Biology subjects

Takashima, E.

Publications and source records attributed to Takashima, E..

8 recordsLinked to original sources

bioRxiv2025

Plasmodium falciparum diacylglycerol acyltransferase maintains phospholipid homeostasis to regulate sexual differentiation, ER stress, and cytoadhesion

Plasmodium falciparum is the causative agent of human malaria, a life-threating infectious disease that imposes a major global health burden. Lipid metabolism is indispensable for this parasites replication and survival, yet most of the molecular components and mechanisms involved remain poorly understood. In eukaryotes, lipid droplets (LDs) serve as dynamic organelles that store neutral lipids (NLs), buffer lipotoxic stress, and regulate signaling pathways, with their biogenesis controlled by diacylglycerol o-acyltransferases (DGATs). Although P. falciparum encodes a putative DGAT (PF3D7_0322300), its role in the parasite life cycle has not been elucidated. We generated conditional PfDGAT-knockout parasites to investigate the enzymes functional significance. PfDGAT deficiency led to parasite death, accompanied by reduced LD formation, elevated phospholipid levels, and induction of ER stress. Moreover, PfDGAT deletion altered protein trafficking, resulting in the decreased cytoadherence of parasite-infected erythrocytes to human brain microvascular endothelial cells, and suppressed parasite sexual differentiation. Thus, PfDGAT deletion affected multiple aspects of the parasites life cycle, highlighting its critical role in parasite survival and pathogenesis. Our findings provide new insights into parasite lipid homeostasis and highlight DGAT as a potential target of antimalarial intervention.

microbiology↗

bioRxiv2025

Navigating parasite antigen genetic diversity in the design of Plasmodium vivax serological exposure markers for malaria

BackgroundPlasmodium vivax poses a major obstacle to malaria elimination because it can lie dormant in the liver for weeks or months before reactivating and causing a relapse of infection. These dormant forms (hypnozoites) cannot be detected using standard diagnostics, but recent P. vivax exposure and by proxy, hypnozoite carriage, can be inferred using antibody-based tests (serological markers). In this study, we examined how genetic variation in P. vivax affects the utility of these antibody markers, and whether redesigned antigens could improve performance. MethodsWe analysed global P. vivax genetic data to assess variation in leading serological markers. Based on this, we produced new antigen versions (haplotypes) that better reflect global sequence diversity, compared to the commonly used reference strain (Sal-1). Antibody responses against these new constructs were then tested using samples from well-characterised cohorts in Brazil and Thailand. Antibody levels were assessed in relation to how recently participants had a qPCR-detectable blood-stage P. vivax infection. We compared the ability of the haplotypes and reference constructs to correctly identify individuals infected within the prior 9-months. FindingsExtensive genetic diversity was identified in two P. vivax antigens, DBPII and MSP5. Several antigens had large numbers of circulating haplotypes globally, with the percentage with similar sequence identity to the reference Sal-1 ranging from 0.4% (MSP5) to 99% (S16). Two antigens exhibited strong differences in immunogenicity by region and construct (RBP2a and DBPII). However, for most proteins (5 out of 8), these differences had little impact on the accuracy of identifying recent exposure. In cases where performance was affected (e.g. RBP2a), this could be overcome by adding multiple antigens into the classification model. InterpretationEven highly diverse antigens can be effective serological exposure markers. Our findings highlight the importance of testing the impact of genetic diversity when designing serological tests and suggest practical strategies, such as using a mix of antigens, to ensure consistent performance across regions.

microbiology↗

bioRxiv2025

An in vivo fitness gene of Toxoplasma, MIC11, is essential for PLP1-mediated egress from host cells

After invasion and replication, intracellular pathogens must egress from infected host cells. Toxoplasma gondii facilitates this process by permeabilizing host cells by releasing perforin-like protein 1 (PLP1) through induced microneme secretion. However, the precise mechanism of host cell permeabilization remains enigmatic. Here, we identified the secretory microneme protein MIC11 as a key factor for membrane disruption. A CRISPR-based in vivo screen revealed several genes including MIC11 as an essential gene for virulence. Deletion of MIC11 resulted in severe defects in both membrane rupture and egress. Scanning mutagenesis identified functional motifs in MIC11, and mechanistic analyses demonstrated that MIC11 directly associates with PLP1, regulating its activity in membrane disruption. The MIC11 paralogue MIC22 compensated for MIC11 deletion, suggesting a conserved mechanism of egress in the feline-restricted stages of T. gondii. The discovery of MIC11 advances the understanding of how parasites disrupt host cells to facilitate rapid egress and successful dissemination.

