Search Results (2,153)

Background: Edwardsiella piscicida is a significant intracellular pathogen of channel catfish (Ictalurus punctatus) and a major threat to U.S. aquaculture. A recently developed recombinant attenuated vaccine strain (χ16016) uses arabinose-regulated murA expression to trigger delayed cell wall lysis in vivo, ensuring biological containment while conferring strong protection against virulent challenge. Although its efficacy has been demonstrated, the host immune programs underlying protection remain incompletely defined. Methods: We used RNA sequencing to characterize tissue-specific transcriptomic responses in the intestines and kidneys of channel catfish at 7 days post-vaccination. Fish were vaccinated with χ16016 by either bath immersion or intracoelomic (IC) injection, and differentially expressed genes and enriched immune pathways were analyzed to determine how the vaccine delivery route shapes systemic and mucosal immune responses. Results: Across comparisons, 19,101 differentially expressed genes revealed pronounced route- and tissue-dependent immune remodeling. As aquaculture vaccination strategies increasingly prioritize scalability and practical deployment, understanding how the delivery route shapes immune outcomes is critical. Here, IC vaccination induced broader systemic transcriptional changes, particularly in the intestine, whereas bath immunization elicited a more focused yet coordinated mucosal response. Overall, intestinal tissue exhibited greater transcriptional responsiveness than kidney tissue, underscoring its central role in early vaccine-induced immunity. Functional enrichment analyses identified the activation of innate recognition pathways, MAPK and calcium signaling cascades, complement components, antigen processing machinery, and cell adhesion networks. Notably, bath immunization enriched the intestinal immune network for IgA production pathway, which represents an orthology-based mapping of conserved mucosal immune components, alongside the upregulation of IL-6, CXCL12–CXCR4, integrins (α4β7), MHC class II, complement C3, and polymeric immunoglobulin receptor (pIgR). Given that catfish rely primarily on IgM in mucosal immunity, these findings indicate the induction of IgM-mediated mucosal defense rather than classical mammalian IgA responses. Concurrent complement and scavenger receptor signatures suggest a transition toward efficient opsonophagocytic clearance with controlled inflammation at this subacute stage. Conclusions: This study provides the first systems-level view of host transcriptomic responses to a regulated-lysis E. piscicida vaccine in channel catfish. The findings demonstrate that immersion vaccination, although transcriptionally less expansive than injection, effectively activates coordinated mucosal innate and adaptive immune programs, supporting its practical use as a scalable vaccination strategy for aquaculture. Full article

The Mycobacterium tuberculosis bacterium encodes two active potassium (K⁺)-uptake transport systems, the Trk and the Kdp. The Trk is the low-affinity K⁺ transporter, consisting of two TrkA proteins, while the Kdp consists of the high-affinity K⁺ transporter KdpFABC and the two-component system KdpDE. Both transporters are utilised by the bacteria for growth and survival. During growth, the bacteria utilise the constitutively expressed Trk and suppress the Kdp, but upregulate both transporters during survival. In the current study, we investigated the interactive effects of these systems on bacterial growth and survival. This was achieved by first constructing a M. tuberculosis mutant strain in which both the Trk and Kdp systems were inactivated by homologous recombination. The mutant was evaluated for its growth kinetics in planktonic cultures, as well as survival in biofilm and macrophage cultures. The constructed M. tuberculosis mutant showed faster growth rates in planktonic cultures, but was attenuated for both biofilm formation and intracellular survival in isolated human monocyte-derived macrophages. These results illustrate that both K⁺-uptake systems are essential to sustain slow rates of bacterial growth, as well as for bacterial persistence in hostile environments via optimisation of biofilm formation, and intracellular survival in macrophages. (Words: 194) Full article

