Staphylococcus aureus is responsible for a wide range of pyogenic infections, food poisoning, and allergic inflammation. S. aureus produces a very wide variety of proteinaceous exotoxins, which are classified by structure as β-barrel pore-forming toxin, oligonucleotide/oligosaccharide-binding (OB)-fold/β-grasp proteins, and triple-helix bundle-containing proteins. We previously reported that three staphylococcal exotoxins belonging to the first two groups activate basophils and mast cells, which are innate immune cells that play a central role in allergic inflammation. In this study, we focused on a group of triple-helix bundle-containing toxins and examined their ability to activate murine bone marrow-derived mast cells and basophils. Extracellular complement-binding protein (Ecb), previously known as a complement inhibitor, was found to induce IL-4 and IL-6 production in basophils but not that of IL-6 and IL-13 in mast cells. Ecb did not induce IL-4 production in murine splenocytes or CD4+ T cells, suggesting that it is a basophil-specific activator. Ecb-induced IL-4 expression in basophils in the absence of serum. The C3-binding-deficient mutant of Ecb also activated basophils, indicating that Ecb activates basophils independently of its complement-inhibiting activity. Dasatinib, a Src kinase inhibitor, prevented Ecb-induced IL-4 production in basophils, suggesting that the activation required a Src family kinase. These findings reveal a novel function of Ecb, that is, the activation of basophils to produce IL-4 in an IgE-independent manner, and suggest a dual Ecb contribution to immune evasion-by interfering with complement activation and skewing Th2 immunity-and a role in the development of allergic inflammation-by inducing IL-4 production.
Many mammalian cells restrict viral replication by utilizing various host restriction factors. We recently demonstrated that CCHC-type zinc-finger-containing protein 3 (ZCCHC3) suppresses human immunodeficiency virus type 1 (HIV-1) replication through multiple mechanisms. We also revealed that single-nucleotide polymorphisms in human ZCCHC3 affect its antiviral function; however, whether similar genetic and functional diversity is present in other species remains unknown. In this study, we investigated the genetic and functional diversity of ZCCHC3 in cynomolgus macaques, a critical animal model for HIV-1-related research. Sequencing analysis of eight independent ZCCHC3 clones per animal revealed substantial amino acid diversity among cynomolgus macaques. We selected 12 representative variants and examined their antiviral activity against several retroviral vectors derived from HIV-1, simian immunodeficiency virus, feline immunodeficiency virus, and murine leukemia virus. Moreover, using replication-competent HIV-1, we showed that selected cynomolgus macaque ZCCHC3 variants can affect both viral production and viral infectivity. These results suggest that the genetic and functional diversity of ZCCHC3 is not limited to humans and underscore the importance of considering ZCCHC3 variation in cynomolgus macaques when using them as animal models for HIV-1-related research.
Toxigenic Corynebacterium ulcerans, a zoonotic pathogen, was isolated repeatedly from a cat in an in-house group in Japan. Although the cat was cured once by antibiotic treatment, clonal isolates of the original pathogenic strain were recovered repeatedly during a 2-year course. Clonal isolates of those isolates were also obtained from other asymptomatic cats in the same shelter, suggesting that environmental circulation of the pathogen had occurred. Care should be taken to monitor and cure the animals if any of their groupmates test positive for the pathogen, even if asymptomatic.
A C-type lectin with specificity for N-acetyl-d-glucosamine was purified from the cutaneous mucus of chub mackerel (Scomber japonicus) by affinity chromatography. The lectin comprises 159 amino acid residues and contains an unusual substitution within the conserved Ca2+-binding motif, in which Trp-Asn-Asp is replaced by Trp-Ser-Asp. Reverse transcription-PCR analysis demonstrated that the lectin gene is specifically expressed in the skin, and immunohistochemistry localized the protein to the outer epidermal layer, predominantly in squamous cells. Agglutinating activity was observed against L. garvieae, but not against any of the other bacterial species tested. An approximately 25-kDa bacterial protein was identified as a potential ligand, although its identity remains unresolved. Collectively, these findings indicate that this lectin (SjCL) functions as a mucosal immune molecule and may contribute to the first line of defense against bacterial pathogens in teleost fish.
