Biology subjects

Kataoka, M.

Publications and source records attributed to Kataoka, M..

At least 19 recordsLinked to original sources

bioRxiv2026

A universal plug-and-display vaccine platform for mucosal and systemic immunity using Bacillus subtilis membrane vesicles

Many bacterial species naturally secrete membrane vesicles (MVs) that mediate the intercellular transport of biomolecules, including nucleic acids, proteins, and metabolites. Beyond their native physiological roles, MVs hold considerable potential for biomedical applications. Here, we demonstrate that MVs from several Bacillus species exhibit potent intrinsic adjuvant activity, efficaciously eliciting immune responses and facilitating antigen-specific antibody production in mice. Exploiting this adjuvanticity, we engineered a highly adaptable universal vaccine platform that uses B. subtilis MVs as self-adjuvanting carriers. This system employs a modular "plug-and-display" architecture that covalently anchors recombinant antigens to the MV surface through a multi-step bioconjugation cascade. After validation of this methodology using a model antigen, we adapted the platform to target Yersinia pestis, the causative agent of plague. We formulated a Y. pestis vaccine by labeling the MV surface with a modified capsule antigen fraction 1 (mCaf1). Intranasal administration of the mCaf1-MV vaccine effectively elicited both systemic and mucosal immunity. Crucially, this vaccine conferred highly efficacious protection against a lethal Y. pestis infection in a murine model. These findings demonstrate the exceptional protective efficacy of the B. subtilis MV platform and highlight its broad potential for the rapid development of mucosal vaccines against diverse emerging pathogens.

bioengineering↗

bioRxiv2026

West Nile virus capsid protein promotes viral replication and pathogenesis through PKCα-dependent lamin phosphorylation and nuclear deformation

The genus Orthoflavivirus comprises several medically important pathogens such as the West Nile virus (WNV), which causes encephalitis in humans. Although viral replication occurs in the cytoplasm, the capsid (C) protein of the orthoflavivirus is localized to both the cytoplasm and nucleus. Nuclear C protein contributes to viral replication and disease progression. However, the underlying mechanisms remain unclear. Here, we investigated whether the WNV C protein induces nuclear deformation and examined the underlying mechanism. We also assessed the contribution of this deformation to viral replication and pathogenesis. WNV infection and C protein expression induced morphological alterations in the nuclear lamina, leading to nuclear deformation. C protein expression enhanced lamin phosphorylation and the disassembly of the polymerized lamin network. In addition, C protein interacted with protein kinase C alpha (PKC) and localized PKC near the nuclear lamina. Downregulation of PKC expression inhibited C protein-induced lamin phosphorylation and nuclear deformation. In addition, both the downregulation of PKC expression and pharmacological inhibition of PKC reduced WNV replication. In contrast, the expression of phosphorylation-deficient lamin mutants attenuated the inhibitory effect of downregulated PKC expression on WNV replication. Furthermore, the pharmacological inhibition of PKC increased the survival rate of WNV-infected mice and suppressed both viral replication and nuclear deformation in the brain. Collectively, these results demonstrate that C protein remodels the nuclear lamina architecture through the PKC-lamin pathway, and that virus-induced nuclear deformation contributes to WNV replication and pathogenesis. Author summaryThe West Nile virus (WNV), a neurotropic orthoflavivirus, causes severe neurological diseases in humans. In host cells, orthoflaviviruses exclusively replicate in the cytoplasm. However, their capsid (C) proteins are localized to both the nucleus and cytoplasm. Although the nuclear C protein has been implicated in viral replication and disease progression, its underlying mechanisms remain unclear. Here, we demonstrate that the WNV C protein induces nuclear deformation, accompanied by the phosphorylation of lamin and disassembly of the nuclear lamina, a structural scaffold that maintains the nuclear shape. The C protein promotes the localization of PKC, a host kinase protein, near the nuclear lamina. Suppression of PKC expression or activity reduces lamin phosphorylation, nuclear deformation, and WNV replication. Importantly, pharmacological inhibition of PKC in WNV-infected mice reduced nuclear deformation and viral replication in the brain and improved survival rates. Collectively, our findings identify the host nucleus as an important site of WNV-host interaction and provide a new perspective that WNV, despite replicating in the cytoplasm, remodels host nuclear architecture to promote viral replication and pathogenesis.

