A C-type lectin-like receptor CD302 in yellow drum (Nibea albiflora) functioning in antibacterial activity and innate immune signaling.

Molecular dissection of disease resistance against Vibrio harveyi infection in yellow drum at the genome-wide level uncovered a C-type lectin-like receptor cluster of differentiation CD302 (named as YdCD302) in our previous study. Here, the gene expression pattern of YdCD302 and its function in mediating the defense response to V. harveyi attack were investigated. Gene expression analysis demonstrated that YdCD302 was ubiquitously distributed in various tissues with the highest transcript abundance in liver. The YdCD302 protein exhibited agglutination and antibacterial activity against V. harveyi cells. Binding assay indicated that YdCD302 can physically interact with V. harveyi cells in a Ca2+-independent manner, and the interaction can activate reactive oxygen species (ROS) production in the bacterial cells to induce RecA/LexA-mediated cell death. After infection with V. harveyi, the expression of YdCD302 can be up-regulated significantly in the main immune organs of yellow drum and potentially further trigger the cytokines involved innate immunity. These findings provide insight into the genetic basis of the disease resistance trait in yellow drum and shed light on the functioning of the CD302 C-type lectin-like receptor in host-pathogen interactions. The molecular and functional characterization of YdCD302 is a significant step towards a better understanding of disease resistance mechanisms and the development of new strategies for disease control.

Transcriptome analysis of Macrobrachium rosenbergii hemocytes reveals in-depth insights into the immune response to Vibrio parahaemolyticus infection.

Macrobrachium rosenbergii as one of the common freshwater prawn species in Southeast Asia, which breeding industry is seriously threatened by vibriosis and causes high mortality. In this study, the RNA-seq was employed for assessing the M. rosenbergii hemocytes transcriptomes following Vibrio parahaemolyticus challenge. After challenge for 6 h (h), there were overall 1849 DEGs or differentially expressed genes, including 1542 up-regulated and 307 down-regulated genes, and there was a total of 1048 DEGs, including 510 up-regulated genes and 538 down-regulated genes, after challenge for 12 h. Mitogen-activated protein kinase (MAPK) immune-related pathways, Toll, immune deficiency (IMD), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) were among the immune pathways where a lot of the DEGs were connected. The expression patterns of 18 chosen immune-related genes were examined utilizing qRT-PCR or quantitative real-time polymerase chain reaction, which revealed that the V. parahaemolyticus infection activated the M. rosenbergii's immune response. Permutational multivariate analysis of variance (PERMANOVA) showed that V. parahaemolyticus infection modulated immune regulation and apoptosis pathways. The gathered information provided new insight into M. rosenbergii's immunity and suggested a novel approach to fight against bacterial infection.

Intestinal microbiome-mediated resistance against vibriosis for Cynoglossus semilaevis.

BACKGROUND: Infectious diseases have caused huge economic loss and food security issues in fish aquaculture. Current management and breeding strategies heavily rely on the knowledge of regulative mechanisms underlying disease resistance. Though the intestinal microbial community was linked with disease infection, there is little knowledge about the roles of intestinal microbes in fish disease resistance. Cynoglossus semilaevis is an economically important and widely cultivated flatfish species in China. However, it suffers from outbreaks of vibriosis, which results in huge mortalities and economic loss. RESULTS: Here, we used C. semilaevis as a research model to investigate the host-microbiome interactions in regulating vibriosis resistance. The resistance to vibriosis was reflected in intestinal microbiome on both taxonomic and functional levels. Such differences also influenced the host gene expressions in the resistant family. Moreover, the intestinal microbiome might control the host immunological homeostasis and inflammation to enhance vibriosis resistance through the microbe-intestine-immunity axis. For example, Phaeobacter regulated its hdhA gene and host cyp27a1 gene up-expressed in bile acid biosynthesis pathways, but regulated its trxA gene and host akt gene down-expressed in proinflammatory cytokines biosynthesis pathways, to reduce inflammation and resist disease infection in the resistant family. Furthermore, the combination of intestinal microbes and host genes as biomarkers could accurately differentiate resistant family from susceptible family. CONCLUSION: Our study uncovered the regulatory patterns of the microbe-intestine-immunity axis that may contribute to vibriosis resistance in C. semilaevis. These findings could facilitate the disease control and selective breeding of superior germplasm with high disease resistance in fish aquaculture. Video Abstract.