ABSTRACT Chlamydia trachomatis (C. trachomatis) is a strictly parasitic pathogen that heavily relies on host cells for generating energy, acquiring nutrients, and evading immune responses. Mitochondrial dynamics—the balance of fusion and fission—are integral to cellular functions, including the maintenance of homeostasis, the regulation of metabolic processes, and the modulation of host innate immune pathways. Accordingly, C. trachomatis can specifically change the host mitochondrial dynamics to promote its intracellular replication. Mitochondrial fragmentation has been observed during the later phases of C. trachomatis infection; Nevertheless, the exact mechanisms remain poorly defined. The research aimed to determine the effect of the C. trachomatis secretory protein pORF5 in this process. In stable pORF5‐expressing Hela cells, we employed confocal microscopy to analyze mitochondrial morphology and Western blotting to measure the expression of key mitochondrial dynamics proteins. Finally, immunofluorescence was used to monitor Drp1 mitochondrial translocation, and the effects of a pathway inhibitor on mitochondrial fission were evaluated. We observed that the plasmid‐encoded protein pORF5 can induce mitochondrial fission. Mechanistically, this process is dependent on the activation of the ERK/Drp1 signaling axis, which indicates the crucial importance of this pathway and its effect on pORF5‐induced mitochondrial fragmentation.
Hepatitis C virus (HCV) establishes persistent infection by rewiring host stress-response pathways. Chaperone-mediated autophagy (CMA) contributes to HCV replication, but it remains unclear whether HCV regulates lysosome-associated membrane protein 2 A (LAMP-2A), the rate-limiting receptor for CMA. Here, we examined LAMP-2A regulation in HCV-infected Huh-7.5 cells. HCV infection increased LAMP-2A promoter activity, mRNA, and protein abundance, indicating transcriptional upregulation. Among candidate stress-responsive transcription factors, nuclear factor erythroid 2-related factor 2 (NRF2), hypoxia-inducible factor 1α (HIF-1α), and nuclear factor of activated T cells 1 (NFAT1) were elevated in infected cells. However, promoter mutagenesis identified NRF2 as the principal direct regulator. Mutation of the NRF2-responsive antioxidant response element markedly reduced basal and HCV-induced LAMP-2A promoter activity. Chromatin immunoprecipitation assays revealed NRF2 association with the LAMP-2A promoter, and HCV infection increased nuclear accumulation and Ser40 phosphorylation of NRF2. Functionally, shRNA-mediated knockdown of LAMP-2A reduced intracellular HCV RNA and protein levels. These findings identify an NRF2-LAMP-2A regulatory axis engaged during HCV infection and support a model in which HCV upregulates LAMP-2A to establish a cellular environment favorable for viral replication.
Human herpesvirus 6B (HHV-6B) entry is mediated by the interaction between gH/gL/gQ1/gQ2 complex (tetramer) and CD134 expressed on activated T cells. In this study, we identified and characterized a panel of monoclonal antibodies targeting the HHV-6B tetramer, with particular focus on three antibodies; gHgL J1, gHgL O2, and gHgL F5; that represent anti-gH neutralizing monoclonal antibodies with binding characteristics distinct from the previously described antibody OHV-3. Immunofluorescence and Western blot analyses confirmed recognition of the HHV-6B tetramer in infected and transfected cells. Biolayer interferometry (BLI) demonstrated that these antibodies exhibited dissociation constants (KD) in the range of 62.52-76.99 nM. We next assessed the effect of antibodies on the interaction between the HHV-6B tetramer and CD134. None of the gH-targeting antibodies inhibited the CD134 binding. Competition analysis using BLI-based antibody-Fab competition assays indicated that gHgL J1, gHgL O2, and gHgL F5 form a competition-defined antigenic cluster distinct from that recognized by OHV-3. Collectively, these findings identify a group of anti-gH antibodies which neutralizes HHV-6B with CD134-binding-independent profiles and suggest the presence of at least two distinguishable antibody-recognition regions within HHV-6B gH. These results expand our understanding of the antigenic organization of the HHV-6B entry complex.