microbiology↗

bioRxiv2024

Pfs230 Domain 12 is a potent malaria transmission-blocking vaccine candidate

Malaria transmission-blocking vaccines (TBV) target sexual stage parasites that are transmitted to mosquitoes and critical for spread of the pathogen. The clinically most advanced TBV candidate contains part of the Pro-domain (Pro) and Domain 1 (D1) of Plasmodium falciparum surface protein Pfs230. Subunit vaccines that contain other domains of Pfs230 have so far failed to induce functional antibodies. Here, we produced eight single domain fragments of Pfs230 in Drosophila melanogaster S2 cells and assessed their immunogenicity in mouse immunizations. In addition to D1-specific antibodies, antibodies raised against Domain 12 (D12) showed strong recognition of Pfs230 on the surface of female parasites. Importantly, D12-specific antibodies showed strong functional transmission-reducing activity in membrane feeding assays with cultured parasites, an activity that was complement-dependent. Murine D12-specific antibodies further reduced mosquito transmission of parasites acquired from naturally infected parasite carriers. The D12 antigen was recognized by sera from an all-age cohort of individuals who had been naturally exposed to Plasmodium falciparum with antibody levels increasing with age. In conclusion, we identified Pfs230D12 as a promising new TBV candidate. Impact statementPfs230 Domain 12 induces antibodies in mice that strongly reduce transmission of lab-cultured and naturally circulating malaria parasites to mosquitoes.

immunology↗

bioRxiv2024

Ultrastructural expansion microscopy (U-ExM) visualization of malaria parasite dense granules using RESA as a representative marker protein

Dense granules (DG) are understudied apical organelles in merozoites, the malaria parasite stage that invades erythrocytes. Only six proteins have been identified which localize to DGs, despite that DG proteins play crucial roles in multiple steps of intraerythrocytic parasite development. To develop a tool for investigating DG structure and function, this study applied ultrastructural expansion microscopy (U-ExM) to visualize the ring-infected erythrocyte surface antigen (RESA) in Plasmodium falciparum merozoites. Merozoites were expanded to approximately four times their original size, allowing the identification of DGs without the need for electron microscopy. RESA localization in merozoite DGs was confirmed by staining with a combination of anti-RESA mAb and protein staining by NHS-ester. The translocation of RESA to the infected erythrocyte membrane was also observed in early ring-stage parasites. These results are in good agreement with the RESA localization reported using immunoelectron microscopy (IEM). By using U-ExM, the identification of novel DG proteins will be facilitated without time-consuming IEM, thereby contributing to describing erythrocyte parasitism by P. falciparum.

microbiology↗

bioRxiv2021

Naturally acquired antibody kinetics against Plasmodium vivax antigens in people from a low malaria transmission region in western Thailand

Plasmodium vivax is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex(R) assay, and identified protein-specific variation in antibody longevity. Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

microbiology↗

bioRxiv2020

A comparison of non-magnetic and magnetic beads for measuring IgG antibodies against P. vivax antigens in a multiplexed bead-based assay using Luminex(R) technology (Bio-Plex(R)200 or MAGPIX(R))

Multiplexed bead-based assays that use Luminex xMAP(R) technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data is lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex(R) 100/200 or Bio-Plex(R) 200), magnetic beads can be run on both these and the newer MAGPIX(R) instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex(R) 200, ii) magnetic beads run on the Bio-Plex(R) 200 versus MAGPIX(R) and iii) non-magnetic beads run on a Bio-Plex(R) 200 versus magnetic beads run on the MAGPIX(R). We also performed an external validation of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were strongly correlated in all three of our comparisons, however higher amounts of protein were required for coupling to magnetic beads. Our external validation indicated that results generated in different laboratories using the same coupled beads are also highly comparable, particularly if a reference standard curve is used.

immunology↗

bioRxiv2018

Development and validation of serological markers for detecting recent exposure to Plasmodium vivax infection

In order to accelerate towards malaria elimination, improved targeting of limited resources is essential. A major gap in our elimination toolkit for Plasmodium vivax malaria is the identification of individuals carrying arrested liver stages, called hypnozoites. These clinically silent but frequently relapsing hypnozoites are key to P. vivax persistence. Whilst hypnozoites cannot be directly detected, individuals who have had recent exposure to P. vivax and have not been treated are likely to harbor these parasites. By measuring IgG antibody responses to over 300 P. vivax proteins, a panel of serological markers capable of detecting exposure to P. vivax infections in the prior 9-month period was identified and validated. Using antibody responses to 8 P. vivax proteins, 80% sensitivity and specificity for detecting recent infections were achieved in three independent studies conducted in Thailand, Brazil and the Solomon Islands. As these individuals have a high likelihood of harboring hypnozoites, the suite of these 8 antibody responses can serve as biomarkers for the identification of individuals who should be targeted for treatment with liver-stage drugs such as primaquine and tafenoquine in mass drug administration programs aimed at controlling and eliminating P. vivax malaria.\n\nOne Sentence SummaryThe manuscript describes identification and validation of a novel panel of P. vivax proteins that can be used to detect recent exposure to P. vivax infections within the prior 9 months.

microbiology↗