Significance of Viable But Non-culturable (VBNC) State in Vibrios and Other Pathogenic Bacteria: Induction, Detection and the Role of Resuscitation Promoting Factors (Rpf).

Still, it remains a debate after four decades of research on surviving cells, several bacterial species were naturally inducted and found to exist in a viable but non-culturable (VBNC) state, an adaptive strategy executed by most bacterial species under different stressful conditions. VBNC state are generally attributed when the cells lose its culturability on standard culture media, diminish in conventional detection methods, but retaining its viability, virulence and antibiotic resistance over a period of years and may poses a risk to marine animals as well as public health and food safety. In this present review, we mainly focus the VBNC state of Vibrios and other human bacterial pathogens. Exposure to several factors like nutrient depletion, temperature fluctuation, changes in salinity and oxidative stress, antibiotic and other chemical stress can induce the cells to VBNC state. The transcriptomic and proteomic changes during VBNC, modification in detection techniques and the most significant role of Rpf in conversion of VBNC into culturable cells. Altogether, detection of unculturable VBNC forms has significant importance, since it may not only regain its culturability, but also reactivate its putative virulence determinants causing serious outbreaks and illness to the individual.

Chemoreceptor MCP4580 of Vibrio splendidus mediates chemotaxis toward L-glutamic acid contributing to bacterial virulence.

Chemotaxis has an essential function in flagellar bacteria that allows them to sense and respond to specific environmental signals, enabling their survival and colonization. Vibrio splendidus is an important opportunistic pathogen that infects a wide range of hosts including fish, bivalve, and sea cucumber. Our study demonstrated that V. splendidus AJ01 exhibited chemotaxis toward L-glutamic acid (L-Glu), an abundant amino acid in the intestinal and respiratory tree tissues of the sea cucumber. Bacterial samples collected from two locations in soft agar swimming plates were subjected to RNA-sequencing (RNA-Seq) analysis to identify the methyl-accepting chemotaxis protein (MCP) respond to L-Glu. Among the 40 annotated chemoreceptors, MCP4580 was identified as the MCP that mediates L-Glu-response. Molecular docking and site-directed mutagenesis revealed that L-arginine at residue 81 (R81) and L-glutamine at residue 88 (Q88) in the ligand-binding domain (LBD) are crucial for L-Glu recognition. Bacterial two-hybrid assay (BTH) showed that MCP4580 forms dimers and interacts with the histidine kinase CheA via the coupling protein CheW1 and CheW2. Phosphorylation analysis showed that the binding of L-Glu to MCP4580 results in the inhibition of CheA phosphorylation mainly via CheW1. Notably, sea cucumbers stimulated with each mutant strain of chemotaxis protein exhibited reduced mortality, highlighting the importance of chemotaxis in V. splendidus virulence. The present study provides valuable insights into the molecular components and signal transduction involved in the chemotaxis of V. splendidus toward L-Glu, and highlights the importance of chemotaxis in its virulence.

Identification of putative coral pathogens in endangered Caribbean staghorn coral using machine learning.

Coral diseases contribute to the rapid decline in coral reefs worldwide, and yet coral bacterial pathogens have proved difficult to identify because 16S rRNA gene surveys typically identify tens to hundreds of disease-associate bacteria as putative pathogens. An example is white band disease (WBD), which has killed up to 95% of the now-endangered Caribbean Acropora corals since 1979, yet the pathogen is still unknown. The 16S rRNA gene surveys have identified hundreds of WBD-associated bacterial amplicon sequencing variants (ASVs) from at least nine bacterial families with little consensus across studies. We conducted a multi-year, multi-site 16S rRNA gene sequencing comparison of 269 healthy and 143 WBD-infected Acropora cervicornis and used machine learning modelling to accurately predict disease outcomes and identify the top ASVs contributing to disease. Our ensemble ML models accurately predicted disease with greater than 97% accuracy and identified 19 disease-associated ASVs and five healthy-associated ASVs that were consistently differentially abundant across sampling periods. Using a tank-based transmission experiment, we tested whether the 19 disease-associated ASVs met the assumption of a pathogen and identified two pathogenic candidate ASVs-ASV25 Cysteiniphilum litorale and ASV8 Vibrio sp. to target for future isolation, cultivation, and confirmation of Henle-Koch's postulate via transmission assays.

Metagenomic analysis of pathogenic bacteria and virulence factor genes in coastal sediments from highly urbanized cities of India.

