Biology subjects

Iwanaga, S.

Publications and source records attributed to Iwanaga, S..

13 recordsLinked to original sources

bioRxiv2026

Transcriptional repression by AP2-Sp3 regulates the mosquito-to-mammal infectivity switch in malaria sporozoites

Malaria sporozoites are a key developmental stage responsible for mosquito-to-mammal transmission. Sporozoites formed within oocysts on the mosquito midgut first mature into a salivary gland-invasive stage and subsequently become liver-infective upon colonizing the salivary glands. This functional switch is essential for successful parasite transmission, yet its underlying molecular mechanisms remain unclear. Here, we identify the AP2-family transcription factor AP2-Sp3 as a key regulator of this process in Plasmodium berghei. AP2-Sp3 is expressed in midgut sporozoites but disappears in salivary gland sporozoites. It directly binds to the cis-regulatory motif CATTG and represses the target genes, which include the majority of known liver infection-related genes. Disruption of ap2-sp3 impairs development of midgut sporozoites and causes a premature shift of their transcriptome toward a salivary gland sporozoite-like state. Together, these findings demonstrate that AP2-Sp3 maintains the midgut sporozoite-specific transcriptome by suppressing the salivary gland sporozoite transcriptional program, thereby controlling the mosquito-to-mammal infectivity switch.

microbiology↗

bioRxiv2026

Immune receptor LILRB1 mediates cis-signalling which is targeted by RIFINs of the malaria parasite

The malaria parasite, Plasmodium falciparum, replicates within human erythrocytes, where it is susceptible to clearance by immune cells. It uses erythrocyte surface proteins, the RIFINs, to signal through immune receptors and to suppress immune cell function. Previous studies identified two groups of RIFINs which bind different sites on inhibitory immune receptor LILRB1. While some RIFINs bind an elongated LILRB1 conformation, triggering inhibitory immune signalling in trans, we show that other RIFINs stabilise a c-shaped LILRB1 conformation. This buckled LILRB1 binds MHC class I found on the same immune cell, which triggers inhibitory cis signalling. Therefore, LILRB1 exists in dynamic equilibrium, with an elongated conformation able to bind to ligands in trans on a target cell, while a buckled conformation signals through MHC class I in cis, setting the signalling threshold. Different clades of RIFINs exist to mediate inhibitory signalling through each of these LILRB1 conformations to prevent parasite destruction.

microbiology↗

bioRxiv2025

Precise regulation of gene expression through transcriptional repression is essential for Plasmodium asexual blood stage development

Malaria is caused by the proliferation of Plasmodium parasites in the vertebrate host blood stream through repeated cycles of asexual multiplication inside erythrocytes. During these cycles, parasites dynamically change their transcriptome at each developmental step to express genes exactly when required; however, the mechanisms regulating these transcriptomic changes remain unclear. In this study, we revealed that the AP2-family transcription factor PbAP2-TR is essential for the asexual blood stage development of the rodent malaria parasite Plasmodium berghei, as a transcriptional repressor. Conditional knockout of pbap2-tr caused developmental arrest at the trophozoite stage, i.e., the cell growth phase of asexual blood stage development. Chromatin immunoprecipitation followed by high-throughput sequencing showed that PbAP2-TR binds to two different DNA motifs and targets genes that are downregulated towards late trophozoites, including ribosome biogenesis-related genes and host-modifying exported protein genes. The expression of these target genes was upregulated in pbap2-tr-knockout parasites, and introduction of mutations into the binding motifs increased the promoter activity of the target genes. These results indicate that PbAP2-TR establishes precise transcription peak patterns of its target genes by repressing their transcription during the trophozoite, thereby being essential for asexual blood stage development. Our data also suggest that PbAP2-TR induces transcriptional repression by recruiting a putative chromatin remodeler, PbMORC, as a co-factor.

microbiology↗

bioRxiv2024

Raman flow cytometry using time delay integration

Raman flow cytometry offers chemically sensitive, label-free measurement of cells and particles; however, the technique suffers from low cell throughput due to the weak Raman signal. Here, we demonstrate the use of time-delay integration to achieve Raman flow cytometry combined with dual-sided line illumination. The use of line illumination from both sides of the cell flow capillary kept the cell stream in the detection area by balancing optical force from the illumination lines. The time-delay integration allowed accumulation of Raman signals from flowing cells without sacrificing the spectrum readout rate. With the developed system, we achieved Raman flow cytometry at throughputs of 32 and 75 events per second for cell and particle detection, respectively. We applied the technique for analyzing lipid uptake in HepG2 cells and degranulation in bone marrow-derived murine mast cells.

