Shiroh Iwanaga

Identification of a transcription factor in the mosquito‐invasive stage of malaria parasites

Gene expression in Plasmodium parasites undergoes significant changes in each developmental stage, but the transcription factors (TFs) regulating these changes have not been identified. We report here a Plasmodium TF (AP2‐O) that activates gene expression in ookinetes, the mosquito‐invasive form, and has a DNA‐binding domain structurally related to that of a plant TF, Apetala2 (AP2). AP2‐O mRNA is pre‐synthesized by intraerythrocytic female gametocytes and translated later during ookinete development in the mosquito. The Plasmodium TF activates a set of genes, including all genes reported to be required for midgut invasion, by binding to specific six‐base sequences on the proximal promoter. These results indicate that AP2 family TFs have important roles in stage‐specific gene regulation in Plasmodium parasites.

Transcription factor AP2‐Sp and its target genes in malarial sporozoites

The malarial sporozoite is the stage that infects the liver, and genes expressed in this stage are potential targets for vaccine development. Here, we demonstrate that specific gene expression in this stage is regulated by an AP2‐related transcription factor, designated AP2‐Sp (APETALA2 in sporozoites), that is expressed from the late oocyst to the salivary gland sporozoite. Disruption of the AP2‐Sp gene did not affect parasite replication in the erythrocyte but resulted in loss of sporozoite formation. The electrophoretic mobility‐shift assay showed that the DNA‐binding domain of AP2‐Sp recognizes specific eight‐base sequences, beginning with TGCATG, which are present in the proximal promoter region of all known sporozoite‐specific genes. Promoter assays demonstrated that these sequences act as cis‐acting elements and are critical for the expression of sporozoite‐specific genes with different expression profiles. In transgenic parasites that express endogenous AP2‐O (APETALA2 in ookinetes), but whose AP2 domain had been swapped with that of AP2‐Sp, several target genes of AP2‐Sp were induced in the ookinete stage. These results indicate that AP2‐Sp is a major transcription factor that regulates gene expression in the sporozoite stage.

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.

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.

Functional Identification of the Plasmodium Centromere and Generation of a Plasmodium Artificial Chromosome

Summary The artificial chromosome represents a useful tool for gene transfer, both as cloning vectors and in chromosome biology research. To generate a Plasmodium artificial chromosome (PAC), we had to first functionally identify and characterize the parasites centromere. A putative centromere (pbcen5) was cloned from chromosome 5 of the rodent parasite P. berghei based on a Plasmodium gene-synteny map. Plasmids containing pbcen5 were stably maintained in parasites during a blood-stage infection with high segregation efficiency, without drug pressure. pbcen5-containing plasmids were also stably maintained during parasite meiosis and mitosis in the mosquito. A linear PAC (L-PAC) was generated by integrating pbcen5 and telomere into a plasmid. The L-PAC segregated with a high efficiency and was stably maintained throughout the parasites life cycle, as either one or two copies. These results suggest that L-PAC behaves like a Plasmodium chromosome, which can be exploited as an experimental research tool.

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.

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.

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.