Vibrio Alginolyticus Research Articles

Roles of S100A1 and S100A10 from hybrid grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀) in the immune response to Vibrio infection

The S100 proteins are highly conserved EF-hand calcium-binding proteins found only in vertebrates. In the current study, two S100 genes (S100A1 and S100A10) were successfully identified and characterized from hybrid grouper Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀. The deduced S100A10 protein contained two EF-hand domains, and S100A1 only possessed the N-terminal EF-hand. Phylogenetic analysis revealed that S100A1 and S100A10 from hybrid grouper were evolutionarily closely related to their counterparts in other selected vertebrates. Quantitative real-time PCR results revealed that the transcripts of S100A1 and S100A10 mRNA were ubiquitously distributed in all the examined tissues. After Vibrio alginolyticus infection, the expression of S100A1 and S100A10 in the spleen increased significantly. Moreover, overexpression of S100A1 and S100A10 could not only regulate the expression of interleukin 8 (IL-8), IL-10, and IL-16 in the head kidney, liver, and spleen, change the activities of acid phosphatase, catalase, lysozyme, and superoxide dismutase in serum, but also reduce the promoter activities of interferon 3 and nuclear factor kappa-B in vitro. Taken together, this study indicated that S100A1 and S100A10 participate in the immune response of hybrid grouper against bacterial infection.

Effect of non-antibiotic factors on conjugative transfer of antibiotic resistance genes in aquaculture water

Aquaculture water with antibiotic resistance genes (ARGs) is escalating due to the horizontal gene transfer. Non-antibiotic stressors specifically found, including those from fishery feed and disinfectants, are potential co-selectors. However, the mechanisms underlying this process remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic-resistant plasmid RP4 were established to examine conjugative transfer frequency under exposure to five widely used non-antibiotic factors in aquaculture water: iodine, oxolinic acid, NO2-N, NO3-N and H2O2 and four different recipient bacteria: E. coli HB101, Citrobacter portucalensis SG1, Vibrio harveyi and Vibrio alginolyticus. The study found that low concentrations of non-antibiotic factors significantly promoted conjugative transfer, whereas high concentrations inhibited it. Moreover, the conjugation transfer efficiencies were significantly different with different bacterial species within (E. coli HB101 ∼ 10-3 %) or cross genera (C. portucalensis SG1 ∼10-5 %, V. harveyi ∼1 %). Besides, excessive exposure concentrations inhibited the expression of related genes and the generation of reactive oxygen species (ROS). Regulation of multiple related genes and ROS-induced SOS responses are common primary mechanisms. However, the mechanisms of non-antibiotic factors differ from those of standard antibiotics, with direct changes in cell membrane permeability potentially playing a dominant role. Additionally, variations among non-antibiotic factors and the specific characteristics of bacterial species contribute to differences in conjugation mechanisms. Notably, this study found that non-antibiotic factors could increase the frequency of intergeneric conjugation beyond that of intrageneric conjugation. Furthermore, non-antibiotic factors influenced by multiple transport systems may raise the risk of unintended cross-resistance, significantly amplifying the potential for resistance gene spread. This study underscores the significance of non-antibiotic factors in the propagation of ARGs, highlighting their role in advancing aquaculture development and protecting human health.

Antibacterial Effect of Fermented Pomegranate Peel Polyphenols on Vibrio alginolyticus and Its Mechanism.