A metagenomic approach was employed to investigate the diversity and distribution of Virulence Factors Genes (VFGs) and Pathogenic Bacteria (PB) in sediment samples collected from highly urbanized cities along the Indian coastline. Among the study locations, Mumbai, Veraval and Paradeep showed a higher abundance of PB, with Vibrio and Pseudomonas as dominant at the genus level, and Escherichia coli and Pseudomonas aeruginosa at the species level. In total, 295 VFGs were detected across all sediment samples, of which 40 VFGs showed a similarity of ≥90 % with the Virulence Database (VFDB) and were focused in this study. Among the virulent proteins, twitching motility protein and flagellar P-ring were found to be prevalent and significantly associated with Vibrio spp., and Pseudomonas spp., indicating potential bacterial pathogenicity. This investigation serves as the basis for future studies and provides insights into the comprehensive taxonomic profiles of PB, VFGs and their associated PB in the coastal sediments of India.

The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence.

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus's virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.

High temperatures increase the virulence of Vibrio bacteria towards their coral host and competing bacteria via type VI secretion systems.

The bacterial pathogen Vibrio coralliilyticus induces severe coral diseases in warming oceans. A study in PLOS Biology reveals that high temperatures activate 2 type VI secretion systems in V. coralliilyticus, enhancing pathogenicity by deploying toxic effectors against competing bacteria and coral cells.

Unveiling the pathogenic and multidrug-resistant profiles of Vibrio alfacsensis: A potential identified threat in turbot (Scophthalmus maximus) aquaculture.

Vibrio alfacsensis is traditionally seen as an environmental symbiont within its genus, with no detailedly documented pathogenicity in marine aquaculture to date. This study delves into the largely unexplored pathogenic potential and emerging antibiotic resistance of V. alfacsensis. The VA-1 strain, isolated from recirculating aquaculture system (RAS) effluent of cultured turbot (Scophthalmus maximus), underwent comprehensive analysis including biochemical identification, antibiotic susceptibility testing and reinfection trials. The results confirmed VA-1's pathogenicity and significant multiple antibiotic resistance. VA-1 could induce systemic infection in turbot, with symptoms like kidney enlargement, exhibiting virulence comparable to known Vibrio pathogens, with an LD50 around 2.36 × 106 CFU/fish. VA-1's remarkable resistance phenotype (14/22) suggested potential for genetic exchange and resistance factor acquisition in aquaculture environments. Phylogenetic analysis based on 16S rDNA sequences and whole-genome sequencing has firmly placed VA-1 within the V. alfacsensis clade, while genome-wide analysis highlights its similarity and diversity in relation to strains from across the globe. VA-1 contained numerous replicons, indicating the possibility for the spread of resistance and virulence genes. This study suggests V. alfacsensis may acquire and transfer pathogenic and resistant traits through horizontal gene transfer, a likelihood intensified by changing environmental and aquaculture conditions, highlighting the need for vigilant pathogen monitoring and new non-antibiotic treatments.

Investigation of vibriosis caused by Vibrio anguillarum in rainbow trout (Oncorhynchus mykiss).

Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Mugla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.

The virulence plasmid associated with AHPND in shrimp appears to have originated from Vibrio owensii through a process of homologous recombination of parental plasmids and the transposable insertion of two large fragments.

Acute hepatopancreatic necrosis disease (AHPND) is a highly contagious and lethal disease of shrimp caused by Vibrio strains carrying the virulence plasmid (pAHPND) containing the pirAB virulence genes. Through analysis of plasmid sequence similarity, clustering, and phylogeny, a horizontal transfer element similar to IS91 was discovered within the pAHPND plasmid. Additionally, two distinct clades of plasmids related to pAHPND (designated as pAHPND-r1 and pAHPND-r2) were identified, which may serve as potential parental plasmids for pAHPND. The available evidence, including the difference in G+C content between the plasmid and its host, codon usage preference, and plasmid recombination event prediction, suggests that the formation of the pAHPND plasmid in the Vibrio owensii strain was likely due to the synergistic effect of the recombinase RecA and the associated proteins RecBCD on the pAHPND-r1 and pAHPND-r2, resulting in the recombination and formation of the precursor plasmid for pAHPND (pre-pAHPND). The emergence of pAHPND was found to be a result of successive insertions of the horizontal transfer elements of pirAB-Tn903 and IS91-like segment, which led to the deletion of one third of the pre-pAHPND. This plasmid was then able to spread horizontally to other Vibrio strains, contributing to the epidemics of AHPND. These findings shed light on previously unknown mechanisms involved in the emergence of pAHPND and improve our understanding of the disease's spread.

The ArgR-Regulated ADI Pathway Facilitates the Survival of Vibrio fluvialis under Acidic Conditions.

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.

Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

Antagonistic and enzymatic activities of Bacillus species isolated from the fish gastrointestinal tract as potential probiotics use in Artemia culture.

Probiotics have been used successfully in aquaculture to enhance disease resistance, nutrition, and/or growth of cultured organisms. Six strains of Bacillus were isolated from the intestinal tracts of fish and recognised by conventional biochemical traits. The six isolated strains were Bacillus cereus and Bacillus subtilis using MALDI-TOF-MS technique. The probiotic properties of these Bacillus strains were studied. The tested bacillus strains exhibit antibacterial activity against the different pathogens. The strain S5 gave the important inhibition zones against most pathogens (20.5, 20.33, 23, and 21 mm against Vibrio alginolyticus, Vibrio parahaemolyticus, Staphylococcus aureus, and Salmonella typhimurium, respectively). According to our results, all Bacillus strains have extracellular components that can stop pathogenic bacteria from growing. The enzymatic characterization showed that the tested strains can produce several biotechnological enzymes such as α-glucosidase, naphtol-AS-BI-Phosphohydrolase, esterase lipase, acid phosphatase, alkaline phosphatase, amylase, lipase, caseinase, and lecithinase. All Bacillus strains were adhesive to polystyrene. The adding Bacillus strains to the Artemia culture exerted significantly greater effects on the survival of Artemia. The challenge test on Artemia culture showed that the protection against pathogenic Vibrio was improved. These findings allow us to recommend the examined strains as prospective probiotic options for the Artemia culture, which will be used as food additives to improve the culture conditions of crustacean larvae and marine fish.

Functional and mechanistic studies of a phytogenic formulation, Shrimp Best, in growth performance and vibriosis in whiteleg shrimp.

Climate change and disease threaten shrimp farming. Here, we studied the beneficial properties of a phytogenic formulation, Shrimp Best (SB), in whiteleg shrimp. Functional studies showed that SB dose-dependently increased shrimp body weight and decreased feed conversion ratio. We found that SB protected against Vibrio parahaemolyticus as evidenced by survival rate, bacterial load, and hepatopancreatic pathology in shrimp. Finally, we explored the likely mechanism by which SB affects growth performance and vibriosis in shrimp. The 16S rRNA sequencing data showed that SB increased 6 probiotic genera and decreased 6 genera of pathogenic bacteria in shrimp. Among these, SB increased the proportion of Lactobacillus johnsonii and decreased that of V. parahaemolyticus in shrimp guts. To dissect the relationship among SB, Lactobacillus and Vibrio, we investigated the in vitro regulation of Lactobacillus and Vibrio by SB. SB at ≥ 0.25 µg/mL promoted L. johnsonii growth. Additionally, L. johnsonii and its supernatant could inhibit V. parahaemolyticus. Furthermore, SB could up-regulate five anti-Vibrio metabolites of L. johnsonii, which caused bacterial membrane destruction. In parallel, we identified 3 fatty acids as active compounds from SB. Overall, this work demonstrated that SB improved growth performance and vibriosis protection in shrimp via the regulation of gut microbiota.

Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.

Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.

Transcriptome Analysis of Host Anti-Vibrio harveyi Infection Revealed the Pathogenicity of V. harveyi to American Eel (Anguilla rostrata).

Vibrio harveyi, a recently discovered pathogenic bacterium isolated from American eels (Anguilla rostrata), poses uncertainties regarding its pathogenesis in American eel and the molecular mechanisms underlying host defense against V. harveyi infection. This study aimed to determine the LD50 of V. harveyi in American eel and assess the bacterial load in the liver, spleen, and kidney post-infection with the LD50 dose. The results showed that the LD50 of V. harveyi via intraperitoneal injection in American eels over a 14d period was determined to be 1.24 × 103 cfu/g body weight (6.2 × 104 cfu/fish). The peak bacterial load occurred at 36 h post-infection (hpi) in all three organs examined. Histopathology analysis revealed hepatic vein congestion and thrombi, tubular vacuolar degeneration, and splenic bleeding. Moreover, quantitative reverse transcription polymerase chain reaction (qRT-PCR) results indicated significant up or downregulation of 18 host immune- or anti-infection-related genes post 12 to 60 hpi following the infection. Additionally, RNA sequencing (RNA-seq) unveiled 7 hub differentially expressed genes (DEGs) and 11 encoded proteins play crucial roles in the anti-V. harveyi response in American eels. This study firstly represents the comprehensive report on the pathogenicity of V. harveyi to American eels and RNA-seq of host's response to V. harveyi infection. These findings provide valuable insights into V. harveyi pathogenesis and the strategies employed by the host's immune system at the transcriptomic level to combat V. harveyi infection.