Vaccination is highly effective in controlling infectious diseases in calves. A prime-boost injection is often administered intramuscularly in the neck and the hip in calves. However, no guidelines have established whether the same vaccination sites could be used for booster vaccinations. This study investigated the influence of intramuscular administration sites for sequential vaccines on immune responses in calves. All calves received the primary live-attenuated viral vaccine into the left side of the neck. Four weeks later, the booster was administered at the same site as the primary vaccine (local boost group) or in the right hip (distal boost group). The neutralizing antibody titers did not differ significantly between the groups. To assess T-cell responses to viral antigens after booster vaccination, the expression levels of lymphocyte activation markers CD25 and CD69 were measured in peripheral blood mononuclear cells (PBMCs). The site of booster administration had no significant effect on the activation of CD4+ and CD8+ T cells. Furthermore, vaccine-induced production of interferon-γ, tumor necrosis factor-α, and interleukin-6 in PBMC culture supernatants did not differ significantly between the groups. This study detected no significant differences in vaccine-specific immune responses between the local and distal booster vaccination sites in calves.
Interleukin-15 (IL-15) is a potential adjuvant in the activation of antitumor and antiviral immunity. Certain fermented food components possess immunomodulatory effects. We previously showed that lactic acid bacteria (LAB) isolated from Nozawana-zuke-the traditional Japanese fermented Brassica rapa L. pickle-activate immune function. Here we aimed to identify the LAB strain in fermented B. rapa L. that can markedly induce the expression of IL-15 mRNA in dendritic cells (DCs). We found that the Levilactobacillus brevis NZ3 strain isolated from B. rapa L. induces IL-15 production in DCs. We also provided mechanistic insight into the mechanism by which the NZ3 strain induces IL-15 production in DCs. We report that the NZ3 strain-induced IL-15 production in DCs is mediated by Toll-like receptor 3 signaling. Furthermore, inhibiting the interferon-α/β receptor alpha chain abrogated the NZ3 strain-induced IL-15 production. Further analysis using a Protein Kinase B (AKT) inhibitor revealed that the NZ3 strain-induced IL-15 production is mediated by AKT signaling, which occurs downstream of Interferon-α/β receptor signaling. Overall, our results indicate that the Levilactobacillus brevis NZ3 strain may serve as a potentially beneficial immunomodulatory bacterial strain. Moreover, due to its ability to induce IL-15 production, the NZ3 strain may serve as a candidate immunomodulatory strain for future studies.
Chlamydia trachomatis (Ct) is an obligate intracellular bacterium that can cause severe reproductive complications, including infertility and pelvic inflammatory disease. Its pathogenicity depends on intracellular maturation, which involves differentiation between infectious elementary (EB) and replicative reticulate bodies within inclusion vacuoles. Ct is known to modulate host PI3K-AKT signaling during this process; however, the molecular basis of this regulation remains unclear. To elucidate this mechanism, we screened host factors linked to the PI3K-AKT axis using a PI3K-AKT-mTOR compound library comprising 319 inhibitors, and identified the adaptor protein Gab2 (GRB2-associated binding protein 2) as a new target molecule of Ct. Gab2 protein levels decreased during the late phase of infection, and Gab2 silencing impaired intracellular replication without affecting EB formation. These findings demonstrate that Ct infection modulates the host adaptor Gab2 during intracellular development and provide new insights into host-pathogen interactions underlying chlamydial maturation.
Knee osteoarthritis (KOA) is a chronic joint disorder characterized by cartilage deterioration. lacking disease-modifying therapies. Aerobic exercise is recommended as a first-line non-pharmacological intervention for KOA, but its molecular mechanisms remain unclear. Recent studies indicate that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a key regulator of inflammation and cartilage homeostasis, may mediate the protective effects of aerobic exercise on joint integrity. Male C57BL/6 mice underwent destabilization of the medial meniscus (DMM) surgery and were assigned to Sham, Aerobic exercise, DMM, and DMM + Aerobic exercise groups. Aerobic treadmill running was performed for 4 weeks, with a STING agonist (DMXAA) and STING inhibitor (C-176) used to verify pathway involvement. Joint function, cartilage histology, inflammatory cytokines, ECM metabolism, chondrocyte apoptosis, and cGAS-STING pathway activation, including phosphorylation levels and 2'3'-cGAMP production, were assessed. DMM induced joint swelling, gait impairment, cartilage degradation, ECM catabolism, chondrocyte apoptosis, inflammation, and robust cGAS-STING pathway activation. Aerobic exercise significantly ameliorated these changes, improving joint function, preserving cartilage and ECM integrity, reducing apoptosis, suppressing inflammatory cytokines, and directly inhibiting cGAS-STING signaling. STING inhibition alone exerted comparable chondroprotective effects to aerobic exercise, and no additional benefit was observed in combination. DMXAA treatment abolished these protective effects. Aerobic exercise alleviates DMM-induced knee osteoarthritis by enhancing joint function and preserving cartilage integrity, effects that are directly mediated through suppression of the cGAS-STING pathway. These findings establish a causal link between aerobic exercise and innate immune regulation in osteoarthritis.