biophysics↗

bioRxiv2024

Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei

Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. The advent of CRISPR enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR-Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked-out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirm the role of CLAMP during invasion of erythrocytes.

microbiology↗

bioRxiv2024

RIFINs displayed on malaria-infected erythrocytes bind both KIR2DL1 and KIR2DS1

To discriminate between our own cells and foreign cells such as a pathogen, natural killer cells are armed with inhibitory and activating immune receptors. In some cases, such as the KIRs, these are found in pairs, with inhibitory and activating receptors containing nearly identical extracellular domains that are coupled to different intracellular signalling domains1. The balance in signalling mediated by these receptors determines whether an NK cell is activated to destroy a target cell. Previous studies have shown that RIFINs, displayed on surfaces of Plasmodium falciparum-infected erythrocytes, can bind to inhibitory immune receptors and dampen NK cell activation2,3, reducing parasite killing. Here, we identify a clade of RIFINs that bind to inhibitory immune receptor KIR2DL1 approximately ten-times more strongly than KIR2DL1 binds its host ligand, MHC class I. We show that this interaction mediates inhibitory signalling and reduces activation of KIR2DL1-expressing NK cells. We reveal the structural basis for KIR2DL1 binding and show that the RIFIN binding surface of KIR2DL1 is conserved in the activating immune receptor KIR2DS1. We find that KIR2DL1-binding RIFINs can also bind to KIR2DS1 and that these RIFINs cause activation of KIR2DS1-expressing NK cells. This highlights the evolutionary battle between pathogen and host, suggesting that activating KIRs may have evolved to allow detection of red blood cells infected with Plasmodium falciparum, helping the host to clear the parasite.

microbiology↗

bioRxiv2023

gARID-associated chromatin remodeling events are essential for gametocyte development in Plasmodium

Gametocyte development of the Plasmodium parasite is a key step for transmission of the parasite from their vertebrate hosts to mosquitoes. Male and female gametocytes are produced from a subpopulation of asexual blood-stage parasites, but the mechanisms that regulate the differentiation of sexual stages are still under investigation. In this study, we investigated the role of gARID, a putative subunit of a chromatin remodeling complex, in transcriptional regulation during the gametocyte development of P. berghei. gARID expression starts in early gametocytes before the manifestation of male and female-specific features, and disruption of its gene results in the complete loss of male gametocytes and the production of abnormal female gametocytes. ChIP-seq analysis of gARID showed that it forms a complex with gSNF2, a core subunit of the SWI/SNF chromatin remodeling complex, associating with the male cis-regulatory element. Moreover, ChIP-seq of gARID in gsnf2-knockout parasites revealed an association of gARID with another cis-regulatory element, which is indicated to play a role in both male and female development. Our results showed that gARID functions in two chromatin remodeling events and that remodeling of chromatin states is essential for both male and female gametocyte development.

molecular biology↗

bioRxiv2023

Coordinated regulation of gene expression in Plasmodium female gametocytes by two transcription factors

Gametocytes play key roles in the Plasmodium lifecycle. They are essential for sexual reproduction as precursors of the gametes. They also play an essential role in parasite transmission to mosquitoes. Elucidation of the gene regulation at this stage is essential for understanding these two processes at the molecular level and for developing new strategies to break the parasite life cycle. We identified a novel Plasmodium transcription factor (TF), designated as a partner of AP2-FG or PFG. In this paper, we report that this TF regulates the gene expression in female gametocytes in concert with another female-specific TF AP2-FG. Upon the disruption of PFG, majority of female-specific genes were significantly downregulated, and female gametocyte lost the ability to produce ookinetes. ChIP-seq analysis showed that it was located in the same position as AP2-FG, indicating that these two TFs form a complex. ChIP-seq analysis of PFG in AP2-FG-disrupted parasites and ChIP-seq analysis of AP2-FG in PFG-disrupted parasites demonstrated that PFG mediates the binding of AP2-FG to the ten-base motif and that AP2-FG binds another motif, GCTCA, in the absence of PFG. In promoter assays, this five-base motif was identified as another female-specific cis-acting element. Genes under the control of the two forms of AP2-FG, with or without PFG, partly overlapped; however, each form had target preferences. These results suggested that combinations of these two forms generate various expression patterns among the extensive genes expressed in female gametocytes.

