. AP2-L-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic P. berghei showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.

Purification and characterization of a thrombin inhibitor from the salivary glands of a malarial vector mosquito, Anopheles stephensi

A coagulation inhibitor was identified and isolated from the salivary glands of a malarial vector mosquito, Anopheles stephensi. The salivary gland extract prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) in assays with human plasma. The inhibition assay of the factors in the coagulation cascade by using synthetic chromogenic substrates showed that the anticoagulant in the mosquito salivary glands is a thrombin inhibitor, but not an inhibitor of factor Xa. The anticoagulant was purified to homogeneity from the mosquito thorax which contains the salivary glands by means of a combination of thrombin affinity and anion exchange chromatography. All of the anticoagulant activity was recovered from the fraction bound to the thrombin affinity column and no activity was detected in the unbound fraction. This result indicated that the thrombin inhibitor is the sole anticoagulant in the salivary glands of A. stephensi. This also suggested a noncovalent, reversible interaction between thrombin and its inhibitor. Size exclusion chromatography and SDS-PAGE estimated the molecular weight of the inhibitor as 45 kDa.

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 γ‐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.

Global transcriptional repression: An initial and essential step for Plasmodium sexual development

Significance In malaria parasites, gametocytogenesis is a process whereby gametocytes, the precursors of gametes, are generated from asexual blood-stage parasites. In this study, we report that the plasmodium transcriptional repressor AP2-G2 plays an essential role in this process. We found that AP2-G2 represses the genes that are required for the proliferation of the asexual stage and, thereby, supports the conversion from the asexual blood stage to the nonreplicative sexual stage. Similarly, during the initial phase of germ-line formation in metazoan animals (including insects), a master transcriptional repressor specifies primordial germ cells among embryonic cells. Thus, our findings suggest that the basic molecular mechanisms that are required to establish the germ line have been conserved in eukaryotes, despite their evolutionary divergence. Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood-stage parasites that proliferate in the circulation. However, little is known about how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates this transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasite Plasmodium berghei did not prevent commitment to the sexual stage but did halt development before the appearance of sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targeted ∼1,500 genes and recognized a five-base motif in their promoters. Most of these target genes are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program that precedes asexual replication to promote conversion to the sexual stage. Microarray analysis showed that the identified targets constituted ∼70% of the up-regulated genes in AP2-G2–depleted parasites, suggesting that AP2-G2 actually functions as a repressor in gametocytes. A promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step in Plasmodium sexual development.

CD8+ T Cells Specific for a Malaria Cytoplasmic Antigen Form Clusters around Infected Hepatocytes and Are Protective at the Liver Stage of Infection

ABSTRACT Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8+ T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8+ T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8+ T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8+ T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8+ T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8+ T cells during liver-stage infection.

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.

Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor

Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.