Biology subjects

Nishi, T.

Publications and source records attributed to Nishi, T..

13 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into multiple BAM-bound conformations of BepA enabling substrate triage at the outer membrane

The outer membrane (OM) of Gram-negative bacteria acts as a selective permeability barrier against toxic compounds, and for this function, requires proper assembly of outer membrane proteins (OMPs) by the {beta}-barrel assembly machinery (BAM) complex. A periplasmic metalloprotease BepA promotes maturation of LptD, an essential OMP, while degrades LptD intermediates aberrantly stalled at BAM. However, how BepA switches between these functions remains unclear. Here, we report cryo-EM structures of BAM- BepA complexes that capture multiple BepA conformations. In vivo crosslinking and cysteine-accessibility analyses show that possible regulatory elements, 6- and 9-loops that cover the proteolytic active site, can assume open conformations in living cells. Functional analyses reveal that 6-loop opening supports substrate interaction, degradation, and membrane association, whereas 9-loop opening is required for proteolytic activation and stabilizes BAM association. These findings provide insights into how distinct loop rearrangements regulate BepA-mediated substrate triage at BAM.

molecular biology↗

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

KCC2 Activation Reverses Neurophysiological and Behavioral Deficits in Female Rett Mice

Rett syndrome is an X-linked neurodevelopmental disorder resulting from mutations in the MeCP2 gene, leading to intellectual disability, impaired motor coordination, decreased sociability, and seizures. Central to the underlying pathophysiology are deficits in synaptic inhibition, which are mediated by hyperpolarizing GABAAR currents. These events develop postnatally and are dependent upon increased neuronal Cl- extrusion mediated by SLC12A5 (KCC2). Therefore, we tested whether its activation modifies the disease phenotypes evident in female MeCP2+/- mice, using OV350, a direct activator of KCC2. OV350 rapidly induced a sustained reduction in EEG power, accompanied by a decrease in the severity of epileptic discharges. Increased motor coordination, sociability, and spatial memory were also observed. Deficits in KCC2 phosphorylation were also seen in MeCP2+/- mice, consistent with reductions in its activity that were also ameliorated by OV350. Thus, KCC2 activation may be efficacious in limiting the impact of Rett syndrome and other neurodevelopmental disorders.

neuroscience↗

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↗

bioRxiv2025

Activation of KCC2 during development alleviates cognitive, behavioral, and neural excitability in adult CDKL5-deficient mice

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is a developmental and epileptic encephalopathy (DEE) characterized by severe drug-resistant epileptic disorders beginning in early childhood, along with cognitive and social impairments in later childhood and adulthood. Existing pharmacological therapies for CDD primarily focus on anti-seizure medications, which often have associated sedative side effects. In addition, there are currently no effective treatments for cognitive or behavioral impairments associated with this disorder. Postnatal development expression of CDKL5 has a similar timeline as the developmental activity of the potassium chloride co-transporter (KCC2), the maturation of which is a prerequisite for the developmental switch to fast synaptic hyperpolarizing inhibition mediated by g-aminobutyric acid type A receptors (GABAAR). This developmental GABA switch is determined by changes in the phosphorylation of multiple residues in KCC2. During this initial postnatal period, dramatic changes occur as major neuronal circuits are formed, laying down the initial pathways important for memory consolidation and behavioral processing. Currently, a knowledge gap exists in understanding KCC2 dysfunction in CDD. In adult Cdkl5 KO mice we found aberrant KCC2 phosphorylation and expression, such that KCC2 phosphorylation profile appeared immature. We examined the developmental changes in KCC2 and observed significant alterations in the phosphorylation of key residues and decreased KCC2 expression from p14 to p21. Because KCC2 loss-of-function has been strongly correlated with excessive neuronal excitation, cognitive and behavioral impairments, we examined seizure susceptibility, spatial memory, and social interaction in adult Cdkl5 KO mice following once daily administration of the KCC2 activator (OV350), or vehicle, to infant Cdkl5 KO mice. We found that adult Cdkl5 KO mice are more susceptible to kainate-induced seizures, show poor sociability and deficits in spatial learning and memory compared to WT mice. Twelve days of OV350 treatment as infants (p10 to p21) prevented the development of benzodiazepine-resistant seizures and alleviated cognitive and behavioral deficits in adult Cdkl5 KO mice. In contrast, 12 days of OV350 treatment in adult Cdkl5 KO mice had limited ability to alleviate cognitive and behavioral deficits. In summary, this study demonstrates that enhancing KCC2 function may be a potential therapeutic target for CDD and other DEEs. However, early intervention during critical developmental windows is crucial for optimal outcomes.

