Dynamics of antimicrobial resistance and genomic characteristics of foodborne Vibrio spp. in Southern China (2013-2022).

Vibrio spp., known as significant marine pathogens, have become more prevalent due to global warming. Antibiotics released into the environment drive Vibrio resistance. The increasing consumption of seafood leads to more interactions between Vibrio and humans. Despite this concerning trend, there remains a lack of large-scale surveillance for Vibrio contamination across various types of food. This study isolated 4027 Vibrio strains, primarily comprising V. parahaemolyticus and V. alginolyticus, in 3581 fresh shrimp and meat products from 2013 to 2022. The Vibrio strains showed increased resistance to important antibiotics, especially ß-lactams used to treat foodborne bacterial infections. Whole genome sequencing of 591 randomly chosen strains showed a strong correlation between antibiotic resistance and genotypes in Vibrio. Notably, various ESBL genes have evolved over the past 8 years, with blaVEBs being the most dominant. Additionally, carbapenemase genes, such as blaNDM-1, have become increasingly prevalent in recent years. Various mobile genetic elements, including IncQ and IncA/C plasmids, recoverable in Vibrio, facilitate the transmission of crucial ß-lactamase genes. These data provide insights into the evolutionary traits of antimicrobial resistance in foodborne Vibrio strains over a decade. Policymakers should consider these findings when devising appropriate strategies to combat bacterial antimicrobial resistance and safeguard human health.

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.

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.

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.

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.

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.

PoIL8-L, a teleost interleukin-8 like, enhances leukocyte cellular vitality and host defense against bacterial infections in Japanese flounder (Paralichthys olivaceus).

Interleukin-8 (IL-8), a CXC chemokine, exerts pivotal effect on cell migration, inflammatory response, and immune regulation. In this study, we examined the immunological characteristics of an IL-8 like homologue (PoIL8-L) in Japanese flounder (Paralichthys olivaceus). PoIL8-L contains a conserved chemokine CXC domain and 105 amino acid residues. PoIL8-L expression in tissues was constitutive, and significantly regulated by V. havieri or E. tarda infection. In vitro, rPoIL8-L could bind to eight tested bacteria, exhibited bacteriostatic and bactericidal effects against certain bacteria, and could bind to the targeted bacterial Ⅳ pilin protein rPilA of E. tarda. Furthermore, rPoIL8-L could attach to peripheral blood leukocytes, and enhance their immune genes expression, respiratory burst, chemotaxis, proliferation, acid phosphatase activity, and phagocytic activity. Additionally, rPoIL8-L induce neutrophils to extrude neutrophil extracellular traps. In vivo, rPoIL8-L could promote host resistance to E. tarda infection. In summary, these findings provide fresh perspectives on the immunological antibacterial properties of IL-8 in teleost.

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.

Vibrio splendidus infection promotes circRNA-FGL1-regulated coelomocyte apoptosis via competitive binding to Myc with the deubiquitinase OTUB1 in Apostichopus japonicus.

Circular RNAs (circRNAs) are involved in various physiological and pathological processes in both vertebrates and invertebrates. However, most studies on circRNAs have focused on their roles as endogenous competitive RNAs. Here, we report a novel function of circRNA derived from the Fibrinogen-like protein 1 gene (circ-FGL1) that inhibits coelomocyte apoptosis via competing with the deubiquitinase AjOTUB1 to bind AjMyc in Apostichopus japonicus during Vibrio splendidus infection. The results showed that circ-FGL1 is significantly downregulated in coelomocytes of V. splendidus-induced A. japonicus and negatively regulates coelomocyte apoptosis through the AjBax-AjCyt c pathway. Mechanistically, the deubiquitinase AjOTUB1 and circ-FGL1 could interact with the transcription factor protein AjMyc in the same region with circ-FGL1/AjMyc having greater affinity. Under normal conditions, high levels of circ-FGL1 bind directly to AjMyc, inhibiting the deubiquitylation of AjMyc by AjOTUB1 and leading to the degradation of AjMyc. After V. splendidus infection, AjMyc disassociates from the depressed expression of circ-FGL1, promoting its deubiquitylation by binding to the induced deubiquitinase AjOTUB1 to inhibit its degradation. AjMyc is then transferred to the nucleus and promotes the transcription of AjCyt c and AjBax to induce coelomocyte apoptosis. The new finding will expand our present outstanding on the functional role of circRNAs and suggest new therapeutic targets for the treatment of echinoderms during bacterial invasion.

A transcription factor ATF3 involves in the phagocytosis of granulocytes in oyster Crassostrea gigas.