Senecavirus A (SVA) has rapidly emerged as a globally distributed swine pathogen, with clinical signs mimicking vesicular diseases such as Foot-and-Mouth Disease, posing challenges for timely detection and control. Here, we analyzed 329 complete SVA genomes spanning multiple continents to provide a comprehensive view of its evolutionary dynamics, recombination patterns, haplotype diversity, and global dissemination. Phylogenetic analyses revealed two major lineages: Lineage 1, consisting mainly of early strains from the United States before 2007, and Lineage 2, which emerged post-2007 and subsequently spread across the Americas and East Asia. Recombination was confined to Lineage 2 and concentrated in nonstructural regions, particularly 2C, highlighting intra-lineage genetic exchange as a driver of recent diversification. Haplotype analysis of the 3AB gene identified 170 distinct haplotypes, revealing a star-like network structure consistent with rapid population expansion from a central ancestral variant, while secondary branches reflect ongoing regional diversification. Despite this high genetic variation, genome-wide dN/dS ratios remained below one, and purifying selection was strongest in the N-terminal domains of structural and nonstructural proteins, indicating functional constraints that maintain viral fitness. Time-scaled phylogenetic reconstruction and Bayesian Skyline analysis revealed rapid lineage diversification and a marked increase in effective population size in the early 2010s. Phylogeographic inference further identified repeated introductions from the Americas into East Asia, likely facilitated by swine trade and other anthropogenic factors. Collectively, SVA evolution is driven by frequent mutation and intra-lineage recombination yet constrained by pervasive purifying selection, generating extensive genetic diversity while maintaining functional integrity, with implications for genomic surveillance and targeted control. Full article

Background: The intracellular pathogen Brucella requires biotin for survival, yet the role of its de novo synthesis intermediate enzyme, BioA, in virulence remains undefined. This study investigates the contribution of BioA to the pathogenicity of Brucella abortus. Methods: We constructed a ΔBioA mutant in Brucella abortus 104M via homologous recombination and characterized its phenotype using growth assays, electron microscopy, macrophage infection models, and murine splenic colonization. Virulence gene expression was quantified by RT-qPCR. Results: The ΔBioA mutant exhibited severe growth auxotrophy in a biotin-deficient medium and displayed damaged outer membrane integrity. Furthermore, intracellular survival in macrophages was reduced by approximately 95% compared to the wild-type strain at 48 h post-infection. Notably, mice infected with the mutant showed a significant decrease in both splenic bacterial loads and spleen weight at 3 weeks, concomitant with a marked downregulation of VirB type IV secretion system (T4SS) genes. Conclusions: This study is the first to identify BioA as a critical nexus linking biotin metabolism to Brucella virulence. We demonstrate that BioA deficiency attenuates pathogenicity by impairing both structural integrity and the transcription of key virulence-related genes (VirB operon), thereby nominating BioA as a novel and promising target for anti-brucellosis interventions. Full article

Oenococcus oeni (O. oeni) can initiate and complete the malolactic fermentation (MLF) process, which significantly improves wine quality. However, stress factors commonly encountered in wine, such as acid stress and ethanol stress, can hinder this process. Overexpression of certain key functional genes using genetic recombination technology can enhance the stress tolerance of O. oeni. In this study, the large-conductance mechanosensitive channel (mscl) gene was overexpressed in O. oeni SD-2a using genetic recombination technology. The results showed that overexpression of this gene enhanced the growth rate of O. oeni under 10% ethanol stress conditions. Physiological index measurements indicated that overexpression of this gene enhanced the control of cell membrane permeability in the recombinant strain at different time points under ethanol stress and altered cell membrane fluidity at these time points. Proteomic analysis after 12 h of treatment under 10% ethanol stress revealed that mscl overexpression significantly altered the protein expression pattern of O. oeni. The most significantly affected proteins included some cell membrane transporters (for sugars, lipids, amino acids, and nucleotides) and proteins involved in cell wall synthesis. These results suggest that mscl overexpression enhances the ethanol stress tolerance of O. oeni by altering its cell membrane properties and affecting the expression levels of proteins related to cell membrane transport and cell wall synthesis. This study provides a theoretical reference for obtaining O. oeni recombinant strains with enhanced stress tolerance through genetic recombination technology. Full article