Vibrio alginolyticus frequently breaks out in aquatic animal breeding operations involving shrimp, and it can endanger human health through food and wound infections. The antibacterial effect and mechanism of fermented pomegranate peel polyphenols (FPPPs) on V. alginolyticus were investigated. The results indicated that FPPPs had a strong inhibitory effect on the growth of V. alginolyticus, and their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 2 and 4 mg/mL. FPPPs significantly reduced biofilm formation and biofilm metabolic activity in V. alginolyticus, down-regulated the expression levels of lafA, lafK, fliS and flaK genes involved in flagellar synthesis and inhibited swimming and swarming motility (p < 0.05). Meanwhile, under the treatment of FPPPs, the activities of catalase (CAT) and superoxide dismutase (SOD) in V. alginolyticus were significantly reduced, and the levels of reactive oxygen species (ROS) and extracellular malondialdehyde (MDA) were significantly increased (p < 0.05). FPPPs also resulted in a significant increase in alkaline phosphatase (AKP) activity, protein and nucleic acid content, as well as conductivity from V. alginolyticus cultures. Scanning electron microscopy (SEM) images further revealed that V. alginolyticus treated with FPPPs showed leakage of intracellular substances, abnormal cell morphology and damage to cell walls and cell membranes, with the severity of the damage in a clear dose-dependent manner. Therefore, FPPPs can be used as a promising food-grade antibacterial agent, notably in seafood to control V. alginolyticus.

Vibrio alginolyticus Reprograms CIK Cell Metabolism via T3SS Effector VopS to Promote Host Cell Ferroptosis.

Vibrio alginolyticus is a Gram-negative pathogen of both marine animals and humans, resulting in significant losses for the aquaculture industry. Emerging evidence indicates that V. alginolyticus manipulates cell death for its pathogenicity, but the underlying molecular mechanisms remain unclear. Here, a gene designated vopS in V. alginolyticus HY9901 was identified, which was predicted to encode the T3SS effector protein. To determine whether VopS contributes to the pathogenesis of V. alginolyticus, the ΔvopS mutant strain was constructed and phenotypically characterized. The deletion of VopS not only reduced the ability to secrete extracellular proteases and virulence but also affected the expression of the T3SS genes. Furthermore, VopS was cytotoxic and induced apoptosis, as confirmed by elevated LDH and the activation of caspase-3. Metabolomic analysis revealed considerable metabolomic disruptions upon V. alginolyticus infection. The VopS effector induced host cell ferroptosis by promoting the synthesis of adrenic acid, depleting cellular glutathione, and subsequently increasing the accumulation of ferrous (Fe2+). Taken together, our findings provide that the VopS effector is an essential virulence factor of V. alginolyticus, which can lead to ferroptosis.

SYNLAC prime probiotics enhances growth performance, and resistance of white shrimp, Penaeus vannamei to Enterocytozoon hepatopenaei and Vibrio alginollyticus: Insights into immune and metabolic pathway modulations

This study explores the impact of SYNLAC Prime probiotics on the growth performance, health status, and metabolic profile of white shrimp, Penaeus vannamei. Shrimp fed with the experimental diets, including the control diet without probiotic supplementation, and the diets supplemented with SYNLAC Prime probiotics at concentrations of 105 CFU (g diet)−1 (P5) and 106 CFU (g diet)−1 (P6) for 56 days. Results indicated a significant enhancement in growth performance in probiotic-treated shrimp relative to the control group, attributed to structural improvements in the digestive tract, particularly the increased abundances of B cells in the hepatopancreas. The administration of dietary probiotics markedly reduced the severity of Enterocytozoon hepatopenaei (EHP) infection and decreased cumulative mortalities following Vibrio alginolyticus challenge. Shrimp in the P6 group exhibited significant elevations in phenoloxidase activity, respiratory burst, lysozyme activity and phagocytic activity compared to control group. Furthermore, there was an upregulation of several immune-related genes in hepatopancreas, including serine protease (SP), prophenoloxidase (proPO) I, proPO II, and penaeidin 3a. Additionally, the expression of β-1, 3-glucan binding protein and SP mRNA was significantly increased in hemocytes. Untargeted metabolomics analysis using LC-MS/MS revealed significant changes in the hepatopancreas metabolic profile, highlighting alterations in energy metabolisms pathways, such as citrate cycle and nicotinate and nicotinamide metabolism, as well as amino acid metabolisms pathways including arginine and proline metabolism, taurine and hypotaurine metabolism, and histidine metabolism. These findings underscore the potential of SYNLAC Prime probiotics in enhancing shrimp growth, immune function, and metabolic pathways, offering valuable insights for advancing health management strategies in shrimp aquaculture.