microbiology↗

bioRxiv2022

PbAP2-FG2 and AP2R-2 function together as a transcriptional repressor complex essential for Plasmodium female development

Gametocyte development is a critical step in the life cycle of Plasmodium. Despite that numbers of studies in the gametocyte development have been conducted, the molecular mechanisms regulating this process remains to be fully understood. This study investigates the functional roles of two female-specific transcriptional regulators, PbAP2-FG2 and AP2R-2, in P. berghei. Knockout of pbp2-fg2 or ap2r-2 impairs female gametocyte development, resulting in developmental arrest during ookinete development. ChIP-seq analyses of these two factors indicated their colocalization on the genome, suggesting they function as a complex. These analyses also revealed that their target genes contained a variety of genes, including both male and female-enriched genes. Moreover, differential expression analyses showed that these target genes were upregulated through the disruption of pbp2-fg2 or ap2r-2, indicating that these two factors function as a transcriptional repressor complex in female gametocytes. Further target analysis demonstrated a significant overlap between the target genes of PbAP2-FG2 and AP2-G, suggesting that repression of early gametocyte genes activated by AP2-G is one of the key roles for this female transcriptional repressor complex. Our results indicate that the PbAP2-FG2-AP2-R2 complex-mediated repression of the target genes supports the female differentiation from early gametocytes. Author SummaryGametocyte development in Plasmodium parasites, a causative agent of malaria, is an essential step for their transmission from vertebrate hosts to mosquitoes. Gametocytes are sexual precursor cells produced from a subpopulation of asexual blood-stage parasites. Upon uptake by mosquitoes through blood feeding, the male and female gametocytes become microgametes and macrogametes, respectively, and then they fertilize and develop into the mosquito midgut invasive stage, called ookinete. Therefore, it is crucial to understand the underlying mechanisms regulating this developmental process. This study revealed that the two female transcriptional regulators, PbAP2-FG2 and AP2R-2, function together as an essential transcriptional repressor complex in P. berghei, the target genes of which include male, female, and early gametocyte genes activated by AP2-G. Our findings suggest that PbAP2-FG2 and AP2R-2 play multiple roles in supporting the development of female gametocytes from early gametocytes.

microbiology↗

bioRxiv2022

Identification of a novel AP2 transcription factor in zygotes with an essential role in Plasmodium ookinete development.

The sexual phase of Plasmodium represents a crucial step in malaria transmission, during which these parasites fertilize and form ookinetes to infect mosquitoes. Plasmodium development after fertilization is thought to proceed with female-stored mRNAs until the formation of a retort-form ookinete; thus, transcriptional activity in zygotes has previously been considered quiescent. In this study, we reveal the essential role of transcriptional activity in zygotes by investigating the function of a newly identified AP2 transcription factor, AP2-Z. ap2-z was previously reported as a female transcriptional regulator gene whose disruption resulted in developmental arrest at the retort stage of ookinetes. In this study, although ap2-z was transcribed in females, we show that it was translationally repressed by the DOZI complex and translated after fertilization with peak expression at the zygote stage. ChIP-seq analysis of AP2-Z shows that it binds on specific DNA motifs, targeting the majority of genes known as an essential component of ookinetes, which largely overlap with the AP2-O targets, as well as genes that are unique among the targets of other sexual transcription factors. The results of this study also indicate the existence of a cascade of transcription factors, beginning with AP2-G, that proceeds from gametocytogenesis to ookinete formation. Author summarySexual development in Plasmodium parasites, a causative agent of malaria, is essential for their transmission from vertebrate hosts to mosquitoes. This important developmental process proceeds as follows: formation of a gametocyte/gamete, fertilization and conversion of the zygote into the mosquito midgut invasive stage, called the ookinete. As a target of transmission blocking strategies, it is important to understand the mechanisms regulating Plasmodium sexual development. In this study, we assessed transcriptional regulation after fertilization by investigating the function of a novel transcription factor, AP2-Z. The results revealed the essential role of de novo transcription activated by AP2-Z in zygotes for promoting ookinete development. As transcriptional activity during the zygote stage has previously been considered silent in Plasmodium, novel genes important for ookinete formation can now be explored in the target genes of AP2-Z. Investigating the functions of these genes can help us understand the mechanisms of Plasmodium zygote/ookinete development and identify new targets for transmission blocking vaccines.

microbiology↗

bioRxiv2022

Targetome analysis of malaria sporozoite transcription factor AP2-Sp reveals its role as a master regulator