microbiology↗

bioRxiv2026

Identification of Novel Gamma Radiation-Responsive Genes in the Thiosulfate-Oxidizing Bacterium Limnobacter thiooxidans

Ionizing radiation induces DNA damage and oxidative stress; however, the genes and molecular mechanisms involved in bacterial stress responses have not been sufficiently identified. In this study, we used Limnobacter thiooxidans strain CS-K2, which is the closest relative to the bacteria detected in torus room water at the Fukushima Daiichi Nuclear Power Plant according to 16S rRNA gene sequences, and evaluated its response to {gamma}-ray irradiation using integrated transcriptomic and proteomic analyses. We identified three previously uncharacterized genes (LT3105, LT3115, and LT3126) that were strongly induced at the mRNA and protein levels. These genes exhibited low basal expression but were markedly upregulated by {gamma}-ray irradiation. Notably, LT3126 encodes a protein containing VIT (vault protein inter--trypsin) and VWA (von Willebrand factor type A) domains and showed the strongest induction. Overexpression of LT3126 increased survival after 500 Gy irradiation by approximately 200-fold compared with the control bacteria, demonstrating a direct contribution to survival under high-dose stress. Comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, this study identified a novel set of {gamma}-ray-responsive genes and demonstrated a functional role for LT3126 in radiation resistance, providing new insights into molecular adaptation in radiation-associated environments. IMPORTANCEWe identified a novel set of {gamma}-ray-responsive genes (LT3105, LT3115, and LT3126) in the non-model bacterium Limnobacter thiooxidans. These genes are located in relatively close genomic proximity and are coordinately induced upon irradiation, suggesting a shared functional role in stress response. Overexpression of LT3126 increased survival by approximately 200-fold after 500 Gy irradiation compared with the control bacteria, demonstrating a substantial contribution to survival under high-dose stress. These genes were also induced by heat shock and oxidative stress, indicating that their function extends beyond radiation-specific responses to broader environmental stress adaptation. Consistent with this, comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, these findings highlight previously unrecognized molecular strategies that may support bacterial survival in radiation-associated environments.