neuroscience↗

bioRxiv2024

SIP2 functions as the master transcription factor of the Plasmodium merozoite formation

Malaria, one of the most serious infectious diseases worldwide, is caused by the proliferation of Plasmodium parasites through repeated cycles of intraerythrocytic development. The parasite replicates via schizogony in host erythrocytes, producing multiple progeny merozoites that invade new erythrocytes to continue the intraerythrocytic developmental cycle. Although merozoite formation is the most crucial step in parasite proliferation and malaria pathogenesis, the molecular mechanism regulating merozoite formation remains unclear. SIP2 is an AP2 transcription factor expressed during schizogony and is particularly conserved among erythrocyte-infecting apicomplexan parasites. Here, we reveal that SIP2 in P. berghei (PbSIP2) functions as a transcriptional activator that regulates merozoite formation. Disruption of pbsip2 using a dimerizable Cre recombinase system resulted in developmental arrest before merozoite formation and significant downregulation of merozoite-related genes. ChIP-seq of PbSIP2 showed that it comprehensively activated merozoite-related genes by binding to previously reported cis-regulatory elements of merozoite invasion-related genes, including the bipartite motif (TGCAN4-6GTGCA). Collectively, our results indicate that SIP2 is a transcription factor that establishes erythrocyte infectivity and may have an evolutionary origin from the common ancestor of erythrocyte-infecting apicomplexan parasites.

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↗

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↗

bioRxiv2019

Transmission network reconstruction for foot-and-mouth disease outbreaks incorporating farm-level covariates

Transmission network modelling to infer who infected whom in infectious disease outbreaks is a highly active area of research. Outbreaks of foot-and-mouth disease have been a key focus of transmission network models that integrate genomic and epidemiological data. The aim of this study was to extend Laus systematic Bayesian inference framework to incorporate additional parameters representing predominant species and numbers of animals held on a farm.\n\nLaus Bayesian Markov chain Monte Carlo algorithm was reformulated, verified and pseudo-validated on simulated outbreaks populated with demographic data Japan and Australia. The modified model was then implemented on genomic and epidemiological data from the 2010 outbreak of foot-and-mouth disease in Japan, and outputs compared to those from the SCOTTI model implemented in BEAST2.\n\nThe modified model achieved improvements in overall accuracy when tested on the simulated outbreaks. When implemented on the actual outbreak data from Japan, infected farms that held predominantly pigs were estimated to have five times the transmissibility of infected cattle farms and be 49% less susceptible. The farm-level incubation period was 1 day shorter than the latent period, the timing of the seeding of the outbreak in Japan was inferred, as were key linkages between clusters and features of farms involved in widespread dissemination of this outbreak. To improve accessibility the modified model has been implemented as the R package BORIS for use in future outbreaks.

microbiology↗

bioRxiv2018

Reverse transcription-PCR using a Primer Set Targeting the 3D Region Detects Foot-and-Mouth Disease Virus with High Sensitivity

Because foot-and-mouth disease (FMD) has the potential to spread extensively, methods used for its diagnosis must be rapid and accurate. Therefore, reverse transcription-PCR (RT-PCR) plays an important diagnostic role. Here we designed the primer set FM8/9 to amplify 644 bases of the conserved 3D region of all seven serotypes of FMD virus (FMDV). We compared the performance of RT-PCR assays using FM8/9 with that using the primer set 1F/R targeting the 5-UTR described in the manual of the World Organization for Animal Health. The detection limits of the RT-PCR assays were determined for 14 strains representing all serotypes. Compared with the sensitivities of the RT-PCR assay using 1F/R, those using FM8/9 were 101-to 104-fold higher for eight strains. To assess the validity of the methods for analyzing clinical samples, sera and saliva samples from pigs and cows infected with FMDV were collected daily and analyzed using the two PCR assays. The FM8/9 assay detected FMDV from all infected pigs and cows for longer times compared with the 1F/R assay, therefore revealing higher sensitivity for the clinical samples. Our results suggest that the FM8/9 RT-PCR assay is highly sensitive and is therefore suitable for the diagnosis of FMD.

microbiology↗