Genetically modified (GM) lactic acid bacteria (LAB) are gaining attention as tools for innovation in the food sector, health applications, and industrial processes. LAB have long been used safely due to their GRAS/QPS status, making them suitable for improving fermentation and synthesizing specific and beneficial metabolites. Advances in genomics and gene editing have significantly expanded the available tools, ranging from classical mutagenesis to site-specific recombination, homologous recombination in non-coding regions, CRISPR-based systems, and food-grade chromosomal integration. These approaches enable the insertion of desired genes and the development of engineered strains with tailored functionalities. GM-LAB are also being studied as live delivery systems for therapeutic molecules, including cytokines, hormones, antimicrobial peptides, and vaccine antigens. Engineered strains of Lactococcus lactis and Lactobacillus spp. have yielded promising outcomes in applications such as mucosal immunization, modulation of inflammatory and metabolic responses, and inhibition of pathogenic microorganisms, including multidrug-resistant bacteria. From an industrial perspective, several studies highlight their potential for cost-effective recombinant protein production and the synthesis of high-value metabolites through fermentation. However, within the European Union, their use is subject to stringent regulatory oversight, requiring comprehensive molecular and environmental risk assessments, careful evaluation of horizontal gene transfer, and a preference for markerless chromosomal integrations. Despite these constraints, GM-LAB offer significant potential to improve food quality, sustainability, and human health. Full article

γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter, is known for its physiological functions in alleviating anxiety and improving sleep. Currently, high-yielding GABA food products are mainly obtained through screening wild-type high-producing strains (e.g., Saccharomyces cerevisiae isolated from Sichuan pickles yielding 0.67 g/L) or employing co-culture systems (e.g., Enterococcus faecium and Lactiplantibacillus plantarum reaching 6.35 g/L). While effective, these methods often rely on natural screening strains or multi-microbial interactions. This study employed CRISPR-Cas9 technology to knockout the UGA1 gene in Saccharomyces cerevisiae, a key gene responsible for GABA degradation. Starting from the low higher alcohol Saccharomyces cerevisiae SY-LH, we successfully constructed the recombinant strain SY-LHU. Remarkably, this study discovered a significant upregulation of GAD1 gene expression following UGA1 knockout, which further enhanced GABA synthesis capacity. Under optimal fermentation conditions (inoculum size 4 × 10⁷ cells/mL, wort concentration 10 °P, sugar addition 60 g/L, 30 °C for 10 days, and mixing the malt broth every 48 h), the validation fermentation was performed and the GABA content in the wort beverage reached 280.36 mg/L, representing a 385.4% increase compared to the pre-optimization level. Furthermore, sensory evaluation by a trained panel yielded a mean score of 88, with no significant off-flavors detected, demonstrating the product’s high consumer acceptance. This pioneering work provides a novel and feasible technical pathway for developing functional alcoholic beverages with sleep-aiding properties. Full article

Streptococcus pneumoniae remains a leading cause of morbidity and mortality worldwide, with current polysaccharide-based vaccines offering limited serotype coverage, high production costs, and reduced efficacy in vulnerable populations. These limitations have prompted the search for conserved pneumococcal proteins as universal vaccine candidates. Among them, pneumococcal surface protein A (PspA) stands out as a major virulence factor, present in virtually all clinically relevant strains, and capable of interfering with complement activation, opsonophagocytosis, and host defense mechanisms. Over three decades of research have demonstrated PspA’s strong immunogenicity, protective efficacy in multiple animal models, and safety in early-phase clinical trials. Here, we critically review advances in PspA-based vaccine development, including recombinant protein fragments, fusion constructs, nanoparticle formulations, and live-vector platforms. We highlight the structural and immunological determinants underlying its protective potential, while discussing major challenges such as antigenic variability and cross-reactivity across pneumococcal strains expressing distinct PspA clades. By integrating recent experimental and translational findings, this review outlines the opportunities and obstacles for the implementation of serotype-independent PspA-based vaccines. Full article