microbiology↗

bioRxiv2026

Hepatic Ketogenesis Attenuates Cardiac Hypertrophy via Metabolic Reprogramming

BackgroundHeart failure with preserved ejection fraction (HfpEF) is increasingly recognized as a multisystem disorder linked to the cardiovascular-kidney-metabolic (CKM) syndrome. While the falling heart undergoes metabolic reprogramming, the interorgan crosstalk regulating myocardial substrate preference in HFpEF remains elusive. We aimed to clarify the role of systemic and local ketogenesis in the pathogenesis of cardiac hypertrophy and HFpEF. MethodsA mouse model of HFpEF was employed using a high-fat diet combined with NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Cardiac hypertrophy and systemic metabolic profiling including ketogenesis were evaluated. To dissect the role of site-specific ketogenesis, we generated inducible cardiomyocyte-specific (Hmgcs2{Delta}iCM) and hepatocyte-specific (Hmgcs2{Delta}Hep) knockout mice of HMG-CoA synthase 2 (Hmgcs2), deficient in the rate-limiting enzyme for ketogenesis. Cardiomyocyte -specific nuclei were isolated for transcriptomic (RNA-seq) and in vitro assays in H9C2 cells were used to elucidate molecular mechanisms. ResultsThe HFpEF model successfully exhibited diastolic dysfunction, impaired exercise capacity and cardiac hypertrophy with elevated circulating ketone body concentration. Myocardial metabolomics and snRNA-seq identified a profound metabolic shift characterized by the accumulation of long-chain fatty acids and Krebs cycle intermediates, coupled with the transcriptional downregulation of insulin signaling and fatty acid degradation pathways. Although circulating ketone body level was upregulated, Hmgcs2{Delta}iCM mice showed no exacerbation of the HFpEF phenotype. In contrast, Hmgcs2{Delta}Hep mice exhibited significantly aggravated cardiac hypertrophy (HW/TL; Hmgcs2flox: 7.41 {+/-} 0.87: Hmgcs2{Delta}Hep: 8.29 {+/-} 0.73; p = 0.0154). Mechanistically, hepatic ketogenesis was required to maintain circulating beta-hydroxybutyrate (BHB) levels, which directly modulated cardiomyocyte metabolism. BHB acted as a metabolic signal to dampen fatty acid overload and facilitate glucose utilization. ConclusionsOur study identifies a critical "liver-heart axis" where hepatic ketogenesis serves as an essential regulator of myocardial metabolic resilience. Impaired hepatic ketogenesis creates a metabolic mismatch that drives pathological cardiac remodeling. These findings highlight the liver as a therapeutic target within the CKM syndrome framework, suggesting that restoring the hepato-cardiac metabolic bridge may ameliorate HFpEF progression. What is New?O_LIThis study identifies a novel liver-adipose-heart axis that governs myocardial metabolic resilience during the development of heart failure with preserved ejection fraction (HFpEF). C_LIO_LIWe demonstrate that while both the liver and heart upregulate ketogenesis under metabolic stress, only hepatic ketogenesis--and not cardiac-intrinsic ketogenesis--is essential for mitigating pathological cardiac remodeling. C_LIO_LIMechanistically, liver-derived {beta} -hydroxybutyrate acts as a critical C_LIO_LIendocrine signal that dampens fatty acid oxidation and facilitates myocardial glucose utilization. C_LI What Are the Clinical Implications?O_LIOur findings highlight the liver as a central therapeutic target within the cardiovascular-kidney-metabolic (CKM) syndrome framework, where hepatic metabolic failure directly drives cardiac dysfunction. C_LIO_LIRestoring the hepato-cardiac metabolic bridge, through either hepatic-targeted therapies or ketone body supplementation, represents a promising strategy to enhance myocardial metabolic flexibility and ameliorate HfpEF in patients with multi-organ metabolic disorders. C_LI

biochemistry↗

bioRxiv2025

Oligomeric coiled-coil adhesins that drive chain-like adhesion diversify surface colonization strategies in Shiga toxin-producing Escherichia coli

Bacteria frequently colonize host and environmental surfaces under fluid flow. Chain-like adherence pattern (CLAP) is an EibG-mediated surface colonization phenotype of certain Shiga toxin-producing Escherichia coli (STEC) that lack the locus of enterocyte effacement (LEE). EibG, an immunoglobulin-binding oligomeric coiled-coil adhesin, drives CLAP, but the temporal dynamics and genetic diversity underlying chain formation remain unclear. Here, we use live-cell time-lapse imaging to show that chains arise from single cells that elongate and divide without separation. Under flow, chains resist detachment and undergo shear-dependent fragmentation at cell-cell junctions, releasing viable clonal units that disperse downstream. Comparative genomics reveals substantial diversity among EibG-related adhesins and identifies distinct lineages, including chain-like adhesins (cla) that mediate CLAP while lacking IgG binding. Screening of 1,354 LEE-negative STEC genomes from England shows that claB is present in 95.6% of strains from major LEE-negative STEC serotypes, highlighting its epidemiological prevalence. Targeted mutagenesis demonstrates that chain formation and IgG binding are mediated by distinct structural domains, revealing the modular functional architecture of these adhesins. In a mouse infection model, deletion of eibG reduced lethality, indicating that EibG contributes to virulence. Collectively, these findings establish CLAP as a dynamic, surface-associated strategy of LEE-negative STEC and reveal previously unrecognized diversification among adhesins that drive this behavior.

microbiology↗

bioRxiv2025

Rhodopsin 7 is indispensable for regulating the firing rates of olfactory sensory neurons in response to extracellular field potential changes in Drosophila melanogaster