Comparative phenotype and transcriptome analysis revealed the role of ferric uptake regulator (Fur) in the virulence of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata).

Vibrio harveyi is commonly found in salt and brackish water and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δfur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δfur mutant exhibited weaker biofilm formation and swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels; compared to the wild-type strain, the mutant strain showed time and diameter differences in growth and haemolysis, respectively. Additionally, the adhesion ability of the mutant strain was significantly decreased. Moreover, there were 15 different biochemical indicators observed between the two strains. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δfur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II and type VI secretion systems (T2SS and T6SS), amino acid synthesis and transport and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δfur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δfur strain. These findings suggest that the virulence of the Δfur strain was significantly decreased, which is closely related to phenotype changing and gene transcript regulation.

Strain-specific quorum-sensing responses determine virulence properties in Vibrio anguillarum.

Bacterial populations communicate using quorum-sensing (QS) molecules and switch on QS regulation to engage in coordinated behaviour such as biofilm formation or virulence. The marine fish pathogen Vibrio anguillarum harbours several QS systems, and our understanding of its QS regulation is still fragmentary. Here, we identify the VanT-QS regulon and explore the diversity and trajectory of traits under QS regulation in Vibrio anguillarum through comparative transcriptomics of two wildtype strains and their corresponding mutants artificially locked in QS-on (ΔvanO) or QS-off (ΔvanT) states. Intriguingly, the two wildtype populations showed different QS responses to cell density changes and operated primarily in the QS-on and QS-off spectrum, respectively. Examining 27 V. anguillarum strains revealed that ~11% were QS-negative, and GFP-reporter measurements of nine QS-positive strains revealed a highly strain-specific nature of the QS responses. We showed that QS controls a plethora of genes involved in processes such as central metabolism, biofilm formation, competence, T6SS, and virulence properties in V. anguillarum, with large strain-specific differences. Moreover, we demonstrated that the QS state is an important driver of virulence towards fish larvae in one of two V. anguillarum strains. We speculate that infections by mixed-strain communities spanning diverse QS strategies optimize the infection efficiency of the pathogen.

The Viable but Non-Culturable (VBNC) State in Vibrio Species: Why Studying the VBNC State Now Is More Exciting than Ever.

During periods that are not conducive for growth or when facing stressful conditions, Vibrios enter a dormant state called the Viable But Non-Culturable (VBNC) state. In this chapter, I will analyse the role of the VBNC state in Vibrio species survival and pathogenesis and the molecular mechanisms regulating this complex phenomenon. I will emphasise some of the novel findings that make studying the VBNC state now more exciting than ever and its significance in the epidemiology of these pathogens and critical role in food safety.

Characterization and the potential immune role of class A scavenger receptor member 4 (SCARA4) in bacterial infection in turbot (Scophthalmus maximus L.).

The class A scavenger receptors play important roles in innate immunity and are distributed on plasma membrane of macrophages and other cell types. Notably, the class A scavenger receptor 4 (SCARA4) contains a typical C-type (calcium-dependent) lectin domain, which belongs to the collectin family of pattern recognition receptors and is involved in the immune response against infection. Here, one turbot SCARA4 gene was identified with a 2,292 bp open reading frame (ORF) encoding 763 amino acid residues. Multiple sequence analysis and phylogenetic analysis confirmed that SmSCARA4 gene was more close to that of P. olivaceus. Gene structure and syntenic analysis showed conserved exon/intron organization pattern and syntenic pattern across selected vertebrate species. Tissue distribution analysis showed SmSCARA4 was expressed in all the tested healthy tissues with the relative high expression levels in skin, gill and spleen. Following both E. tarda and V. anguillarum challenge in vivo, SmSCARA4 was significantly repressed in gill and intestine. Remarkably, SmSCARA4 showed the strongest binding ability to LPS and strongest upregulation in turbot head kidney macrophages in response to LPS. Knockdown and overexpression of SmSCARA4 revealed its interactions with the two pro-inflammatory cytokines, TNF-α and IL-1ß. Finally, repression of SmSCARA4 via combined treatment of LPS and overexpression of SmSCARA4 construct in turbot head kidney macrophages further indicated an inhibitory role of SmSCARA4 in LPS-stimulated inflammation. Taken together, turbot SmSCARA4 plays an important role in turbot immunity, especially in the mucosa-related systems; SmSCARA4 possesses strong binding specificity to LPS, and exerts protective roles in response to LPS infection by reducing the release of pro-inflammatory cytokines. The mechanisms of inhibitory role of SmSCARA4 in LPS-elicited inflammation await further investigation.