West Nile virus (WNV) remodels the endoplasmic reticulum (ER) membrane in cells to aid virus production. However, the molecular mechanisms underlying this process remain unclear. PDZD8 is an ER-resident protein that mediates ER-mitochondria contact and lipid transfer, both of which are associated with membrane remodeling. In this study, we found that WNV infection suppressed the expression of PDZD8, leading to a reduction in ER-mitochondria contact sites. By contrast, PDZD8 suppressed WNV production by inhibiting the processes of viral particle assembly and release. These findings suggest that WNV suppresses PDZD8 expression to partially counteract its inhibitory effect on WNV production.
Acne vulgaris is a chronic inflammatory disease of the pilosebaceous unit driven by Cutibacterium acnes colonization and the ensuing inflammatory response. Neutrophil extracellular traps (NETs) play a critical role in acne inflammation. Pipa Qingfei Decoction (PPQFY), a classic Traditional Chinese Medicine formula for acne, exhibits antibacterial and anti-inflammatory effects, but whether it regulates NETs to alleviate Cutibacterium-induced acne remains unclear. In vitro (keratinocytes/neutrophils) and in vivo (C57BL/6 mice) acne models were established using Cutibacterium acnes and treated with PPQFY, DNase I, or GSK484 (NET inhibitors). Neutrophil purity and Ly6G+CD11b+ cell ratio were assessed by flow cytometry. Cell viability and proliferation were evaluated using CCK-8 and EdU assays. Levels of IL-1β, TNF-α, and MPO-DNA were measured by ELISA. Protein levels of PADI4, CitH3, and MPO were determined by western blotting. Skin pathological changes were examined by H&E staining. PPQFY reversed Cutibacterium acnes-induced reductions in keratinocyte viability and proliferation and suppressed the elevation of IL-1β and TNF-α. PPQFY also inhibited NET release, as evidenced by decreased levels of MPO-DNA, CitH3, and PADI4. In mice, PPQFY alleviated skin inflammation, reducing keratin thickening, inflammatory infiltration, and Ly6G+CD11b+cell accumulation. These effects were comparable to those of NET inhibitors. PPQFY ameliorates Cutibacterium acnes-induced acne in mice by regulating NETs, providing a novel mechanistic basis for its clinical use and identifying NETs as a potential therapeutic target for acne.
Surveillance of tick-associated viruses may contribute to our understanding of viral diversity and evolution. Here, we identified a novel victorivirus in a sample derived from a human-biting tick, Amblyomma testudinarium, in Japan. The viral sequence was identified by metatranscriptomic sequencing of total RNA extracted from Vero cells treated with tick homogenate. The viral sequence was 4621 bp in length and contained two major open reading frames predicted to encode a putative coat protein (CP) and a putative RNA-dependent RNA polymerase (RdRp), respectively. The two open reading frames overlapped at the tetranucleotide sequence AUGA. The C-terminal region of putative CP was enriched in alanine, glycine, and proline residues. All these features are similar to those commonly observed in victoriviruses. Phylogenetic analyses based on the amino acid sequences of the putative CP and RdRp showed that the virus belongs to the genus Victorivirus of the family Pseudototiviridae. We therefore designated this putative virus as Amblyomma testudinarium-associated victorivirus 1 (ATaVV1). These findings expand our current knowledge of hidden viral diversity in tick-associated samples.