Although extracellular field potential changes are commonly observed in the nervous systems, it remains controversial if extracellular electrical activity contributes to neural processing or whether it is an epiphenomenon associated with neural activity. We previously reported that the extracellular field potential change in compound eyes in response to light stimulation induces firing rate changes in olfactory sensory neurons in female Drosophila melanogaster. Through further investigation, we found that the extracellular field potential within the olfactory sensillum is regulated by octopaminergic neurons in response to the light stimulation and that rhodopsin 7 mediates the firing rate changes in the olfactory sensory neurons in response to field potential changes in a light-independent manner. Structural analysis suggests a voltage-dependent gating mechanism for rhodopsin 7 to respond to the field potential change. This study reveals that the nervous system actively controls the field potential in response to sensory input, resulting in alteration of behavioral patterns as well as neural firing patterns in a context-dependent manner. Significance statementAlthough extracellular electrical activity has been recorded to diagnose neuropsychiatric disorders, it remains uncertain how it can be controlled by the nervous system. Moreover, it is difficult to investigate how neurons change their excitability by responding to the change in the extracellular field potential, as synaptic communication interferes in the ability to isolate the function of extracellular electrical activity. We here show that the extracellular field potential within the olfactory sensillum in Drosophila melanogaster is actively regulated by octopaminergic neurons in response to sensory input. We also provide evidence that rhodopsin, a major light sensor protein, mediates responses to extracellular electrical signals, resulting in alternation of behavioral patterns as well as neural firing patterns in a context-dependent manner.

neuroscience↗

bioRxiv2025

Integrated structural dynamics uncover new modes of B12 photoreceptor activation

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The molecular mechanism of B12 photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B12 photoreceptor CarH4,5 from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co-C bond triggers a pathway of structural changes that propagate throughout CarH from the B12 chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B12 enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B12-dependent photoreceptors for future optogenetic applications.

biophysics↗

bioRxiv2025

Generative AI-driven artificial DNA design for enhancing inter-species gene activation and enzymatic degradation of PET

Conventional approaches to heterologous gene expression rely on codon optimization, which is limited to swapping synonymous codons and often fails to capture deeper adaptive changes. In contrast, naturally evolved orthologous genes between species often differ by more than just synonymous substitutions - they can include non-synonymous mutations, insertions, and deletions that confer functional adaptation to different host contexts. Here we present OrthologTransformer, a Transformer-based deep learning model that converts orthologous genes between species by learning from large-scale orthologous gene datasets curated for high-quality sequence alignments. The model recapitulates the full spectrum of evolutionary differences - from synonymous codon swaps to amino acid-changing mutations and indels - to predict a coding sequence optimized for a target species while preserving the proteins function. In extensive validation across diverse bacterial species pairs, OrthologTransformer significantly increased the conversion accuracy of generated genes to native target sequences compared to the original source genes, even for pairs with stark disparities in GC content and optimal growth temperature. The Transformers context-aware designs also favored conservative amino acid substitutions, maintaining protein functional integrity. As a proof-of-concept, an OrthologTransformer-designed PETase gene for Bacillus subtilis from the host species Ideonella sakaiensis was synthesized and expressed, yielding robust PET plastic-degradation activity that surpassed synonymous codon-optimized controls. These results establish OrthologTransformer as a powerful tool for de novo cross-species gene adaptation, transcending the limits of traditional codon optimization and enabling more effective heterologous gene performance in synthetic biology applications.

bioinformatics↗

bioRxiv2025

Membrane vesicles of Shewanella oneidensis MR-1 enhance denitrification growth in a species-selective manner

Denitrification, a fundamental bacterial respiratory process that occurs in anoxic environments, plays a pivotal role in energy synthesis and the global nitrogen cycle. Although the significance of this process is well-recognized, its regulation within polymicrobial communities remains poorly understood, particularly concerning interspecies interactions. In this study, we investigated the role that bacterial membrane vesicles (MV) play in modulating denitrification across bacterial species. MV is known to carry specific biomolecules such as secondary metabolites, proteins, and nucleic acids, therefore considered to be a secretion pathway. We found that MV produced by Shewanella oneidensis enhanced denitrification in a species-specific manner. Bacteria with highly hydrophobic surfaces tended to respond to denitrification enhancement, suggesting that the MV-bacteria attachment process is the key to generating species specificity. Transcriptome analysis and isotopic metabolite tracking indicated that the MV influenced denitrifying activities, rather than the transcription of denitrification-related genes. We further demonstrated that c-type cytochromes in MV act as key components that enhance denitrification. These insights expand our understanding of bacterial ecology, highlighting the role of membrane vesicles in facilitating respiratory competition and cooperation in polymicrobial communities.