AIF-1 and RNASET2 are involved in the inflammatory response in the Mediterranean mussel Mytilus galloprovincialis following Vibrio infection.

Filter-feeding bivalves, such as the Mytilus species, are exposed to different types of bacteria in the surrounding waters, in particular of the Vibrio genus. Mussels lack an adaptive immune system and hemocytes can recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) to activate intracellular signaling pathways to trigger the antimicrobial effectors synthesis. Among the areas of bivalve immunity that deserve study include the role of hemocyte subpopulations. Since little information are available on immune responses at the tissue level to human pathogenic vibrios commonly detected in coastal waters involved in seafood-borne diseases, in this work, immunological parameters of the hemocytes from the Mediterranean mussel M. galloprovincialis were evaluated in response to in vivo challenge with Vibrio splendidus. The histological approach has been first used in order to identify the hemocytes recruitment at the infection site and the morphological change of muscular fibers. In addition, using immunolabeling with specific antibody we detected the production of molecules involved in the inflammatory activated cascade: Toll-like receptors 4 (TLR4), the myeloid differentiation factor 88 (MyD88), the Allograft inflammatory factor-1 (AIF-1) and the ribonucleases RNASET2, belonging to the T2 family, that in vertebrates are involved in the recruitment and activation of macrophages. Our results indicate the activation of TLR4 during bacterial infection preparatory to the recruitment of the MyD88 adapter with a putative role in recognition and intracellular signalling. Furthermore, the data presented in this work suggest that challenging with Gram-negative bacteria causes a massive migration of AIF-1+ hemocytes and that the ribonuclease RNASET2 could play a key role in the recruitment of these activated hemocytes. Our approach is useful for further understanding the complex molecular defence mechanisms of the host in invertebrates, especially in relation to the need to develop methods to evaluate the immunological response of bivalve molluscs used in aquaculture.

Comparative Mucous miRomics in Cynoglossus semilaevis Related to Vibrio harveyi Caused Infection.

Epidermal mucus is an important barrier and regulating mediator in fish. MicroRNAs (miRNAs) are proved to be involved in various biological processes, also as promising biomarkers for disease diagnosis. Vibrio harveyi has long been a noticeable bacterial pathogen in Cynoglossus semilaevis aquaculture. To find the evidence whether there are indicating miRNAs in mucus and whether the miRNAs are related to infections caused by V. harveyi, miRNA profiles of mucus from V. harveyi infected fish and healthy controls were screened by small RNA sequencing and verified by quantitative real-time PCR. This is the first report about miRNA profiling of flatfish mucus, aiming at illustrating the pathogenesis of V. harveyi caused infection and developing disease-related biomarkers. The results revealed significant differences in expression levels of some miRNAs between infected fish and healthy ones. Three hundred differentially expressed miRNAs were obtained after filtering through FC > 2 or FC < 0.5 and most of the differential miRNAs were downregulated. After verification through qRT-PCR, four unique miRNAs, dre-miR-451, dre-miR-184, dre-miR-205-5p > ssa-miR-205b-5p, and dre-miR-181a-5p > ssa-miR-181a-5p, were identified as V. harveyi infection-related signatures, consistent with sequencing trend. The expression levels of these four miRNAs in the infected fish were all significantly lower than controls. These miRNAs in mucus could be used to differentiate diseased and healthy fish in a non-invasive way with practical value for large-scale disease screening. They also provided new insights into the mechanism underlying the bacterial infections in fish.

Analysis of Gene Expression Profiles, Cytokines, and Bacterial Loads Relevant to Alcoholic Liver Disease Mice Infected With V. vulnificus.