Novel function of single-target regulator NorR involved in swarming motility and biofilm formation revealed in Vibrio alginolyticus

NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus.

Existence of Thermotolerant and Salt-Loving Diarrheagenic Vibrio alginolyticus in Non-Saline Potable Water System: A Novel Finding from India.

We present a novel report of abundance of halophilic Vibrio alginolyticus with thermotolerant and enterotoxigenic characteristics from community water system of an inland-focus of India causing diarrheal outbreak as an index pathogen. Though, Vibrio alginolyticus causing diarrhea after exposure to marine water and consumption of seafood was reported globally, its existence in non-saline drinking-water sources with pathogenic viability was unknown. A 'matched-pair-case-control' study identified the primary source of V. alginolyticus infection as 'tap-water' distributed by the municipality, used for drinking (MOR: 8.33; 95% CI 2.51-27.6) and household chores (MOR: 3.75; 95% CI 1.24-11.3). Cardinal toxin gene 'tdh' and other pathogenicity markers viz.tlh, vppC, toxR, VPI, T3SS1 and sxt were detected in V. alginolyticus isolates. Expression potential of the hemolytic genes are demonstrated by hemolysis assay and transcriptome analysis. Altogether 30.55% of isolates exhibited strong hemolytic potential in vitro. RT-PCR revealed uninterrupted virulence gene expression in outbreak strains under heat stress. Surprisingly, ~ 100% of V. alginolyticus from the outbreak focus were sensitive/partially sensitive to all group of antibiotics except β-lactums, carbapenem and quinolones. High drug-sensitivity suggested lack of previous human gut exposure and indicated a fresh dissemination from the environmental niche to the community domain. The maximum likelihood phylogeny depicted multiple clades in V. alginolyticus strains from Pan India sources. Isolated outbreak strains shared common ancestry with the strains from nearby riverine system, a source of 'drinking water' supplied to the affected community, confirming its environmental origin. V. alginolyticus, traditionally a fish-pathogen, is steadily gaining an emerging epidemiological relevance alongside other waterborne diarrheagenic bacteria and its 'thermotolerant' attribute poses additional threat under the canvas of climate change.

Biofabrication, characterization and unveiling the biological potential of zinc oxide nanoparticles from bacterium Bacillus pacificus KR A1

The utilization of cell free supernatant for synthesizing nanomaterials has been considered as an innovative approach. This investigation aims to fabricate a ZnO NPs using the cell free supernatant of Bacillus pacificus KR A1. The prepared nanomaterials were systematically characterized by UV-DRS, FT-IR, XRD, HR-TEM, XPS and elemental mapping (EDX) techniques. The optical band gap energy of Bacillus pacificus ZnO NPs (Bp-ZnO NPs) was observed as 3.25 eV. The XRD analysis revealed that Bp-ZnO NPs were hexagonal wurtzite structures with 50 nm crystallite size. Furthermore, XPS data revealed the elements present in Bp-ZnO NPs. The HR-TEM analysis revealed that most of the particles were spherical in shape with an average calculated particle size of 20 nm with a lattice fringe range of 0.246 nm. In addition, Bp-ZnO NPs showed promising antibiofilm properties against Bacillus cereus and Vibrio alginolyticus at 100 μg/mL concentration. Furthermore, MDA-MB-231 cells treated with an IC50 concentration showed changes in cell morphology including apoptosis, cell shrinkage and nuclear fragmentation. However, HUVEC cells treated with higher concentration (40 µg/mL) of Bp-ZnO NPs displayed less toxicity and 86.3 % of cells are viable. In summary, Bp-ZnO NPs could serve as a promising material for biomedical claims.