microbiology↗

bioRxiv2024

Implication of S-1 biomarkers on aging and prognosis in elderly patients with lung cancer treated by adjuvant S-1 chemotherapy: Accompanying results of the Setouchi Lung Cancer Group Study 1201

BACKGROUNDSThe management of elderly patients presents several challenges due to age-related declines; however recent recommendations advocate for age not being the sole determinant for adjuvant treatment decisions in patients with non-small cell lung cancer (NSCLC). Aging may alter expression levels of 5-fluorouracil (5-FU) biomarkers. METHODSExpression changes with aging were explored using The Cancer Genome Atlas (TCGA) database. 5-FU-related biomarker expressions, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase, epidermal growth factor receptor (EGFR), and excision repair cross-complementation group-1 (ERCC1), were assessed by the quantitative reverse-transcription PCR assays in 89 NSCLCs elderly patients ([≥] 75 years old) receiving adjuvant S-1, an oral fluoropyrimidine agent, therapy in a SLCG1201 trial. RESULTS: TCGA database analysis (n=955) indicated decreased TS expression with aging, particularly in those 75 or older. In the SCLG1201 trial, univariate analysis revealed that high EGFR and low TS expressions correlated with favorable recurrence-free survival (RFS)(p=0.0264) and overall survival (OS)(p=0.0365), respectively. Multivariable analysis confirmed pathological stage as an independent prognostic factor for both RFS and OS (p=0.0052 and 0.0352, respectively). EGFR mutant tumors (n=27) showed significantly higher DPD (p=0.0066) and EGFR (p<0.0001) expressions, and lower TS(p=0.0125) and ERCC1(p=0.0015) expressions. CONCLUSIONDespite pathological stage being an independent prognostic factor, high EGFR and low TS expressions may predict better clinical outcomes in elderly NSCLC patients using adjuvant S-1. The age-related decrease in TS expression supports the potential benefit of 5-FU-based therapy in them compared to younger patients. Further research is warranted to validate these clinical implications.

pharmacology and toxicology↗

bioRxiv2024

Drp1-mediated mitochondrial fission protects macrophages from mtDNA/ZBP1-mediated sterile inflammation and inhibits post-infarct cardiac remodeling

BackgroundIschemic heart disease is a leading cause of death worldwide, and heart failure after myocardial infarction (MI) is a growing issue in this aging society. Macrophages play central roles in left ventricular (LV) remodeling after MI. Mitochondria consistently change their morphology, including fission and fusion, but the role of these mitochondrial morphological changes, especially in macrophages, is unknown. This study aims to illuminate the effects and mechanisms of Dynamin-related protein 1 (Drp1), a molecule mediating mitochondrial fission, in macrophages for cardiac remodeling after MI. MethodsThis study utilized genetically altered mice lacking Drp1 in monocytes/macrophages (Drp1-KO) to elucidate the specific role of macrophage Drp1 in post-infarct LV remodeling. ResultsDeletion of Drp1 in macrophages exacerbated LV remodeling, including reduced ejection fraction and increased LV diameters, which resulted in decreased survival after MI. Histological analysis indicated increased fibrosis and sustained macrophage accumulation in Drp1-KO mice. Blockade of Drp1 in macrophages decreased mitochondrial fission and impaired mitophagy, leading to the subsequent release of mitochondrial DNA (mtDNA) to the cytosol and induction of inflammatory cytokines. This induction was abrogated by an autophagy inducer, Tat-beclin1, or siRNA-mediated knockdown of Z-DNA Binding Protein 1 (ZBP1). Deletion of ZBP1 in bone marrow-derived cells abrogated LV remodeling induced by Drp1 inhibitor, Mdivi-1. ConclusionMacrophage Drp1 plays a critical role in the pathobiology of LV remodeling after MI, especially mitochondria quality control mechanisms. Macrophage Drp1 could be a novel therapeutic molecule to mitigate the progression of LV remodeling and consequent heart failure after MI.