Background/Objectives: The efficacy of inactivated foot-and-mouth disease virus (FMDV) vaccines depends on the structural integrity of the 146S virions. However, instability of 146S antigens during vaccine manufacturing and storage can compromise vaccine quality. Despite its high immunogenicity, the Korean serotype O strain O Jincheon (O JC) exhibits poor physical stability. Methods: To enhance antigenic stability while preserving strain-specific antigenicity, we engineered a VP1-substituted recombinant virus, (R) O1 M–O JC_VP1, by integrating the VP1 coding region of O JC into the O1 Manisa (O1 M) backbone. Results: The resulting chimeric virus exhibited significantly improved capsid stability, as demonstrated by an increased melting temperature and enhanced resistance to thermal stress, chloroform exposure, and long-term storage. Importantly, the recombinant antigen maintained its immunogenicity and induced antibody responses comparable to those induced by the parental O JC strain in vaccinated pigs. Conclusions: These findings demonstrate that VP1-direct chimeric engineering can improve capsid stability without compromising antigenicity and provide a practical approach for developing a stable FMDV vaccine. Full article

This study developed a dual-antigen enzyme-linked immunosorbent assay (ELISA) based on σB protein and genotype 5-specific σC protein of avian reovirus (ARV). First, σB and σC proteins were expressed and purified using recombinant technology. Through optimization of coating conditions, the optimal antigen combination was determined to be a mixture of the two proteins at a 1:3 molecular ratio (total concentration: 0.8 μg/mL). Key parameters of the indirect ELISA were optimized via checkerboard titration. Validation confirmed that the dual-antigen ELISA exhibited a sensitivity of 1:3200 against genotype 5 ARV-positive sera, with no cross-reactivity and a coefficient of variation of 2.9–8.6%, demonstrating excellent reproducibility. In application testing, the method specifically detected serum antibodies against genotype 5 ARV variant strains, achieving a 100% positive detection rate in experimental chickens within the first week post-challenge and effectively monitoring dynamic antibody changes in infected flocks. Furthermore, the detection rate for genotype 5-positive serum samples (100%) was significantly higher than that of a commercial kit (75%). This dual-antigen indirect ELISA overcomes the sensitivity limitations associated with conventional genotype 5 ARV detection methods and provides a reliable tool for epidemiological surveillance and infection monitoring. Full article

The protozoan parasite Leishmania mexicana serves as a widely used model for studying trypanosomatid biology, yet the demand for stable, high-intensity fluorescent tools for precise subcellular protein localization remains unmet. In this study, we developed a versatile molecular toolbox by re-engineering the pLEXSY-hyg2.1 vector to express mNeonGreen (mNG), a next-generation fluorophore with superior brightness and photostability. Using a modular cloning strategy, we introduced a customized multiple cloning site (MCS) upstream of the mNG sequence to facilitate seamless N-terminal tagging of target proteins. The construct was integrated into the 18S rRNA locus via homologous recombination to ensure stable, constitutive expression. As a proof-of-concept, we fused a flagellar marker to the mNG reporter, resulting in a transgenic line exhibiting robust and specific subcellular fluorescence without compromising cellular fitness. Our results demonstrate that this integration-based system provides a highly efficient and stable platform for visualizing protein distribution within Leishmania. This tool significantly simplifies the generation of reporter strains and will facilitate high-resolution imaging studies of parasite organelle dynamics and functional genomics. Full article

Deoxynivalenol (DON), a Group III carcinogenic mycotoxin frequently detected in cereals and animal-derived food products, poses serious health risks to animals and humans. In this study, we developed a genetically engineered Saccharomyces cerevisiae strain as a proof-of-concept platform for DON detoxification. The yeast was engineered to co-express two detoxification genes, YTDepA and YTDepB (homologs of DepA and DepB from Devosia mutans 17-2-E-8) originally identified in Youhaiella tibetensis. Concurrently, the pyrroloquinoline quinone (PQQ) biosynthesis gene cluster from Klebsiella pneumoniae was integrated to supply the essential cofactor. Gene expression was verified by qRT-PCR and Western blot. The recombinant strain demonstrated a significant 13.98% detoxification of DON after 72 h of fermentation (p < 0.05), as confirmed by HPLC–MS, while the strain expressing only the PQQ cluster showed no detoxification activity. This study establishes an integrated yeast cell factory for DON detoxification and highlights key limitations to guide future optimization efforts. Full article

