Yibei Zhang

Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, poses a significant threat to the large yellow croaker (Larimichthys crocea) aquaculture industry. Developing live attenuated vaccines (LAVs) requires a fine balance between safety and immunogenicity, which remains a primary challenge. In this study, we developed a stable and effective LAV by constructing a markerless triple-gene deletion mutant ΔmlaEΔompRΔdeoR of P. plecoglossicida. The mutant exhibited high genetic stability and maintained growth kinetics compared to the wild-type (WT) strain under host-relevant conditions. Vaccination trials demonstrated that the triple mutant was significantly attenuated and safe for L. crocea. Following WT challenge, the triple mutant provided a relative percent survival (RPS) of 80.43% via intraperitoneal (i.p.) injection and 76.92% via immersion. For i.p.-vaccinated group, a significant reduction in splenic bacterial colonization and the absence of macroscopic white nodules were observed post-challenge. Furthermore, i.p.-vaccination triggered a coordinated immune response, characterized by the early upregulation of pro-inflammatory cytokines followed by the sustained activation of adaptive immune-related genes and elevated serum-specific IgM levels. These findings demonstrate that the ΔmlaEΔompRΔdeoR mutant is a promising LAV candidate, providing a viable strategy for the integrated control of P. plecoglossicida infection.

Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, poses a significant threat to the large yellow croaker (Larimichthys crocea) aquaculture industry. Developing live attenuated vaccines (LAVs) requires a fine balance between safety and immunogenicity, which remains a primary challenge. In this study, we developed a stable and effective LAV by constructing a markerless triple-gene deletion mutant ΔmlaEΔompRΔdeoR of P. plecoglossicida. The mutant exhibited high genetic stability and maintained growth kinetics compared to the wild-type (WT) strain under host-relevant conditions. Vaccination trials demonstrated that the triple mutant was significantly attenuated and safe for L. crocea. Following WT challenge, the triple mutant provided a relative percent survival (RPS) of 80.43% via intraperitoneal (i.p.) injection and 76.92% via immersion. For i.p.-vaccinated group, a significant reduction in splenic bacterial colonization and the absence of macroscopic white nodules were observed post-challenge. Furthermore, i.p.-vaccination triggered a coordinated immune response, characterized by the early upregulation of pro-inflammatory cytokines followed by the sustained activation of adaptive immune-related genes and elevated serum-specific IgM levels. These findings demonstrate that the ΔmlaEΔompRΔdeoR mutant is a promising LAV candidate, providing a viable strategy for the integrated control of P. plecoglossicida infection.

Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA–CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl−1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA–CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.

Two-component system (TCS) histidine kinases enable bacterial pathogens to sense environmental signals and regulate adaptive responses during infection. The EnvZ/OmpR TCS, known for its role in osmolarity/pH-dependent regulation of outer membrane porins across bacterial species, is also a central virulence regulator. However, the environmental cues that activate EnvZ/OmpR to trigger pathogenicity have remained unclear, limiting our understanding of host–pathogen interactions. Here, we demonstrate that in Vibrio parahaemolyticus , a major etiological agent of seafood-associated gastroenteritis, EnvZ functions as a direct ferric iron (Fe ³⁺ ) sensor governing virulence programs. Fe ³⁺ -EnvZ interaction triggers kinase phosphorylation and activation, enabling transcriptional control of biofilm formation, swarming motility, and type 3/6 secretion systems. An iron-binding-deficient EnvZ mutant (EnvZ ^Q103A ) abrogated Fe ³⁺ responsiveness and downstream signaling pathways. In an infant rabbit infection model, Fe ³⁺ enhanced V. parahaemolyticus intestinal colonization and virulence through EnvZ/OmpR signaling. This study identifies Fe ³⁺ as the physiological ligand activating the EnvZ/OmpR virulence regulon and provides insight into how enteric pathogens exploit host-derived iron cues to promote infection.

Recombinase polymerase amplification (RPA) is a powerful isothermal nucleic acid amplification technique, yet its efficiency is critically dependent on the catalytic efficiency of the recombinase UvsX, a key enzyme mediating homologous DNA pairing and strand exchange. To address this limitation, in this study, we developed a specific, sensitive, and robust RPA detection method by optimizing the UvsX enzyme through protein engineering and refining the RPA reaction system. By conducting comparative structural and functional analysis of UvsX orthologs from 13 Myoviridae phages, we identified critical determinants of recombinase activity within the Loop 2 domain of T4 UvsX. Furthermore, we systematically optimized the stoichiometric ratios of core enzymes and crowding agents to establish a robust RPA system. This system was subsequently integrated with lateral flow strips for point-of-need detection of highly lethal Vibrio parahaemolyticus in shrimp. Our results demonstrated that the engineered UvsXv1 variant exhibited significantly improved strand displacement activity, leading to enhanced RPA amplification efficiency and stability.

Pseudomonas plecoglossicida (P. plecoglossicida) is the causative agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea) and it causes severe economic loss to its industry. Biofilm formation, related to intracellular cyclic bis (3′–5′) diguanylic acid (c-di-GMP) levels, is essential for the lifestyle of P. plecoglossicida. This research aims to investigate the role of YfiR—a key regulator of the diguanylate cyclase YfiN to regulate c-di-GMP levels and reveal its regulatory function of bacterial virulence expression in P. plecoglossicida. A genetic analysis was carried out to identify the yfiBNR operon for c-di-GMP regulation in P. plecoglossicida. Then, we constructed a yfiR mutant and observed increased c-di-GMP levels, enhanced biofilm formation, increased exopolysaccharides, and diminished swimming and swarming motility in this strain. Moreover, through establishing a yolk sac microinjection infection model in zebrafish larvae, an attenuated phenotype of yfiR mutant that manifested as restored survival and lower bacterial colonization was found. YfiR is the key regulator of virulence in P. plecoglossicida, which contributes to c-di-GMP level, biofilm formation, exopolysaccharides production, swimming, swarming motility, and bacterial colonization in zebrafish model.

The viable but nonculturable (VBNC) state is a dormant state of nonsporulating bacteria that enhances survival in adverse environments. Systematic genome-w