A novel small non-coding RNA 562 mediates the virulence of Pseudomonas plecoglossicida by regulating the expression of fliP, a key component of flagella T3SS.

Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.

A bacterial ghost vaccine against Aeromonas salmonicida infection in turbot (Scophthalmus maximus).

Aeromonas salmonicida is one of the most prevalent pathogens that causes huge economic losses to aquaculture. Effective vaccination is the first choice for preventing infection. Bacterial ghost (BG), an empty bacterial shell devoid of cytoplasm, is a promising vaccine antigen with distinct advantages. Herein, we established strategies for producing a substantial yield of A. salmonicida ghost (ASG) and investigated the immune-protective properties of it. As a result, 2.84 mg/ml NaOH was discovered to be capable of inducing considerable amounts of ASG. Furthermore, the ASG vaccine elicited adaptive immunity in turbots after rapid activation of innate immunity. Even though formalin-killed cells (FKC) produced a few more antibodies than ASG, ASG ultimately provided a much stronger immune protection effect because it strengthened cellular immunity, with a relative percentage survival (RPS) of 50.1 % compared to FKC. These findings demonstrated that ASG effectively activated cell-mediated immunity, which helped get rid of microorganisms inside cells. Therefore, this study presented novel perspectives for future research on furunculosis vaccine products based on ASG as an antigen.

Identification and genomic analysis of a pathogenic circovirus associated with maricultured Scophthalmus maximus L. in China.

In China, a novel pathogen within the genus Circovirus has been identified as a causative agent of the 'novel acute hemorrhage syndrome' (NAHS) in aquacultured populations of turbot (Scophthalmus maximus L.). Histopathological examination using light microscopy revealed extensive necrosis within the cardiac, splenic, and renal tissues of the afflicted fish. Utilizing transmission electron microscopy (TEM), we detected the presence of circovirus particles within the cytoplasm of these cells, with the virions consistently exhibiting a spherical morphology of 20-40 nm in diameter. TEM inspections confirmed the predominance of these virions in the heart, spleen, and kidney. Subsequent molecular characterization through polymerase chain reaction (PCR) analysis corroborated the TEM findings, with positive signals in the aforementioned tissues, in stark contrast to the lack of detection in gill, fin, liver, and intestinal tissues. The TEM observations, supported by PCR electrophoresis data, strongly suggest that the spleen and kidney are the primary targets of the viral infection. Further characterization using biophysical, biochemical assays, and genomic sequencing confirmed the viral classification within the genus Circovirus, resulting in the nomenclature of turbot circovirus (TurCV). The current research endeavors to shed light on the pathogenesis of this pathogen, offering insights into the infection mechanisms of TurCV in this novel piscine host, thereby contributing to the broader understanding of its impact on turbot health and aquaculture.

Erratum: A Bacterial Pathogen Senses Host Mannose to Coordinate Virulence.

[This corrects the article DOI: 10.1016/j.isci.2019.09.028.].

Long-chain unsaturated fatty acids sensor controlling the type III/VI secretion system is essential for Edwardsiella piscicida infection.

Edwardsiella piscicida is an acute marine pathogen that causes severe damage to the aquaculture industry worldwide. The pathogenesis of E. piscicida is dependent mainly on the type III secretion system (T3SS) and type VI secretion system (T6SS), both of which are critically regulated by EsrB and EsrC. In this study, we revealed that fatty acids influence T3SS expression. Unsaturated fatty acids (UFAs), but not saturated fatty acids (SFAs), directly interact with EsrC, which abolishes the function of EsrC and results in the turn-off of T3/T6SS. Moreover, during the in vivo colonization of E. piscicida, host fatty acids were observed to be transported into E. piscicida through FadL and to modulate the expression of T3/T6SS. Furthermore, the esrCR38G mutant blocked the interaction between EsrC and UFAs, leading to dramatic growth defects in DMEM and impaired colonization in HeLa cells and zebrafish. In conclusion, this study revealed that the interaction between UFAs and EsrC to turn off T3/T6SS expression is essential for E. piscicida infection.

Advance in Hippophae rhamnoides polysaccharides: Extraction, structural characteristics, pharmacological activity, structure-activity relationship and application.

Hippophae rhamnoides (Sea buckthorn) is an excellent medicinal and edible plant owing to its high nutritional and health-promoting properties. As an important bioactive component, H. rhamnoides polysaccharides (HRPs) have aroused wide attention due to their various pharmacological activities, including hepatoprotective, immuno-modulatory, anti-inflammatory, anti-oxidant, anti-tumor, hypoglycemic, anti-obesity, and so on. Nevertheless, the development and utilization of HRP-derived functional food and medicines are constrained to a lack of comprehensive understanding of the structure-activity relationship, application, and safety of HRPs. This review systematically summarizes the advancements on the extraction, purification, structural characteristics, pharmacological activities and mechanisms of HRPs. The structure-activity relationship, safety evaluation, application, as well as the shortcomings of current research and promising prospects are also highlighted. This article aims to offer a comprehensive understanding of HRPs and lay a groundwork for future research and utilization of HRPs as multifunctional biomaterials and therapeutic agents.

Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies.

Gene function verification is a crucial step in studying the molecular mechanisms regulating various plant life activities. However, a stable and efficient homologous genetic transgenic system for herbaceous peonies has not been established. In this study, using virus-induced gene silencing technology (VIGS), a highly efficient homologous transient verification system with distinctive advantages was proposed, which not only achieves true "intact-plant" infiltration but also minimizes the operation. One-year-old roots of the representative species, Paeonia lactiflora Pall., were used as the materials; prechilling (4 °C) treatment for 3-5 weeks was applied as a critical precondition for P. lactiflora to acquire a certain chilling accumulation. A dormancy-related gene named HOMEOBOX PROTEIN 31 (PlHB31), believed to negatively regulate bud endodormancy release (BER), was chosen as the target gene in this study. GFP fluorescence was detected in directly infiltrated and newly developed roots and buds; the transgenic plantlets exhibited remarkably earlier budbreak, and PlHB31 was significantly downregulated in silenced plantlets. This study established a homologous transient silencing system featuring intact-plant infiltration and minimized manipulation for gene function research, and also offers technical support and serves as a theoretical basis for gene function discovery in numerous other geophytes.

Complete genome sequence and genome-wide transposon mutagenesis enable the determination of genes required for sodium hypochlorite tolerance and drug resistance in pathogen Aeromonas veronii GD2019.

Aeromonas veronii, a significant pathogen in aquatic environments, poses a substantial threat to both human and animal health, particularly in aquaculture. In this study, we isolated A. veronii strain GD2019 from diseased largemouth bass (Micropterus salmoides) during a severe outbreak of aeromonad septicemia in Guangdong Province, China. The complete genome sequence of A. veronii GD2019 revealed that GD2019 contains a single chromosome of 4703,168 bp with an average G+C content of 58.3%. Phylogenetic analyses indicated that GD2019 forms a separate sub-branch in A. veronii and comparative genomic analyses identified the existence of an intact Type III secretion system. Moreover, to investigate the genes that are required for the conditional fitness of A. veronii under various stresses, a high-density transposon insertion library in GD2019 was generated by a Tn5-based transposon and covers 6311 genomic loci including 4155 genes and 2156 intergenic regions. Leveraging this library, 630 genes were classified as essential genes for growth in rich-nutrient LB medium. Furthermore, the genes GE001863/NtrC and GE002550 were found to confer tolerance to sodium hypochlorite in A. veronii. GE002562 and GE002614 were associated with the resistance to carbenicillin. Collectively, our results provide abundant genetic information on A. veronii, shedding light on the pathogenetic mechanisms of Aeromonas.

Fur-mediated regulation of hydrogen sulfide synthesis, stress response, and virulence in Edwardsiella piscicida.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

Toxicity, physiological response, and biosorption mechanism of Dunaliella salina to copper, lead, and cadmium.

Background: Heavy metal pollution has become a global problem, which urgently needed to be solved owing to its severe threat to water ecosystems and human health. Thus, the exploration and development of a simple, cost-effective and environmental-friendly technique to remove metal elements from contaminated water is of great importance. Algae are a kind of photosynthetic autotroph and exhibit excellent bioadsorption capacities, making them suitable for wastewater treatment. Methods: The effects of heavy metals (copper, lead and cadmium) on the growth, biomolecules accumulation, metabolic responses and antioxidant response of Dunaliella salina were investigated. Moreover, the Box-Behnken design (BBD) in response surface methodology (RSM) was used to optimize the biosorption capacity, and FT-IR was performed to explore the biosorption mechanism of D. salina on multiple heavy metals. Results: The growth of D. salina cells was significantly inhibited and the contents of intracellular photosynthetic pigments, polysaccharides and proteins were obviously reduced under different concentrations of Cu2+, Pb2+ and Cd2+, and the EC50 values were 18.14 mg/L, 160.37 mg/L and 3.32 mg/L at 72 h, respectively. Besides, the activities of antioxidant enzyme SOD and CAT in D. salina first increased, and then descended with increasing concentration of three metal ions, while MDA contents elevated continuously. Moreover, D. salina exhibited an excellent removal efficacy on three heavy metals. BBD assay revealed that the maximal removal rates for Cu2+, Pb2+, and Cd2+ were 88.9%, 87.2% and 72.9%, respectively under optimal adsorption conditions of pH 5-6, temperature 20-30°C, and adsorption time 6 h. Both surface biosorption and intracellular bioaccumulation mechanisms are involved in metal ions removal of D. salina. FT-IR spectrum exhibited the main functional groups including carboxyl (-COOH), hydroxyl (-OH), amino (-NH2), phosphate (-P=O) and sulfate (-S=O) are closely associated with the biosorption or removal of heavy metalsions. Discussion: Attributing to the brilliant biosorption capacity, Dunaliella salina may be developed to be an excellent adsorbent for heavy metals.

