Vibrio Research Articles

The potential role of eyestalk in the immunity of Litopenaeus vannamei to Vibrio infection

The X-organ-sinus gland complex (XO-SG) in the eyestalk is an important neuroendocrine regulatory organ of crustaceans such as Litopenaeus vannamei, a prominent aquaculture species. The current study found significant changes in the enzyme activities of ALP, ACP, and T-SOD of hepatopancreatic in response to Vibrio parahaemolyticus exposure following eyestalk ablation, indicating that they were all involved in the immunological regulation of shrimps against V. parahaemolyticus infection. A total of 52,656 unigenes were obtained after RNA-Seq, with an average length of 1036 bp and an N50 of 1847 bp. Subsequently, 1899 eyestalk positive regulation genes (EPRGs), 745 eyestalk negative regulation genes (ENRGs), and 2077 non-eyestalk regulatory genes (NEGs) were identified. KEGG analysis of EPRGs revealed that eyestalk ablation might activate the neuroendocrine-immune (NEI) system. The RNA-Seq data were validated using quantitative real-time PCR (qRT-PCR). The findings suggested that eyestalk ablation might affect the expression of genes involved in the prophenoloxidase-activating system, the TLR signaling pathway, and numerous other immune-related genes in L. vannamei. All of these findings revealed that the eyestalk might have a role in the immune response of L. vannamei. The genes and pathways discovered in this study will help to elucidate the molecular mechanisms of hemocytes’ immune response to V. parahaemolyticus following eyestalk ablation in shrimp, as well as provide the framework for building crustacean immunity theory.

Molecular cloning and expression analysis of mytilin-like antimicrobial peptides from Asian green mussel Perna viridis

Mytilin is one of the most important CS-αβ peptides involved in innate immune response in Mytilidae. In this study, we successfully identified four mytilin-like antimicrobial peptides (pernalins) from Asian green mussel Perna viridis by aligning the P. viridis transcriptome with 186 mytilins and myticins related sequences collected from the transcriptome data of six Mytilus species. Analysis on gene structure showed that pernalin genes had high conservation with mytilin B of Mediterranean mussel Mytilus galloprovincialis. Interestingly, all pernalin genes have a similar tissue expression feature, evidenced by the highest transcription level observed in the hemocytes and followed by the mantle. The lowest transcription level was observed in the foot and gills. qRT-PCR analysis showed that all pernalin genes were significantly down-regulated at each time points from 3 h to 48 h after Vibrio parahaemolyticus infection, suggesting their timely immune responses after bacterial infection.

<i>Vibrio vulnificus</i> Disease Prevalence

Vibrio vulnificus is a deadly disease that has been increasing in prevalence. In this study, we review both primary and secondary data to discuss the factors that are contributing to the increase of vibrio disease, causing a 41% increase between 1996 and 2005. It has also been shown that public health campaigns to limit vibrio infections have focused on raw seafood consumption. However, an estimated 42% of infections are now being caused by wound infection, rather than through contaminated seafood. This shows the disparities in addressing vibrio contamination through contaminated seawater and open wounds. This is particularly stressing as global warming is causing an increase in risk of contaminated seawater. Reasons for this increase are discussed, and also possible solutions are presented for public health interventions to help mitigate this rise in vibrio infections.

Genome-wide characterization of gap junction (connexins and pannexins) genes in turbot (Scophthalmus maximus L.): evolution and immune response following Vibrio anguillarum infection.

Gap junction (GJ), a special intercellular junction between different cell types, directly connects the cytoplasm of adjacent cells, allows various molecules, ions and electrical impulses to pass through the intercellular regulatory gate, and plays vital roles in response to bacterial infection. Up to date, the information about the GJ in turbot (Scophthalmus maximus L.) is still limited. In current study, 43 gap junction genes were identified in turbot, phylogeny analysis suggested that gap junctions from turbot and other species were clustered into six groups, GJA, GJB, GJC, GJD, GJE and PANX, and turbot GJs together with respective GJs from Japanese flounder, half-smooth tongue sole and large yellow croaker, sharing same ancestors. In addition, these 43 GJ genes distributed in different chromosomes unevenly. According to gene structure and domain analysis, these genes (in GJA-GJE group) were highly conserved in that most of them contain the transmembrane area, connexin domain (CNX) and cysteine-rich domain (connexin CCC), while PANXs contain Pfam Innexin. Although only one tandem duplication was identified in turbot gap junction gene, 235 pairs of segmental duplications were identified in the turbot genome. To further investigate their evolutionary relationships, Ka/Ks was calculated, and results showed that most ratios were lower than 1, indicating they had undergone negative selection. Finally, expression analysis showed that gap junction genes were widely distributed in turbot tissues and significantly regulated after Vibrio anguillarum infection. Taken together, our research could provide valuable information for further exploration of the function of gap junction genes in teleost.