Patients with liver disease are susceptible to infection with Vibrio vulnificus (V. vulnificus), but the specific reasons remain elusive. Through RNA-seq, we found that when mice with alcoholic liver disease (ALD) were infected with V. vulnificus by gavage, compared with the Pair group, the small intestinal genes affecting intestinal permeability were upregulated; and the number of differentially expressed genes related to immune functions (e.g., such as cell chemotaxis, leukocyte differentiation, and neutrophil degranulation) decreased in the liver, spleen, and blood. Further analysis showed that the number of white blood cells decreased in the Pair group, whereas those in the ALD mice did not change significantly. Interestingly, the blood bacterial load in the ALD mice was about 100 times higher than that of the Pair group. After the ALD mice were infected with V. vulnificus, the concentrations of T cell proliferation-promoting cytokines (IL-2, IL-23) decreased. Therefore, unlike the Pair group, ALD mice had weaker immune responses, lower T cell proliferation-promoting cytokines, and higher bacterial loads post-infection, possibly increasing their susceptibility to V. vulnificus infection. These new findings we presented here may help to advance the current understanding of the reasons why patients with liver disease are susceptible to V. vulnificus infection and provides potential targets for further investigation in the context of treatment options for V. vulnificus sepsis in liver disease patient.

The Evolution and Diversity of Interleukin-17 Highlight an Expansion in Marine Invertebrates and Its Conserved Role in Mucosal Immunity.

The interleukin-17 (IL-17) family consists of proinflammatory cytokines conserved during evolution. A comparative genomics approach was applied to examine IL-17 throughout evolution from poriferans to higher vertebrates. Cnidaria was highlighted as the most ancient diverged phylum, and several evolutionary patterns were revealed. Large expansions of the IL-17 repertoire were observed in marine molluscs and echinoderm species. We further studied this expansion in filter-fed Mytilus galloprovincialis, which is a bivalve with a highly effective innate immune system supported by a variable pangenome. We recovered 379 unique IL-17 sequences and 96 receptors from individual genomes that were classified into 23 and 6 isoforms after phylogenetic analyses. Mussel IL-17 isoforms were conserved among individuals and shared between closely related Mytilidae species. Certain isoforms were specifically implicated in the response to a waterborne infection with Vibrio splendidus in mussel gills. The involvement of IL-17 in mucosal immune responses could be conserved in higher vertebrates from these ancestral lineages.

Characterization and functional study of a chimera galectin from yellow drum Nibea albiflora.

Galectins are protein that participates in a variety of immune responses in the process of pathogenic infections. In the present study, a chimera galectin gene was screened from the transcriptome database of Nibea albiflora, which was named as YdGal-3. The results of qRT-PCR showed that the mRNA transcripts of YdGal-3 were ubiquitously distributed in all the detected tissues. After infection with Vibrio harveyi, the expression of YdGal-3 in liver, spleen, and head kidney increased significantly. Immunohistochemistry showed that YdGal-3 protein was widely expressed in the head kidney. The purified YdGal-3 protein by prokaryotic expression agglutinated red blood cells. Sugar inhibition assay showed that the agglutinating activity of YdGal-3 protein was inhibited by different sugars including lactose, D-galactose, and lipopolysaccharide. In addition, we mutated YdGal-3 His 294 into proline (P), alanine (A), glycine (G), and aspartic acid (D), it was further proved that the residue plays a key role in agglutination. YdGal-3 agglutinated some gram-negative bacteria including Pseudomonas plecoglossicida, Vibrio parahemolyticus, V. harveyi, and Aeromonas hydrophila, and exhibited antibacterial activity. These results suggested that YdGal-3 protein played an important role in the innate immunity of N. albiflora.

Reduced virulence of the MARTX toxin increases the persistence of outbreak-associated Vibrio vulnificus in host reservoirs.

Opportunistic bacteria strategically dampen their virulence to allow them to survive and propagate in hosts. However, the molecular mechanisms underlying virulence control are not clearly understood. Here, we found that the opportunistic pathogen Vibrio vulnificus biotype 3, which caused an outbreak of severe wound and intestinal infections associated with farmed tilapia, secretes significantly less virulent multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which is the most critical virulence factor in other clinical Vibrio strains. The biotype 3 MARTX toxin contains a cysteine protease domain (CPD) evolutionarily retaining a unique autocleavage site and a distinct ß-flap region. CPD autoproteolytic activity is attenuated following its autocleavage because of the ß-flap region. This ß-flap blocks the active site, disabling further autoproteolytic processing and release of the modularly structured effector domains within the toxin. Expression of this altered CPD consequently results in attenuated release of effectors by the toxin and significantly reduces the virulence of V. vulnificus biotype 3 in cells and in mice. Bioinformatic analysis revealed that this virulence mechanism is shared in all biotype 3 strains. Thus, these data provide new insights into the mechanisms by which opportunistic bacteria persist in an environmental reservoir, prolonging the potential to cause outbreaks.

Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor.

Acute hepatopancreatic necrosis disease (AHPND) caused by PirABVP-producing strain of Vibrio parahaemolyticus, VPAHPND, has seriously impacted the shrimp production. Although the VPAHPND toxin is known as the VPAHPND virulence factor, a receptor that mediates its action has not been identified. An in-house transcriptome of Litopenaeus vannamei hemocytes allows us to identify two proteins from the aminopeptidase N family, LvAPN1 and LvAPN2, the proteins of which in insect are known to be receptors for Cry toxin. The membrane-bound APN, LvAPN1, was characterized to determine if it was a VPAHPND toxin receptor. The increased expression of LvAPN1 was found in hemocytes, stomach, and hepatopancreas after the shrimp were challenged with either VPAHPND or the partially purified VPAHPND toxin. LvAPN1 knockdown reduced the mortality, histopathological signs of AHPND in the hepatopancreas, and the number of virulent VPAHPND bacteria in the stomach after VPAHPND toxin challenge. In addition, LvAPN1 silencing prevented the toxin from causing severe damage to the hemocytes and sustained both the total hemocyte count (THC) and the percentage of living hemocytes. We found that the rLvAPN1 directly bound to both rPirAVP and rPirBVP toxins, supporting the notion that silencing of LvAPN1 prevented the VPAHPND toxin from passing through the cell membrane of hemocytes. We concluded that the LvAPN1 was involved in AHPND pathogenesis and acted as a VPAHPND toxin receptor mediating the toxin penetration into hemocytes. Besides, this was the first report on the toxic effect of VPAHPND toxin on hemocytes other than the known target tissues, hepatopancreas and stomach.

Medicinal applications and molecular targets of dequalinium chloride.

For more than 60 years dequalinium chloride (DQ) has been used as anti-infective drug, mainly to treat local infections. It is a standard drug to treat bacterial vaginosis and an active ingredient of sore-throat lozenges. As a lipophilic bis-quaternary ammonium molecule, the drug displays membrane effects and selectively targets mitochondria to deplete DNA and to block energy production in cells. But beyond its mitochondriotropic property, DQ can interfere with the correct functioning of diverse proteins. A dozen of DQ protein targets have been identified and their implication in the antibacterial, antiviral, antifungal, antiparasitic and anticancer properties of the drug is discussed here. The anticancer effects of DQ combine a mitochondrial action, a selective inhibition of kinases (PKC-α/ß, Cdc7/Dbf4), and a modulation of Ca2+-activated K+ channels. At the bacterial level, DQ interacts with different multidrug transporters (QacR, AcrB, EmrE) and with the transcriptional regulator RamR. Other proteins implicated in the antiviral (MPER domain of gp41 HIV-1) and antiparasitic (chitinase A from Vibrio harveyi) activities have been identified. DQ also targets α -synuclein oligomers to restrict protofibrils formation implicated in some neurodegenerative disorders. In addition, DQ is a typical bolaamphiphile molecule, well suited to form liposomes and nanoparticules useful for drug entrapment and delivery (DQAsomes and others). Altogether, the review highlights the many pharmacological properties and therapeutic benefits of this old 'multi-talented' drug, which may be exploited further. Its multiple sites of actions in cells should be kept in mind when using DQ in experimental research.

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival.

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence.

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpAL10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.

Comparative Metabolomics and Proteomics Reveal Vibrio parahaemolyticus Targets Hypoxia-Related Signaling Pathways of Takifugu obscurus.