Ross River virus (RRV), a mosquito-borne alphavirus of the Togaviridae family, is the causative agent of Ross River fever a debilitating infectious disease characterized by fever, rash, and persistent polyarthritis. The virus remains endemic across Australia and the Pacific regions, with sporadic outbreaks reported in new geographic areas due to climate change and increased mosquito activity. Despite its growing public health impact, no licensed vaccine or antiviral therapy currently exists, underscoring the urgent need for an effective preventive strategy. In this study, an immunoinformatics-based approach was employed to design a novel multi-epitope vaccine (MEV) against RRV, where conserved and antigenic cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes were predicted from structural proteins (capsid and envelope glycoproteins E1 and E2) as well as selected non-structural protein regions, followed by their rational assembly into a vaccine construct using appropriate linkers and an immune-stimulatory adjuvant. Conserved and antigenic cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes were predicted from structural and non-structural proteins and linked with appropriate linkers, while β-defensin was incorporated as an adjuvant to enhance immunogenicity. The vaccine construct underwent modeling, refinement, and structural validation using PSIPRED, AlphaFold, and GalaxyRefine. Molecular docking analyses with immune receptors (TLR8 and HLA) and molecular dynamics (MD) simulations evaluated receptor binding affinity and structural stability. The C-ImmSim server was used to simulate immune responses, and codon optimization ensured suitability for Escherichia coli expression. The designed MEV exhibited strong antigenicity (0.79), stability, and solubility, with a molecular weight of ~30 kDa and theoretical pI of 9.98. Structural validation revealed over 95% of residues in favored Ramachandran regions, and docking analysis showed predicted interaction with TLR8 (- 1712.2 kJ/mol). MD simulations confirmed stable complex formation, while immune simulations predicted robust and long-lasting humoral and cellular immune responses with elevated IgG, IFN-γ, and memory B/T-cell populations. The designed MEV demonstrates promising antigenic, structural, and immunological properties, making it a potential vaccine candidate for further in vitro and in vivo evaluation against Ross River virus infection.
Escherichia albertii is an emerging diarrheagenic pathogen that causes foodborne outbreaks. Several selective media for E. albertii have been developed for outbreak investigation or surveillance of a potential contamination source. Although these selective media contain potassium tellurite (PT) as a selective agent, the genetic determinants for PT resistance in E. albertii are unclear. Here, we analyzed the whole genomes of our 22 E. albertii isolates and found three that harbored the IncHI2-type plasmid encoding the tellurite resistance operon (ter-operon). The minimum inhibitory concentration (MIC) value of PT against ter-positive E. albertii was 640 μg/mL for all three strains, which is higher than those against other E. albertii isolates (5 to 40 μg/mL). The conjugation assay using three ter-positive E. albertii strains found that two of the three ter-positive plasmids transferred into a PT-susceptible E. coli recipient when tested individually, resulting in an increase in the MIC of PT to more than 640 μg/mL. The three ter-positive plasmids had sequence diversity, especially in the region coding antimicrobial resistance genes. The results of a plasmid database (PLSDB) search implied the circulation of ter-positive IncHI2 plasmids among species belonging to several genera within the order Enterobacterales. Phylogenetic analysis with genomes downloaded from a public database identified the ter-operon in 93 of 751 E. albertii genomes (12.4%) belonging to multiple E. albertii lineages. These data suggest that the plasmid-encoded ter-operon plays an important role in the dissemination and acquisition of high PT resistance in E. albertii.
Innate immune responses trigger the development of adaptive immune responses through the production of cytokines. In the present study, we compared the cytokine production in mice inoculated with a novel replicon mRNA COVID-19 vaccine. Biphasic and prolonged IFN-γ production was observed after the first dose, and sustained production was observed after the second dose, whereas conventional mRNA vaccines induced a peak of IFN-γ production only on Day 1 after both first and second inoculations. These findings may be associated with the self-replicating properties of the replicon mRNA vaccine following inoculation.