Metallothionein (MT) is a multifunctional metal-binding protein with broad applications in medicine, healthcare, and food industries, but its large-scale use is limited by inefficient industrial synthesis. To address this and obtain optimal fermentation parameters for large-scale MT production, this study used the recombinant marine-derived MT-producing Pichia pastoris strain SMD1168-MT. We first optimized the strain’s growth and induced fermentation conditions, then constructed a Back Propagation (BP) neural network model for in-depth parameter optimization and accurate MT expression prediction. Results showed the optimal growth conditions for SMD1168-MT were: 30 °C, initial pH 8.0, shaking speed 220 r/min, and 4% inoculum size. The BP model exhibited high accuracy (training set: R² = 0.8430, MAE = 0.0129, RMSE = 0.0175; validation set: R² = 0.8337, MAE = 0.0144, RMSE = 0.0174). Combined with Particle Swarm Optimization (PSO), the optimal fermentation conditions were: 7.7% methanol, initial OD₆₀₀ 8.2, 240 r/min, 50 h induction, and 125 μmol/L Zn²⁺. Validation confirmed MT expression reached 0.2141 mg/mL (2.93-fold). This study demonstrates that the BP neural network effectively optimizes recombinant P. pastoris-based marine-derived MT fermentation, improving yield and providing a basis for industrial scale-up. Full article

RNA interference (RNAi) provides a sequence-specific strategy for pest management, but efficient and stable double-stranded RNA (dsRNA) delivery remains a key challenge. Here, we established a plant-probiotic-based gene silencing system using the endophytic fungus Fusarium commune G3-29 as a dsRNA delivery vector against western flower thrips (WFTs, Frankliniella occidentalis). Recombinant G3-29 strains expressing dsRNA targeting the essential WFT genes ACT and SNF were constructed and confirmed to colonize kidney bean leaves without pathogenicity. Bioassays showed that feeding on leaves colonized by dsRNA-expressing G3-29 significantly decreased survival and downregulated target gene expression in both WFT larvae and adults. Within 4 days, survival of both larvae and adults fell below 10%. In larvae, target gene expression decreased by 63% (ACT) and 33% (SNF), while in adults, reductions of 74% (ACT) and 65% (SNF) were observed. In contrast, in vitro-synthesized dsRNA failed to induce significant gene silencing or mortality in larvae, and its control efficacy against adults was also inferior to that of endophytic fungus-mediated dsRNA delivery. Our findings establish endophytic fungus F. commune G3-29 as an effective and sustainable dsRNA delivery vehicle for RNAi-based pest control, offering distinct advantages over existing strategies such as HIGS and SIGS. This approach provides a promising new direction for managing WFTs and other insect pests. Full article

Heterologous protein secretion in filamentous fungi is often constrained by limitations in signal peptide recognition and intracellular trafficking. Aspergillus oryzae, a food-grade industrial fungus, has a robust native secretory system. However, its capacity for recombinant protein secretion remains suboptimal. Here, we developed a two-step, carrier-free engineering strategy to enhance protein secretion in A. oryzae. We identified endogenous signal peptides among highly secreted proteins using a green fluorescent protein (GFP) reporter. The oryzin signal peptide SPAoalp1 increased GFP secretion 5.50-fold compared with a no-signal-peptide control. We co-overexpressed Aosly1, a Sec1/Munc18 family protein that regulates soluble N-ethylmaleimide-sensitive factor attachment protein receptor–mediated vesicle trafficking, which, in combination with SPAoalp1, increased secretion approximately two-fold compared with SPAlp1 control and ten-fold with no-SP control. Applying the engineered platform for genetic improvement of heterologous bovine κ-casein increased secretion from 0.11 to 0.24 mg/L. Physiological optimization further increased secretion. The developed system provided initial evidence for secretion of a ~12 kDa band consistent with Aopafb transcription, with MIC₉₀ values of 4.56–8.24% (v/v) against two Candida albicans strains and 4.68% (v/v) against Aspergillus niger. The system offers a modular framework for engineering fungal secretion and expands the utility of A. oryzae for recombinant protein production. Full article