Coronavirus disease 2019 (COVID-19) raises the issue of how hypoxia destroys normal physiological function and host immunity against pathogens. However, there are few or no comprehensive omics studies on this effect. From an evolutionary perspective, animals living in complex and changeable marine environments might develop signaling pathways to address bacterial threats under hypoxia. In this study, the ancient genomic model animal Takifugu obscurus and widespread Vibrio parahaemolyticus were utilized to study the effect. T. obscurus was challenged by V. parahaemolyticus or (and) exposed to hypoxia. The effects of hypoxia and infection were identified, and a theoretical model of the host critical signaling pathway in response to hypoxia and infection was defined by methods of comparative metabolomics and proteomics on the entire liver. The changing trends of some differential metabolites and proteins under hypoxia, infection or double stressors were consistent. The model includes transforming growth factor-ß1 (TGF-ß1), hypoxia-inducible factor-1α (HIF-1α), and epidermal growth factor (EGF) signaling pathways, and the consistent changing trends indicated that the host liver tended toward cell proliferation. Hypoxia and infection caused tissue damage and fibrosis in the portal area of the liver, which may be related to TGF-ß1 signal transduction. We propose that LRG (leucine-rich alpha-2-glycoprotein) is widely involved in the transition of the TGF-ß1/Smad signaling pathway in response to hypoxia and pathogenic infection in vertebrates as a conserved molecule.

Gut-Liver Immune Response and Gut Microbiota Profiling Reveal the Pathogenic Mechanisms of Vibrio harveyi in Pearl Gentian Grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀).

Vibrio harveyi causes vibriosis in nearly 70% of grouper (Epinephelus sp.), seriously limiting grouper culture. As well as directly inhibiting pathogens, the gut microbiota plays critical roles in immune homeostasis and provides essential health benefits to its host. However, there is still little information about the variations in the immune response to V. harveyi infection and the gut microbiota of grouper. To understand the virulence mechanism of V. harveyi in the pearl gentian grouper, we investigated the variations in the pathological changes, immune responses, and gut bacterial communities of pearl gentian grouper after exposure to differently virulent V. harveyi strains. Obvious histopathological changes were detected in heart, kidney, and liver. In particular, nodules appeared and huge numbers of V. harveyi cells colonized the liver at 12 h postinfection (hpi) with highly virulent V. harveyi. Although no V. harveyi was detected in the gut, the infection simultaneously induced a gut-liver immune response. In particular, the expression of 8 genes associated with cellular immune processes, including genes encoding inflammatory cytokines and receptors, and pattern recognition proteins, was markedly induced by V. harveyi infection, especially with the highly virulent V. harveyi strain. V. harveyi infection also induced significant changes in gut bacterial community, in which Vibrio and Photobacterium increased but Bradyrhizobium, Lactobacillus, Blautia, and Faecalibaculum decreased in the group infected with the highly virulent strain, with accounting for 82.01% dissimilarity. Correspondingly, four bacterial functions related to bacterial pathogenesis were increased by infection with highly virulent V. harveyi, whereas functions involving metabolism and genetic information processing were reduced. These findings indicate that V. harveyi colonizes the liver and induces a gut-liver immune response that substantially disrupts the composition of and interspecies interactions in the bacterial community in fish gut, thereby altering the gut-microbiota-mediated functions and inducing fish death.

The sterol regulatory element binding protein homolog of Penaeus vannamei modulates fatty acid metabolism and immune response.

The sterol regulatory element binding proteins (SREBPs) transcription factors family, which regulate the expression of genes involved in cellular lipid metabolism and homeostasis, have recently been implicated in various physiological and pathophysiological processes such as immune regulation and inflammation in vertebrates. Consistent with other invertebrates, we identified a single SREBP ortholog in Penaeus vannamei (designated PvSREBP) with transcripts ubiquitously expressed in tissues and induced by lipopolysaccharide (LPS), Vibrio parahaemolyticus and Streptococcus iniae. In vivo RNA interference (RNAi) of PvSREBP attenuated the expression of several fatty acid metabolism-related genes (i.e., cyclooxygenase (PvCOX), lipoxygenase (PvLOX), fatty acid binding protein (PvFABP) and fatty acid synthase (PvFASN)), which consequently decreased the levels of total polyunsaturated fatty acids (ΣPUFAs). In addition, PvSREBP silencing decreased transcript levels of several immune-related genes such as hemocyanin (PvHMC) and trypsin (PvTrypsin), as well as genes encoding for heat-shock proteins (i.e., PvHSP60, PvHSP70 and PvHSP90). Moreover, in silico analysis revealed the presence of SREBP binding motifs on the promoters of most of the dysregulated genes, while shrimp depleted of PvSREBP were more susceptible to V. parahaemolyticus infection. Collectively, we demonstrated the involvement of shrimp SREBP in fatty acids metabolism and immune response, and propose that PvSREBP and PvHMC modulate each other through a feedback mechanism to establish homeostasis. The current study is the first to show the dual role of SREBP in fatty acid metabolism and immune response in invertebrates and crustaceans.