Alginolyticus Virulence: An Overview

Introduction: Marine habitats harbor the gram-negative, halophilic Vibrio alginolyticus. Usually connected with aquatic life, it poses a serious health risk to humans, especially seafood and marine workers. Marine economic animals like prawns, fish, and shellfish can develop serious illnesses with rapid progression and significant fatality rates. Pathogenic relatives such as Vibrio cholerae and Vibrio parahaemolyticus, which carry genes for several virulence factors, have inherited its virulence, thereby increasing its clinical importance. No effective treatments exist to stop V. alginolyticus dissemination and infection; therefore, studying its virulence mechanisms is crucial to understanding its pathogenicity and improving prevention and therapy. Objectives: To analyze the virulence factors and antibiotic resistance mechanisms of Vibrio alginolyticus to improve prevention and control strategies for managing infections in aquaculture and public health settings. Methods: This study is a literature review synthesising findings from recent research on V. alginolyticus. Sources include peer-reviewed articles, clinical studies, and microbiological research focusing on virulence factors and antibiotic resistance. Data collection involved searching databases like PubMed, Scopus, and Web of Science, filtering for studies on V. alginolyticus’s virulence mechanisms, antibiotic resistance, and impacts on public health and aquaculture. We selected key studies to gain a comprehensive understanding of the pathogenic characteristics and resistance profiles of the bacterium. Results: The results show that V. alginolyticus uses many ways to be strong, like quorum sensing, making virulence proteins, secreting haemolysin, and making biofilms, which make it more dangerous to humans and marine animals. Several resistance genes and efflux systems contribute to its antibiotic resistance, thereby impeding the effectiveness of treatment. Studies also show that biofilm formation and quorum sensing facilitate survival in hostile environments, making infections more challenging to manage. Conclusions: Understanding the virulence factors and antibiotic resistance mechanisms of Vibrio alginolyticus is crucial for developing effective control strategies. This bacterium’s rapid spread and severe impact on marine life and human health necessitate new approaches in aquaculture management and clinical interventions. Enhanced monitoring and targeted research are essential to curb its spread and prevent infection, ultimately improving aquaculture productivity and reducing public health risks associated with this pathogen.

Antibacterial activity of Kefa forest honey against Vibrio alginolyticus and Aeromonas hydrophila and study of its physical and chemical characteristics

Antibacterial activity of Kefa forest honey against Vibrio alginolyticus and Aeromonas hydrophila and study of its physical and chemical characteristics

Molecular Characteristics Demonstrate the Occurrence of Phylogenetic Similar Isolates of &lt;i&gt;Vibrio alginolyticus&lt;/i&gt; and &lt;i&gt;Vibrio parahaemolyticus&lt;/i&gt; in Aquatic Environments

Vibrio alginolyticus is an opportunistic fish pathogen with a potential to cause septicemia and often associated with other Vibrio infections, particularly Vibrio parahaemolyticus, and can pose a significant threat to aquatic health. This study investigated the biochemical and phylogenetic characteristics of V. alginolyticus isolated from various aquaculture farms, focusing on its prevalence, hemolysin genes, genetic relatedness, and antibiotic susceptibility. Out of 92 brackish water aquaculture farm samples, 16 isolates were biochemically confirmed as V. alginolyticus, with 12 subsequently confirmed by polymerase chain reaction targeting collagenase gene. Molecular analysis of the thermolabile hemolysin gene (tlh) via specific PCR amplified a 450 bp fragment in 8 isolates, confirming the presence of the tlh gene. Pulsed Field Gel Electrophoresis (PFGE) typing differentiated tlh-positive and tlh-negative V. alginolyticus isolates with 92% genetic similarity. The isolates exhibited proteolytic, lipolytic, and lecithinase activities. Notably, the isolates showed intermediate resistance to most of the tested antibiotics, indicating exposure to antimicrobial agents. This study provides evidence of the presence of similar phylogenetical isolates of V. alginolyticus and V. parahaemolyticus in aquatic environments. Furthermore, this study emphasizes the importance of expanding surveillance programs by incorporating strain-specific characterization to better understand and control vibriosis in aquaculture.