Phagocytosis is a major cellular mechanism for mollusk granulocytes to eliminate nonself substances and dead cells, and thus to preserve the immune homeostasis. The knowledge of the regulatory mechanisms controlling phagocytic capacity is vital to understanding the immune system. In the present study, an ATF3 homolog (CgATF3) with a typical bZIP domain was identified in the Pacific oyster Crassostrea gigas. Its highly conserved bZIP domain consisted of two structural features, a basic region for DNA binding and a leucine zipper region for dimerization. Its transcript was found to be abundantly expressed in haemocytes, which was induced by Vibrio splendidus stimulation and recombinant CgTNF-2 treatment, along with an increase of its protein content in the nucleus. Moreover, CgATF3 showed a consistent and specific high expression in granulocytes, and CgATF3+ granulocytes were characterized morphologically by the largest diameter, smaller nucleus to cytoplasmic ratio, and abundant cytoplasmic granules, and functionally by a higher capacity for phagocytosis. When CgATF3 expression was inhibited by RNAi, the expression levels of CgRab1, CgRab33 and CgCathepsin L1, as well as the phagocytic rate and index of granulocytes all decreased after V. splendidus stimulation. These results together demonstrated the involvement of CgATF3 in regulating the expressions of Rabs and Cathepsin L1, as well as the phagocytosis of granulocytes in oyster C. gigas.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus.

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5'-untranslated region (UTR), and a 233 bp 3'-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37-335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Potential therapeutic use of dermaseptin S4 from the frog Phyllomedusa sauvagii and its derivatives against bacterial pathogens in fish.

AIM: Dermaseptins are one of the main families of antimicrobial peptides (AMPs) derived from the skin secretions of Hylidae frogs. Among them, dermaseptin S4 (DS4) is characterized by its broad-spectrum of activity against bacteria, protozoa, and fungi. In this study, the physicochemical properties of the native peptide DS4 (1-28) and two derivatives [DS4 (1-28)a and DS4 (1-26)a] isolated from the skin of the frog Phyllomedusa sauvagii were investigated and their antimicrobial properties against two marine pathogenic bacteria (Vibrio harveyi and Vibrio anguillarum) were examined. METHODS AND RESULTS: The results indicate that the peptide DS4 (1-26)a has high-antibacterial activity against the tested strains and low-hemolytic activity (<30% lysis at the highest tested concentration of 100 µg/mL) compared to the other two peptides tested. In addition, all three peptides affect the membrane and cell wall integrity of both pathogenic bacteria, causing leakage of cell contents, with DS4 (1-26)a having the most severe impact. These skills were corroborated by transmission electron microscopy and by the variation of cations in their binding sites due to the effects caused by the AMPs. CONCLUSIONS: These results suggest that DS4 and its derivatives, in particular the truncated and amidated peptide DS4 (1-26)a could be effective in the treatment of infections caused by these marine pathogenic bacteria. Future studies are required to validate the use of DS4  in vivo for the prevention of bacterial diseases in fish.

Bacterial recognition and inhibition activities of an LRR-only protein in the Chinese mitten crab, Eriocheir sinensis.

LRR-only protein (LRRop) is an important class of immune molecules that function as pattern recognition receptor in invertebrates, however, the bacterial inhibitory activity of this proteins remain largely unknown. Herein, a novel LRRop was cloned from Eriocheir sinensis and named as EsLRRop2. The EsLRRop2 consists of six LRR motifs and formed a horseshoe shape three-dimension structure. EsLRRop2 was mainly expressed in intestine and hepatopancreas. The transcripts of EsLRRop2 in the intestine and hepatopancreas were induced by Vibrio parahaemolyticus and Staphylococcus aureus, and displayed similar transcriptional profiles. The expression levels of EsLRRop2 responded more rapidly and highly to V. parahaemolyticus than S. aureus in the intestine and hepatopancreas. Although the basal expression level of EsLRRop2 in hemocytes was relatively low, its transcripts in hemocytes were significantly induced by V. parahaemolyticus and S. aureus. The recombinant proteins of EsLRRop2 (rEsLRRop2) displayed a wide range of binding spectrum against vibrios, including V. parahaemolyticus, V. alginolyticus, and V. harveryi. The rEsLRRop2 showed dose- and time-dependent inhibitory activity against V. parahaemolyticus and S. aureus, and it could agglutinate the two bacteria. Furthermore, the inhibitory activities of rEsLRRop2 against V. parahaemolyticus, V. alginolyticus, V. harveryi and S. aureus was slightly affected by pH and salinity, and the rEsLRRop2 displayed the strongest inhibitory activity against all the three vibrios when the salinity was 20 ‰ and pH was 8.0. Collectively, these results elucidate the bacterial binding and inhibitory activities of EsLRRop2, and provide theoretical foundations for the application of rEsLRRop2 in prevention and control of vibrio diseases in aquaculture.