Malaria transmission to humans begins with sporozoite infection of the liver. The elucidation of gene regulation during the sporozoite stage will promote the investigation of mechanisms of liver infection by this parasite and contribute to the development of strategies for preventing malaria transmission. AP2-Sp is a transcription factor (TF) essential for the formation of sporozoites or sporogony, which takes place in oocysts in the midgut of infected mosquitoes. To understand the role of this TF in the transcriptional regulatory system of this stage, we performed ChIP-seq analyses using whole mosquito midguts containing late oocysts as starting material and explored its genome-wide target genes. We identified 697 target genes, comprising those involved in distinct processes parasites experience during this stage, from sporogony to development into the liver-stage, and representing the majority of genes highly expressed in the sporozoite stage. These results suggest that AP2-Sp determines basal patterns of gene expression by targeting a broad range of genes directly. The ChIP-seq analyses also showed that AP2-Sp maintains its own expression by a transcriptional auto-activation mechanism (positive feedback loop) and induces all TFs reported to be transcribed at this stage, including AP2-Sp2, AP2-Sp3, and SLARP. The results showed that AP2-Sp exists at the top of the transcriptional cascade of this stage and triggers the formation of this stage as a master regulator.

microbiology↗

bioRxiv2021

Chromosome splitting of Plasmodium berghei using the CRISPR/Cas9 system.

Spatial arrangement of chromosomes is responsible for gene expression in Plasmodium parasites. However, methods for rearranging chromosomes have not been established, which makes it difficult to investigate its role in detail. Here, we report a method for splitting chromosome in rodent malaria parasite by CRISPR/Cas9 system using fragments in which a telomere and a centromere were incorporated. The resultant split chromosomes segregated accurately into daughter parasites by the centromere. In addition, elongation of de novo telomeres were observed, indicating its proper function. Furthermore, chromosome splitting had no effect on development of parasites. Splitting of the chromosome is expected to alter its spatial arrangement, and our method will thus be useful for investigating its biological role related with gene expression.

microbiology↗

bioRxiv2020

Species composition and risk of transmission of Aedes-borne arboviruses around some Yellow hotspot areas in Northern Ghana

Aedes-borne viral diseases mainly Yellow Fever (YF), Dengue (DEN), Zika (ZIK) and Chikungunya (CHK) have contributed to many deaths in the world especially in Africa. There have been major outbreaks of these diseases in West Africa. YF outbreaks have occurred in Ghana. Although Ghana has not recorded any outbreak of DEN, ZIK and CHK, the risk is high due to its proximity to West African countries where outbreaks have been recently been recorded. This study assessed the risk of transmission of Yellow fever (YFV), Dengue (DENV), Chikungunya (CHKV) and Zika (ZIKV) viruses in Larabanga and Mole Game Reserve areas in Northern Ghana. The immature and adult stages of Aedes mosquitoes were collected from Larabanga and Mole Game Reserve area. There was a significant (P>0.001) number of mosquitoes collected during the rainy season than the dry season. A total of 1,930 Aedes mosquitoes were collected during the rainy season and morphologically identified. Of these, 1,915 (99.22%) were Aedes aegypti and 15 (0.22%) were Aedes vittatus. During the dry season, 27 Aedes aegypti mosquitoes were collected. A total of 415 Ae. aegypti mosquitoes were molecularly identified to subspecies level of which Aedes (Ae) aegypti aegypti was the predominant subspecies. Both Ae. aegypti aegypti and Ae aegypti formosus exist in sympatry in the area. All Aedes pools (75) were negative for DENV, ZIKV and CHKV when examined by RT-PCR. Three Larval indices namely House Index, HI (percentage of houses positive for Aedes larvae or pupae), Container Index, CI (the percentage of containers positive for Aedes larvae or pupae) and Breteau Index, BI (the number of positive containers (with larvae and/or pupae per 100 inspected houses) were assessed as a measure for risk of transmission. The HI, CI and BI for both sites were as follows; Mole game reserve (HI, 42.1%, CI, 23.5% and BI, 100 for rainy season and 0 for all indices for dry season) and Larabanga (39%, 15.5% and 61 for rainy season and 2.3%, 1.3% and 2.3 for dry season). The spatial distribution of Aedes breeding sites in both areas indicated that Aedes larvae were breeding in areas with close proximity to humans. Lorry tires were the main source of Aedes larvae in all the study areas. Information about the species composition and the potential role of Aedes mosquitoes in future outbreaks of the diseases that they transmit is needed to design efficient surveillance and vector control tools.

ecology↗