biochemistry↗

bioRxiv2023

Characterization of carotenoid-producing Muricauda sp. strains isolated from the reef-building coral Galaxea fascicularis and subtropical seawater

Bacterial symbionts in corals and coral-associated zooxanthellae are attracting increasing attention as potential probiotics. Some members of the family Flavobacteriaceae are zooxanthellae-associated bacteria, which are known to protect zooxanthellae from stresses such as heat and light by producing carotenoids that reduce reactive oxygen species production. This study characterized two Flavobacteriaceae bacteria, Muricauda sp. strains ORYM1 (NBRC115792) and ORYM2 (NBRC115793), isolated from the reef-building coral Galaxea fascicularis and its surrounding seawater in Okinawa, Japan, respectively. The Muricauda sp. strain ORYM2 was isolated from subtropical seawater, and carotenoid production was assessed using ORYM2 as well as ORYM1, which was previously isolated from G. fascicularis. De novo genome sequencing revealed that both strains contain complete sets of zeaxanthin biosynthesis genes, similar to those found in other Muricauda spp. Thin-layer chromatography and high-performance liquid chromatography analyses demonstrated that ORYM1 and ORYM2 produce several carotenoids. The bacterial strains and carotenoids identified in this study provide insights into the biological roles of zooxanthellae-associated bacteria in protecting zooxanthellae and reef-building corals from environmental stresses. Statements and declarationsCompeting interests: The authors declare no competing interests.

microbiology↗

bioRxiv2023

Cyclophilin D induces necrotic core formation by promoting mitochondria-mediated macrophage apoptosis in advanced atherosclerotic lesions

BackgroundIn advanced atherosclerotic lesions, apoptotic cell death of plaque macrophages results in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. MethodsTo clarify the specific role of CypD, apolipoprotein-E/CypD-double knockout (ApoE-/-CypD-/-) mice were generated. These mice were fed a high-fat diet containing 0.15% cholesterol for 24 weeks to accelerate atherosclerotic lesion development. ResultsThe deletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control ApoE-/- mice. In RAW264.7 cells treated with endoplasmic reticulum stress inducer thapsigargin, the release of cytochrome c to the cytosol was attenuated by siRNA-mediated knockdown of CypD. Ly-6Chigh inflammatory monocytes in the peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by the deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-{gamma}, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage. ConclusionsThis is the first report showing that CypD induces macrophage death and promotes necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.

molecular biology↗

bioRxiv2022

Identification of inosine monophosphate dehydrogenase as a potential target for anti-monkeypox virus agents

Monkeypox virus (MPXV) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.032-1.40 M) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the post-entry step to reduce the intracellular production of virions. Knockdown of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. These evidences propose that IMPDH is a potential target for the development of anti-MPXV agents. ImportanceMonkeypox is a zoonotic disease caused by infection with the monkeypox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against monkeypox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against monkeypox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-monkeypox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited monkeypox virus propagation, exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested inosine monophosphate dehydrogenase as a potential target for the development of anti-monkeypox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-monkeypox virus activity than mycophenolic acid.

microbiology↗

bioRxiv2022

Isolation and characterization of bacteriophages against IMP-6-producing Klebsiella pneumoniae isolated from clinical settings in Japan