Genetic and virulence characterisation of Vibrio parahaemolyticus isolated from Indian coast.

BACKGROUND: V. parahaemolyticus is autochthonous to the marine environment and causes seafood-borne gastroenteritis in humans. Generally, V. parahaemolyticus recovered from the environment and/or seafood is thought to be non-pathogenic and the relationship between environmental isolates and acute diarrhoeal disease is poorly understood. In this study, we explored the virulence potential of environmental V. parahaemolyticus isolated from water, plankton and assorted seafood samples collected from the Indian coast. RESULTS: Twenty-two V. parahaemolyticus isolates from seafood harboured virulence associated genes encoding the thermostable-direct haemolysin (TDH), TDH-related haemolysin (TRH), and Type 3 secretion systems (T3SS) and 95.5% of the toxigenic isolates had pandemic strain attributes (toxRS/new+). Nine serovars, with pandemic strain traits were newly identified and an O4:K36 tdh-trh+V. parahaemolyticus bearing pandemic marker gene was recognised for the first time. Results obtained by reverse transcription PCR showed trh, T3SS1 and T3SS2ß to be functional in the seafood isolates. Moreover, the environmental strains were cytotoxic and could invade Caco-2 cells upon infection as well as induce changes to the tight junction protein, ZO-1 and the actin cytoskeleton. CONCLUSION: Our study provides evidence that environmental isolates of V. parahaemolyticus are potentially invasive and capable of eliciting pathogenic characteristics typical of clinical strains and present a potential health risk. We also demonstrate that virulence of this pathogen is highly complex and hence draws attention for the need to investigate more reliable virulence markers in order to distinguish the environmental and clinical isolates, which will be crucial for the pathogenomics and control of this pathogen.

Influence of different oil sources on growth, disease resistance, immune response and immune-related gene expression on the hybrid grouper (♀ Epinephelus fuscoguttatus × â™‚ E. lanceolatu), to Vibrio parahaemolyticus challenge.

The aim of this study was to investigate the effects of feeding alternative dietary oils to hybrid grouper fish (♀Epinephelus fuscoguttatus × â™‚E. lanceolatu) on their growth, histological morphology of hepatocytes, disease resistance, immune response, and expression of immune-related genes. Seven experimental fish meal-based isonitrogenous and isolipidic diets were formulated containing 5% fish oil (FO; acting as controls) and various vegetable oils (VOs): corn oil (CO), sunflower oil (SO), tea oil (TO), olive oil (OO), rice oil (RO), and mixed oil (MO); comprising equal amounts of these oils). Each diet was fed to triplicate groups of 40 fish (initial mean body weight ± standard error = 15.09 ± 0.01 g) for eight weeks. The results show that 1) alternative dietary oils had no significant effects on weight gain rate, specific growth rate, protein efficiency ratio, and survival rate compared with controls (P > 0.05). The weight gain rate (WGR) and specific growth rate (SGR) of the SO group were lower than in the CO and OO groups. 2) These were no differences in morphological indexes among groups; except for the CO group, in which the condition factor and hepatosomatic index were lower than those in other groups. 3) Compared with controls, the whole-body moisture and crude protein contents in the VO groups were higher, while their crude lipid contents were lower. 4) The fatty acid contents in liver and muscle were affected by lipid type, and the contents of eicosapentaenoic acid and docosahexaenoic acid in liver and muscle in the VO groups were markedly lower than in controls. 5) Compared with control group, VO groups damaged the histological morphology of hepatocytes. 6) After a challenge with the Vibrio parahaemolyticus bacterium, there were no differences in mortality among groups. However, VO enhanced the activity of non-specific immune enzymes while down-regulating the expression of Nrf2 and inducing the expression of pro-inflammatory factors (IL1ß, TNFα, TLR22, and MyD88) in the kidney. It can be concluded that dietary VO substitution does not affect the growth of fish but damaged the histological morphology of hepatocytes and induced the expression of pro-inflammatory factors in tissues. Finally, OO and CO were recommended as the appropriate lipid replacement for FO.

Melatonin restores Muc2 depletion induced by V. vulnificus VvpM via melatonin receptor 2 coupling with Gαq.

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.