Development and optimization of immunomagnetic bead separation-convective PCR (IMBS-CPCR) for on-site detection of four common Vibrio species in aquaculture

Foodborne infectious diseases, particularly those caused by pathogenic Vibrio species in aquaculture, are a significant concern in the field of seafood safety. They not only hinder the economic development of the industry but also pose a major threat to public health. Traditional detection methods are often too slow, insensitive, or impractical for portable use, highlighting the urgent need for a sensitive, on-site detection method for Vibrio that prioritizes convenience and speed. This study developed a visual IMBS-CPCR method for the detection of four common pathogenic Vibrio species: Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, and Vibrio harveyi. The method achieved high capture rates for target bacteria (82.6%–83.3%) and low capture rates for non-target bacteria (below 11%), demonstrating good sensitivity by detecting bacteria at concentrations as low as 10 CFU/mL. Optimal amplification conditions were determined for each species, with specificity tests confirming that only target bacteria produced amplification bands. Sensitivity tests revealed detection limits of 101 CFU/mL for V. alginolyticus, V. anguillarum, and V. fluvialis, and 102 CFU/mL for V. harveyi. The combined techniques improved detection sensitivity in simulated fish samples by 10–100 fold compared to direct CPCR or PCR methods.

Identification of a cellular role of hemolysin co-regulatory protein (Hcp) in Vibrio alginolyticus modulating substrate metabolism and biofilm formation by cAMP-CRP

Cyclic AMP (cAMP) and cAMP receptor protein (CRP) system controls catabolic enzyme expression based on metabolite concentrations in bacteria. Hemolysin co-regulatory protein (Hcp) is well known as a molecular chaperone for virulence factor secretion of the type VI secretion system (T6SS). However, the intracellular role of Hcp involving in bacterial physiological processes remains unknown. To clarify that, we constructed a single hcp mutant strain and analyzed their effects on the physiological processes of Vibrio alginolyticus. The omics results revealed the extensive involvement of Hcp in the catabolic metabolism in bacteria. Simultaneously, Hcp1 and Hcp2 played opposing regulatory roles on the bacterial growth, biofilm formation, and intracellular cAMP-CRP levels during cultivation in a glucose medium. Furthermore, the interacting protein screening and co-immunoprecipitation (Co-IP) assays confirmed that the glucose-specific phosphoenolpyruvate (PEP)-phosphotransferase system (PTS) enzyme IIA component (EIIAglc) was a key interacting partner with Hcp proteins as well as class I adenylyl cyclase (AC-I) in Vibrio alginolyticus. These results indicated that, to achieve cellular homeostasis, Hcp1 and Hcp2 might exert antagonistic and synergistic effects, respectively, on the interaction between EIIAglc and AC thus cooperatively regulating intracellular cAMP-CRP production.

Screening and functional characterization of isocitrate lyase AceA in the biofilm formation of Vibrio alginolyticus.

Biofilm is a well-known sessile lifestyle for bacterial pathogens, but a little is known about the mechanism on biofilm formation in Vibrio alginolyticus. In this study, we screened V. alginolyticus strains with strong biofilm formation ability from coastal seawater. The antibiotic resistance of the biofilm cells (BFs) was higher than that of the planktonic cells (PTs). To study the genes and pathways involved in biofilm formation, we performed transcriptome analysis of the BFs and PTs of V. alginolyticus R9. A total of 685 differentially expressed genes (DEGs) were upregulated, and 517 DEGs were downregulated in the BFs. The upregulated DEGs were significantly enriched in several pathways including glyoxylate and dicarboxylate metabolism, while the downregulated genes were significantly enriched in the flagellar assembly pathways. The key gene involved in glyoxylate shunt, aceA, was cloned, and ΔaceA mutant was constructed to determine the function of AceA in carbon source utilization, biofilm formation, and virulence. Real-time reverse transcription PCR showed that the expression of aceA was higher at the mature stage but lower at the disperse stage of biofilm formation, and the expression of the flagellar related genes was upregulated in ΔaceA. This is the first study to illustrate the global gene expression profile during the biofilm formation of V. alginolyticus, and isocitrate lyase AceA, the key enzyme involved in glyoxylate shunt, was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the late stage.IMPORTANCEBiofilms pose serious public problems, not only protecting the cells in it from environmental hazard but also affecting the composition and abundance of bacteria, algae, fungi, and protozoa. The important opportunistic pathogen Vibrio alginolyticus is extremely ubiquitously present in seawater, and it also exhibited a strong ability to form biofilm; thus, investigation on the biofilm formation of V. alginolyticus at molecular level is fundamental for the deeper exploration of the environmental concerns arose by biofilm. In this study, transcriptome analysis of biofilm cells (BFs) and planktonic cells (PTs) from V. alginolyticus was performed and AceA was screened to play an important role in biofilm formation. AceA was shown to maintain biofilms accompanied by downregulation of flagellation but promoted dispersal of BFs at the disperse stage. This method was helpful to further understand the ability and mechanism of V. alginolyticus biofilm formation and provide clues for prevention of V. alginolyticus infection.