Carbapenemase-producing Enterobacteriaceae (CPE) are one of the most detrimental species of antibiotic-resistant bacteria worldwide. Phage therapy has emerged as an effective strategy for the treatment of infections caused by CPE pathogens. In west Japan, the increasing occurrence of Klebsiella pneumoniae harboring the pKPI-6 plasmid, which encodes blaIMP-6, is a growing concern. To manage such major antimicrobial-resistant pathogens, we isolated 29 novel phages from sewage in Japan, targeting 31 strains of K. pneumoniae and one strain of Escherichia coli harboring the pKPI-6 plasmid. Electron microscopy analysis indicated that of the 29 isolated phages, 21 (72.4%), 5 (17.2%), and 3 (10.3%) belonged to Myoviridae, Siphoviridae, and Podoviridae, respectively. Host range analysis revealed that 20 Myoviridae members in isolated phages infected 25-26 strains of K. pneumoniae, indicating that most of the isolated phages have a broad host range. The K. pneumoniae Kp21 can only be infected by phage oKp_21, while Kp22 can be infected by more than 20 phages. We applied a phage cocktail, which consists of 10 phages, against Kp21 and Kp22 and found that the phage cocktail delayed the emergence of phage-resistant bacteria for Kp21 strain but not for the Kp22 strain. Furthermore, phage-resistant Kp21 (Kp21r) became prone to be infected from other bacteriophages as a "trade-off" of resistance to phage oKp_21. Our proposed phage set has an adequate number of phages to combat the K. pneumoniae strain isolated in Japan. Notably, our work demonstrates how a suitable phage cocktail diminishes the occurrence of phage-resistant bacteria. ImportanceKlebsiella pneumoniae harboring the plasmid carrying blaIMP-6 is becoming an increasingly hazardous species in Japan. We collected and characterized 29 novel bacteriophages that infect K. pneumoniae carrying the pKPI-6 plasmid, isolated in clinical settings of west Japan. Our phages showed broad host ranges. We applied a phage cocktail treatment constructed from 10 phages against two host strains, Kp21 and Kp22, which show different phage susceptibility patterns each other. Although the phage cocktail delayed phage-resistant Kp21 emergence, the emergence of phage-resistant Kp22 could not be delayed. Moreover, phage-resistant Kp21 became sensitive to other phages, which did not originally infect wild-type Kp21. Our study demonstrates how a suitable phage cocktail can diminish the occurrence of phage-resistant bacteria.

microbiology↗

bioRxiv2022

Potential anti-monkeypox virus activity of atovaquone, mefloquine, and molnupiravir, and their potential use as treatments

Monkeypox virus (MPXV) is a zoonotic orthopoxvirus that causes smallpox-like symptoms in humans and caused an outbreak in May 2022 that led the WHO to declare global health emergency. In this study, from a screening of approved-drug libraries using an MPXV infection cell system, atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 M, which is more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted post-entry process to impair intracellular virion accumulation. Inhibitors of dihydroorotate dehydrogenase, an atovaquones target enzyme, showed conserved anti-MPXV activities. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by seven days at clinically relevant drug concentrations. Moreover, atovaquone and molnupiravir exhibited pan-Orthopoxvirus activity against vaccinia and cowpox viruses. These data suggest that atovaquone would be potential candidates for treating monkeypox.

microbiology↗

bioRxiv2021

Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro

Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 M and 99% at 15 M, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 M), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

microbiology↗

bioRxiv2019

Field evaluation of a quantitative, and rapid malaria diagnostic system using a fluorescent Blue-ray optical device

We improved a previously developed quantitative malaria diagnostic system based on fluorescent Blue-ray optical device. Here, we first improved the diagnostic system to enable fully automated operation and the field application was evaluated in Kenya. We detected Plasmodium falciparum in blood samples collected from 288 individuals aged 1-16 years using nested polymerase chain reaction (nPCR), rapid diagnostic test (RDT), and automated system. Compared to RDT, the automated system exhibited a higher sensitivity (100%; 95% confidence interval [CI], 93.3-100%) and specificity (92.8%; 95%CI, 88.5-95.8%). The limit of detection was 0.0061%. Linear regression analysis revealed a correlation between the automated system and microscopic examination for detecting parasitemia (adjusted R2 value=0.63, P=1.13x10-12). The automated system exhibited a stable quantification of parasitemia and a higher diagnostic accuracy for parasitemia than RDT. This indicates the potential of this system as a valid alternative to conventional methods used at local health facilities, which lack basic infrastructure.

physiology↗