Microbial and transcriptional response of Acropora valida and Turbinaria peltata to Vibrio coralliilyticus challenge: insights into corals disease resistance.

BACKGROUND: Coral diseases are significant drivers of global coral reef degradation, with pathogens dominated by Vibrio coralliilyticus playing a prominent role in the development of coral diseases. Coral phenotype, symbiotic microbial communities, and host transcriptional regulation have been well-established as factors involved in determining coral disease resistance, but the underlying mechanisms remain incompletely understood. METHODS: This study employs high-throughput sequencing to analyse the symbiotic microbial and transcriptional response of the hosts in order to evaluate the disease resistance of Acropora valida and Turbinaria peltata exposed to Vibrio coralliilyticus. RESULTS: A. valida exhibited pronounced bleaching and tissue loss within 7 h of pathogen infection, whereas T. peltata showed no signs of disease throughout the experiment. Microbial diversity analyses revealed that T. peltata had a more flexible microbial community and a higher relative abundance of potential beneficial bacteria compared to A. valida. Although Vibrio inoculation resulted in a more significant decrease in the Symbiodiniaceae density of A. valida compared to that of T. peltata, it did not lead to recombination of the coral host and Symbiodiniaceae in either coral species. RNA-seq analysis revealed that the interspecific differences in the transcriptional regulation of hosts after Vibrio inoculation. Differentially expressed genes in A. valida were mainly enriched in the pathways associated with energy supply and immune response, such as G protein-coupled receptor signaling, toll-like receptor signaling, regulation of TOR signaling, while these genes in T. peltata were mainly involved in the pathway related to immune homeostasis and ion transport, such as JAK-STAT signaling pathway and regulation of ion transport. CONCLUSIONS: Pathogenic challenges elicit different microbial and transcriptional shifts across coral species. This study offers novel insights into molecular mechanisms of coral resistance to disease.

A coordinated attack by a bacterial secretion system and a small molecule drives prey specificity.

Vibrio species are recognized for their role in food- and water-borne diseases in humans, fish, and aquatic invertebrates. We screened bacterial strains isolated from raw food shrimp for those that are bactericidal to Vibrio strains. Here we identify and characterize Aeromonas dhakensis strain A603 which shows robust bactericidal activity specifically towards Vibrio and related taxa but less potency toward other Gram-negative species. Using the A603 genome and genetic analysis, we show that two antibacterial mechanisms account for its vibriocidal activity -- a highly potent Type Six Secretion System (T6SS) and biosynthesis of a vibriocidal phenazine-like small molecule, named here as Ad-Phen. Further analysis indicates coregulation between Ad-Phen and a pore-forming T6SS effector TseC, which potentiates V. cholerae to killing by Ad-Phen.

Epidemiological and environmental investigation of the 'big four' Vibrio species, 1994 to 2021: a Baltic Sea retrospective study.

BackgroundThe Vibrio genus comprises several bacterial species present in the Baltic Sea region (BSR), which are known to cause human infections.AimTo provide a comprehensive retrospective analysis of Vibrio-induced infections in the BSR from 1994 to 2021, focusing on the 'big four' Vibrio species - V. alginolyticus, V. cholerae non-O1/O139, V. parahaemolyticus and V. vulnificus - in eight European countries (Denmark, Estonia, Finland, Germany, Latvia, Lithuania, Poland and Sweden) bordering the Baltic Sea.MethodsOur analysis includes data on infections, Vibrio species distribution in coastal waters and environmental data received from national health agencies or extracted from scientific literature and online databases. A redundancy analysis was performed to determine the potential impact of several independent variables, such as sea surface temperature, salinity, the number of designated coastal beaches and year, on the Vibrio infection rate.ResultsFor BSR countries conducting surveillance, we observed an exponential increase in total Vibrio infections (n = 1,553) across the region over time. In Sweden and Germany, total numbers of Vibrio spp. and infections caused by V. alginolyticus and V. parahaemolyticus positively correlate with increasing sea surface temperature. Salinity emerged as a critical driver of Vibrio spp. distribution and abundance. Furthermore, our proposed statistical model reveals 12 to 20 unreported cases in Lithuania and Poland, respectively, countries with no surveillance.ConclusionsThere are discrepancies in Vibrio surveillance and monitoring among countries, emphasising the need for comprehensive monitoring programmes of these pathogens to protect human health, particularly in the context of climate change.

Bioemulsifier from sponge-associated bacteria reduces staphylococcal biofilm.

Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Molecular identification and functional characterization of a C-type lectin gene in Meretrix meretrix.