Identification of a Mnlrig-1 involved in testis reproductive immunity in the oriental river prawn Macrobrachium nipponense

The testis evolves a highly organized testicular microenvironment to support spermatogenesis. However, the knowledge about it is limited in crustacean. In this study, we identified a member of immunoglobulin superfamily (IgSF) from Macrobrachium nipponense testis and explored its roles as a potential pattern recognition receptor (PRR) involved in reproductive immunity. Based on the domains it contains and homology analysis result, we designate it as leucine-rich repeats and immunoglobulin-like domains protein-1 (MnLrig-1). The Mnlrig-1 comprises a 3288 bp open reading frame (ORF) encoding a 1095 amino acid protein. MnLrig-1 is consisted of one signaling peptide; one LRR_NT domain; eight LRR domains; five LRR_TYP domains; one LRR_CT domain; three IGc2 regions; one transmembrane region, and C-terminal cytoplasmic tail, sharing similar domains with orthologs in other crustacean species. MnLrig-1 is widely expressed in various tissues of M. nipponense. Mnlrig-1 is significantly induced by LPS, PGN, Aeromonas hydrophila, and Vibrio alginolyticus challenge in the testis at 3 h and maintained a high level from 3 h to 24 h. Additionally, two recombinant immunoglobulin domains of MnLrig-1 are obtained, while only one domain shows direct binding affinity towards LPS, PGN, Escherichia coli, A. hydrophila, Staphylococcus aureus, and Bacillus subtilis in vitro. Moreover, silencing Mnlrig-1 results in a significant upregulation of three anti-lipopolysaccharide factors (ALFs) in the testis. These results reveal the potential role of MnLrig-1 as a PRR involved in the testis reproductive immunity in M. nipponense. The insights gained from this study will expand our understanding of immune system in crustacean and may have implications for aquaculture and disease management in crustaceans.

In vitro efficacy of aquaculture antimicrobials and genetic determinants of resistance in bacterial isolates from tropical aquaculture disease outbreaks.

Understanding the efficacy of antimicrobials against pathogens from clinical samples is critical for their responsible use. The manuscript presents in vitro efficacy and antimicrobial resistance (AMR) genes in seven species of fish pathogens from the disease outbreaks of Indian aquaculture against oxytetracycline, florfenicol, oxolinic acid, and enrofloxacin. In vitro efficacy was evaluated by minimum inhibitory concentration and minimum bactericidal concentration. The gene-specific PCR screened AMR genes against quinolones (qnrA, qnrB, and qnrS) and tetracyclines (tetM, tetS, tetA, tetC, tetB, tetD, tetE, tetH, tetJ, tetG, and tetY). The results showed that Aeromonas veronii (45%) showed the maximum resistance phenotype, followed by Streptococcus agalactiae (40%), Photobacterium damselae (15%), Vibrio parahaemolyticus (10%), and Vibrio vulnificus (5%). There was no resistance among Vibrio harveyi and Vibrio alginolyticus against the tested antimicrobials. The positive association between tetA, tetB, tetC, tetM, or a combination of these genes to oxytetracycline resistance and qnrS to quinolone resistance indicated their potential in surveillance studies. The prevalence of resistance phenotypes (16.43%) and evaluated AMR genes (2.65%) against aquaculture antimicrobials was low. The resistance phenotype pattern abundance was 0.143. All the isolates showed susceptibility to florfenicol. The results help with the appropriate drug selection against each species in aquaculture practices.