Clinical Application of Serum Interleukin-6 Combined with Inflammatory Cytokines in the Dynamic Monitoring of Patients with Acute Cholecystitis.

Objective: To investigate the dynamic fluctuations of serum interleukin-6 (IL-6), procalcitonin (PCT), and neutrophil counts in individuals diagnosed with acute cholecystitis. Additionally, the research seeks to investigate the potential clinical significance of these biomarkers in the early stages of acute cholecystitis. Methods: This retrospective cohort study included one hundred patients with acute cholecystitis (60 with mild acute cholecystitis and 40 with severe cholecystitis) admitted to our hospital between January 2022 and December 2022 were included. The levels of various cytokines, PCT and neutrophils in serum on days 1, 3, 5, and 7 were dynamically detected. The difference in each indicator between the two groups was analysed, and the diagnostic value of each indicator for acute cholecystitis was evaluated using a receiver operating characteristic (ROC) curve. Results: IL-6 and PCT levels and neutrophil counts were significantly higher in patients with moderate and severe cholecystitis than those in those with mild cholecystitis (P <0.01). The AUC values for the three indicators were all greater than 60%, and the AUC value for the joint diagnosis of the three indicators reached 90%. Conclusion: Serum interleukin-6 combined with PCT and neutrophil count is helpful to determine the degree of disease development in patients with acute cholecystitis. The advantage of dynamic monitoring of the three indicators is that the detection is simple and worthy of clinical promotion.

Type III secretion system effector YfiD inhibits the activation of host poly(ADP-ribose) polymerase-1 to promote bacterial infection.

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida's proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1's activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection.

An aluminium adjuvant compound with ginseng stem leaf saponins enhances the potency of inactivated Pseudomonas plecoglossicida vaccine in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) farm industry in China suffered from huge economic loss caused by Pseudomonas plecoglossicida infection. Due to multi-antibiotic resistance, efficient vaccines are urgent to be developed to combat this pathogen. In this study, an inactivated vaccine was developed with an aluminium adjuvant (Alum) plus ginseng stem and leaf saponins (GSLS). As a result, the relative percentage survival (RPS) against P. plecoglossicida was up to 67.8 %. Comparatively, RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum or Montanide™ 763A as adjuvant were 21.8 %, 32.2 % and 62.1 %, respectively. Assays for total serum protein and serum lysozyme activity in group vaccinated with inactivated vaccine plus Alum + GSLS adjuvant were significantly higher than that in control group. Moreover, specific antibody in serum elicited a rapid and persistent level. According to the expression of some immune related genes, inactivated vaccine plus Alum + GSLS adjuvant induced a stronger cellular immune response which was vital to defend against P. plecoglossicida. In conclusion, our study demonstrated that the compound Alum and GSLS adjuvant is a potential adjuvant system to develop LYC vaccine.

The QseE-QseF two-component system: A key mediator of epinephrine-regulated virulence in the marine pathogen Edwardsiella piscicida.

Edwardsiella piscicida is a widespread pathogen that infects various fish species and causes massive hemorrhagic septicemia, resulting in significant property damage to the global aquaculture industry. Type III and VI secretion systems (T3/T6SS), controlled by the master regulator EsrB, are important virulence factors of E. piscicida that enable bacterial colonization and evasion from host immune clearance. In this study, we demonstrate that the QseE-QseF two-component system negatively regulated esrB expression by reanalysis of Tn-seq data. Moreover, the response regulator QseF directly bound to esrB promoter and inhibited the expression of T3/T6SS genes, especially in the presence of epinephrine. Furthermore, in response to the prompt increasing of epinephrine level, the host immune genes were delayed repressed and QseE-QseF timely inhibited the expression of T3/T6SS genes to evade immune clearance. In summary, this study enhances our understanding and knowledge of the conditional pathogenesis mechanism and virulence regulation network of E. piscicida.

Lysine acetylation regulates the AT-rich DNA possession ability of H-NS.

H-NS, the histone-like nucleoid-structuring protein in bacteria, regulates the stability of the bacterial genome by inhibiting the transcription of horizontally transferred genes, such as the type III and type VI secretion systems (T3/T6SS). While eukaryotic histone posttranslational modifications (PTMs) have been extensively studied, little is known about prokaryotic H-NS PTMs. Here, we report that the acetylation of H-NS attenuates its ability to silence horizontally transferred genes in response to amino acid nutrition and immune metabolites. Moreover, LC-MS/MS profiling showed that the acetyllysine sites of H-NS and K120 are indispensable for its DNA-binding ability. Acetylation of K120 leads to a low binding affinity for DNA and enhances T3/T6SS expression. Furthermore, acetylation of K120 impairs the AT-rich DNA recognition ability of H-NS. In addition, lysine acetylation in H-NS modulates in vivo bacterial virulence. These findings reveal the mechanism underlying H-NS PTMs and propose a novel mechanism by which bacteria counteract the xenogeneic silencing of H-NS.