C-type lectins (CTLs) are a kind of Ca2+-dependent immunoreactive factors, which participated in pathogens recognition and defense. The present study identified a new CTL from hard clam Meretrix meretrix (designated as MmCTL4). The full-length of MmCTL4 cDNA was 608 bp, encoding a presumed signal peptide of 19 bp and a carbohydrate recognition domain (CRD) of 131 bp. The tertiary structure of recombinant MmCTL4 protein (rMmCTL4) was the typical long double-ring structure with three conserved disulfide bonds, and the motifs in Ca2+-binding sites of MmCTL4 were QPN and WSD. The SYBR Green real-time PCR analysis indicated that MmCTL4 was widely expressed in the hemocytes, hepatopancreas and mantle of healthy clams. After Vibrio splendidus stimulation, the temporal expression profile of MmCTL4 mRNA in hemocytes and hepatopancreas increased by 7.8-fold at 6 hpi and 3.9-fold at 12 hpi, respectively. The cDNA fragments encoding MmCTL4 were recombined into pET-32a (+) vectors, and transformed into Escherichia coli BL21 (DE3). The rMmCTL4 with the presence of Ca2+ performed obvious hemagglutination activity, and could agglutinate E. coli, Bacillus subtilis, and Staphylococcus aureus, while it only weakly agglutinate Vibrio parahaemolyticus and fungi P. pastoris. The agglutination activity of rMmCTL4 were significantly inhibited by D-mannose, D-xylose, D-lactose, maltose and lipopolysaccharides. These results indicated that MmCTL4, as a class of typical pattern recognition receptors (PRRs), could protect the host against pathogen invasion in the innate immunity of clams.

MiR-210, not let-7, regulates Akt gene expression against Vibrio splendidus infection in the sea cucumber Apostichopus japonicus.

The immune regulatory roles of microRNAs (miRNAs) have recently attracted considerable attention. Bioinformatics prediction revealed that both let-7 and miR-210 provide potential binding sites for the Akt (rac-alpha serine/threonine-protein kinase) gene sequence in the sea cucumber Apostichopus japonicus (termed AjAkt). In this study, we first used a dual-luciferase reporter assay and functional validation techniques to verify the interactions between these two miRNAs (let-7 and miR-210) and AjAkt, and then investigated the functions of the validated miRNA/mRNA pair as part of the innate immune response against Vibrio splendidus infection. We found that AjAkt interacts with miR-210 rather than let-7, and miR-210 negatively regulates the expression of AjAkt. From 8 to 48 h after infection with V. splendidus, opposite trends were observed in the expression levels of miR-210 and AjAkt (mRNA and protein) in coelomocytes, suggesting that the miR-210/AjAkt pair is involved in immune regulation during this period after infection. Both AjAkt silencing and miR-210 overexpression enhanced the phagocytic capacity and reduced the infectivity of A. japonicus after pathogen infection, suggesting that the miR-210/AjAkt pair may regulate the innate immune response of A. japonicus by altering phagocytic capacity. The findings of this study enrich our knowledge of the role of miRNA/mRNA pairs in immune regulation in sea cucumbers and provide insights into the molecular mechanisms of the innate immune response in marine echinoderms.

An innovative mixture sampling strategy with uniform design: Application to global sensitivity analysis of mixture toxicity.

Global sensitivity analysis combined with quantitative high-throughput screening (GSA-qHTS) uses random starting points of the trajectories in mixture design, which may lead to potential contingency and a lack of representativeness. Moreover, a scenario in which all factor levels were at stimulatory effects was not considered, thereby hindering a comprehensive understanding of GSA-qHTS. Accordingly, this study innovatively introduced an optimised experimental design, uniform design (UD), to generate non-random and representative sample points with smaller uniformity deviation as starting points of multiple trajectories. By combining UD with the previously optimised one-factor-at-a-time (OAT) method, a novel mixture design method was developed (UD-OAT). The single toxicity tests showed that three pyridinium and five imidazolium ionic liquids (ILs) exerted stimulatory effects on Vibrio qinghaiensis sp.-Q67; thus, four stimulatory effective concentrations of each IL were selected as factor levels. The UD-OAT generated 108 mixture samples with equal frequency and without repetition. High-throughput microplate toxicity analysis revealed that all 108 mixtures exhibited inhibitory effects. Among these, type B mixtures exhibited increasing toxicities that subsequently decreased, unlike type C mixtures, which consistently increased over time. GSA successfully identified three of the eight ILs as important factors influencing the toxicities of the mixtures. When individual ILs produced stimulatory effects, mixtures containing two to three ILs exhibited either stimulatory effects or none. In contrast, mixtures containing five to eight ILs exhibited inhibitory effects, while those containing four ILs showed a transition from stimulatory to inhibitory effects. This study provides a novel mixture design method for studying mixture toxicity and fills the application gap of GSA-qHTS. The phenomenon of individuals being beneficial while mixtures can be harmful challenges traditional mixture risk assessments.