Myeloid differentiation protein 2 regulates the innate immunity and the disease resistant against Vibrio alginolyticus in Scylla paramamosain

Myeloid differentiation protein 2 (MD2), generally functions as a coreceptor of Toll-like receptor 4 (TLR4), facilitating the activation of TLR4 and the recognition of lipopolysaccharides (LPS) in host organisms. While the role of MD2 in immune activation is well-documented across various species, the specific role of the MD2 homolog in Scylla paramamosain (SpMD2) remains unidentified. In this study, we applied RNA interference to reduce SpMD2 expression, aiming to elucidate its role in immune system of mud crabs. Notably, SpMD2 interference leded to decrease in the hemocyte counts and phagocytic activity, along with increase in apoptosis rates and level of reactive oxygen species (ROS). Furthermore, the activities of key enzymes related to immune, such as superoxide dismutase (SOD), catalase (CAT), phenoloxidase (PO), peroxidase (POD), lysozyme (LZM), and acid phosphatase (ACP), were reduced by SpMD2 knockdown. Following infection with Vibrio alginolyticus, increase of SpMD2 expression level was observed. This was accompanied by alterations in the expression levels of genes related to immune in mud crabs. Challenge experiment with Vibrio alginolyticus showed a higher mortality rate after SpMD2 interference. Our study underscore the critical role of SpMD2 in enhancing the innate immunity and disease resistant in S. paramamosain, advancing our understanding of the innate immune regulatory mechanisms in crustaceans.

The Effect of the Lysine Acetylation Modification of ClpP on the Virulence of Vibrio alginolyticus.

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus.

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Investigating dynamics, etiology, pathology, and therapeutic interventions of Caligus clemensi and Vibrio alginolyticus co-infection in farmed marine fish

This study investigated a disease outbreak characterized by caligid copepod infestations and subsequent secondary bacterial infections in European seabass (Dicentrarchus labrax) and flathead grey mullet (Mugil cephalus) cultivated at a private facility in the Deeba Triangle region of Egypt. Moribund fish displayed brown spots on the skin, tongue, and gills, along with lethargy and excess mucus. The fish suffered severe infections, exhibiting external hemorrhages, ulcers, and ascites. The fish had pale, enlarged livers with hemorrhaging. Comprehensive parasitological, bacteriological, molecular, immunity and histopathological analyses were conducted to identify the etiological agents and pathological changes. Caligid copepod infestation was observed in wet mounts from the buccal and branchial cavities of all examined fish, and the caligids were identified as Caligus clemensi through COI gene sequencing and phylogenetic analysis. Vibrio alginolyticus was confirmed as a secondary bacterial infection through biochemical tests, recA gene sequencing, and phylogenetic analyses. Antibiotic susceptibility testing revealed resistance to β-lactams, aminoglycosides, and trimethoprim-sulfamethoxazole in V. alginolyticus isolates. Upregulation of the inflammatory marker IL-1β in gill and skin tissues indicated a robust cell-mediated immune response against the pathogens. Histopathological examination revealed severe tissue damage, hyperplasia, hemorrhage, and congestion in the gills, along with hepatocellular degeneration and steatosis in the liver, providing initial insights into this outbreak. A comprehensive therapeutic regimen was implemented, comprising prolonged hydrogen peroxide immersion baths, followed by the application of the nature-identical plant-based compound Lice-less and probiotic Sanolife Pro-W supplementation. This integrated approach effectively eliminated C. clemensi infestations, controlled secondary bacterial infections, and restored fish health, reducing morbidity and mortality rates to minimal levels.