India along with other countries in the World Health Organization (WHO) Southeast Asia region has set a goal to eliminate rubella by December 2026 through active tracking and vaccination program. However, the incidence of sporadic events and outbreaks in recent years has posed a significant challenge to this goal. To access the ongoing infections and the effectiveness of immunization efforts, we evaluated the presence of anti-rubella IgM and IgG antibodies, which serve as indicators of ongoing infection and immune protection, respectively. Serum samples from individuals presenting with suspected rubella symptoms were collected and referred to the State Level Virus Research and Diagnostic Laboratory (SL-VRDL) at ICMR-NIRTH, Jabalpur between 1st May 2023 to 30th April 2024 for anti-rubella IgM and IgG analysis. Concurrently, samples from healthy pregnant women, attending Netaji Subhash Chandra Bose Medical College & Hospital, Jabalpur were obtained during the same period to assess the prevalence of maternal immunity against rubella. Collected samples were tested for rubella specific IgM and IgG antibody using ELISA. Rubella IgM seropositivity rate during the study period was 7.2% across all age groups, indicating ongoing transmission of rubella in central India. Rubella IgG seropositivity, reflecting immunity, was 81.0%, among all age groups, demonstrating a high level of immunity against the rubella virus in this region. Among pregnant women, rubella IgG seropositivity was 89.6%, suggesting that most pregnant women of reproductive age are having protection against rubella infection. The finding indicates that rubella elimination efforts are on the right track, and that the vaccination program is broadly effective. However, continuous detection of IgM positive cases, particularly in young children, highlights, immunity gaps, that may hinder timely elimination. These gaps could be due to logistical, cultural, and educational barriers and require further intensive efforts towards active immunization. Targeted and intensified immunization efforts are essential to achieve the elimination goal by 2026.
Chronic rhinosinusitis with nasal polyps (CRSwNP) exhibits pronounced endotypic heterogeneity, with macrophages serving as key drivers of sustained mucosal inflammation. In this study, we identify S100A9 as a macrophage-derived alarmin that is markedly elevated in CRSwNP tissues. Integrative analyses of public bulk transcriptomic datasets and single-cell RNA-sequencing atlases demonstrated that S100A9 expression was predominantly enriched in macrophage clusters, where it showed strong co-expression with canonical M1-associated markers, while exhibiting limited expression in epithelial cell subsets. Spatial and correlation analyses further supported a close association between S100A9⁺ macrophages and epithelial barrier-related gene signatures. Functionally, shRNA-mediated silencing of S100A9 attenuated M1-like macrophage polarization, as evidenced by reduced expression of pro-inflammatory mediators and polarization markers, accompanied by a shift toward a less inflammatory macrophage phenotype. Conditioned media derived from S100A9-deficient macrophages significantly mitigated epithelial injury, leading to restoration of epithelial barrier integrity, as indicated by enhanced expression of tight junction proteins, including occludin and claudins. Importantly, S100A9 knockdown disrupted the pathogenic macrophage-epithelial inflammatory feedback loop, thereby dampening sustained inflammatory signaling and limiting epithelial barrier breakdown that perpetuates tissue damage in CRSwNP. Clinically, elevated S100A9 levels correlated with disease severity indices and effectively distinguished a macrophage-enriched inflammatory endotype of CRSwNP, highlighting S100A9 as both a mechanistic driver and a potential biomarker for disease stratification. Collectively, these findings position S100A9 as a mechanistic mediator and a promising therapeutic target for CRSwNP.
Rotavirus A infects many animal species, including humans, and causes severe diarrhea and acute gastrointestinal diseases. This virus also infects various bird species including pigeons; however, information on avian rotavirus A (ARVA) infection in pigeon populations in Japan is limited. Here, we retrospectively report the genetic characterization based on whole-genome sequences of two Japanese ARVA isolates from pigeons in 2012. The isolates were obtained by inoculation into primary chicken kidney cell cultures and subsequently subjected to near-complete genome sequencing using an NGS system. The genotype constellation of the two ARVA isolates was G18-P[17]-I4-R4-C4-M4-A4-N4-T4-E19-H4, which were identical to those derived from several pigeons. Genetic analysis based on each segment revealed that the genomic sequences of all 11 genes from these ARVAs are closely related to those of ARVAs from foreign countries such as Germany, Italy, or China rather than those of the Japanese ARVAs in previous reports. These data suggest an ancestor common to the ARVA analyzed in this study might be distributed and maintained worldwide. This information will help researchers better understand the evolution and epidemiology of ARVA in feral birds.