A Global Perspective of Vibrio Species and Associated Diseases: Three-Decade Meta-Synthesis of Research Advancement.

Outbreaks of Vibrio infections have a long history of global public health concern and threat to the aquaculture industry. This 3-decade (1990-2019) meta-synthesis of global research progress in Vibrio species and associated disease outbreaks was undertaken to generate the knowledge needed to design effective interventions with policy implications. Using PRISMA protocol, we obtained data on the online version of the Institute for Scientific Information (ISI), Web of Science (WOS), and Scopus from January 1990 to September 2021 by title search of the keywords “Vibrio species OR Vibrio spp. OR vibriosis.” On the 3-decade survey, the result has shown that a total of 776 publications document types were published on the subject, with an average of 24.25 ± 13.6 published documents per year with an annual growth rate of 4.71%. The year 2020 recorded the highest output of 52 published documents accounting for 6.70% of the total. The most prolific author, Blanch A., published 12 articles on the subject and has received citations of 1003 with an h-index of 10. While the most global cited paper author is the journal of J. Bacteriol (Bassler et al), receiving total citation (TC) (550) and per Year (22). The top active corresponding authors country is the United States of America with (92) articles, freq. 12.40%; TC of 3103. The observations in this study, such as the collaborations network map, and index, which have outlined a big difference between countries based on economic status, have underscored the need for a sustained research mentorship program that can define future policies.

Identification and characterization of mkk genes and their expression profiles in rainbow trout (Oncorhynchus mykiss) symptomatically or asymptomatically infected with Vibrio anguillarum

Mitogen-activated protein kinase kinases (MKKs) are intermediate kinases of mitogen-activated protein kinases (MAPKs) signaling pathways. MKKs are activated by mitogen-activated protein kinase kinase kinase (MKKK) and then the activated MKKs trigger the activation of downstream MAPKs. MAPK signaling pathways play an important role in regulating immune functions including apoptosis and inflammation. However, studies on identification and characterization of mkk repertoire in rainbow trout (Oncorhynchus mykiss) are still limited. Trout experienced 4 rounds (4R) of whole genome duplication (WGD), thus exhibiting increased paralogs of mkks with potentially functional diversity. In this study, we identified 17 mkk genes in trout and the following bacterial challenge (Vibrio anguillarum) studies showed functional diversity of different mkk subtypes. Vibrio anguillarum infection resulted in significantly up-regulated mkk2 subtypes in spleen and liver, and mkk4b3 in spleen, suggesting immunomodulation was regulated by activation of ERK, p38 and JNK pathways. Compared to other mkk subtypes, mkk6s were down-regulated in symptomatic group, rather than asymptomatic group. The organisms present negative feedback on MAPK activation, thus reducing extra damage to cells. We observed down-regulated mkk6s with up-regulated genes (dusp1 &dusp2) involved in negative feedback of MAPK activation. Based on these results, we might propose the distinct expression patterns of genes associated with MAPK pathways resulted in different phenotypes and symptoms of trout in response to bacterial challenge.

First report of the presence of Vibrio vulnificus in the Gulf of Gdansk.

Vibrio infections are becoming more frequent in the Baltic Sea region, which is caused by an increase in the sea surface temperature. Climate change creates the conditions for the emergence of new environmental niches that are beneficial for Vibrio spp., especially in the summer months. Vibrio vulnificus, which causes wound infections and septicaemia, represents a particularly dangerous species of Vibrio spp. There are numerous publications on the prevalence of V. vulnificus in various regions of the Baltic Sea, but there is a lack of such data for the Polish coast. This prompted us to conduct a pilot study into the prevalence of the bacteria in the Gulf of Gdansk. The study aimed to detect Vibrio spp. in the coastal waters and the wet sand at the beaches and bathing areas in the Gulf of Gdansk. During the period from June 16th to September 23rd 2020, 112 samples of seawater and 105 samples of wet sand were collected at 16 locations along the coast of the Gulf of Gdansk and Hel peninsula. Isolation of Vibrio spp. was conducted by filtering method and the isolated bacteria was cultured on CHROM agar Vibrio and TCBS agar. Final genus identification was performed by the MALDI TOF technique. In the present study, 10 isolates of Vibrio spp. were obtained from seawater and wet sand samples collected in the Gulf of Gdansk and Hel peninsula coast. Three of the isolates were identified as V. vulnificus; the presence of the species was confirmed in the seawater samples which had been collected in Hel (1 isolate), Jastarnia (1 isolate), and Chalupy (1 isolate). One strain of Vibrio alginolyticus was isolated from the seawater sample collected in Hel. Moreover, identification was incomplete for 6 of the isolated strains, these were identified as Vibrio cholerae/mimicus These strains were collected in Jastarnia (1 isolate), Kuznica (1 isolate), Gdansk-Brzezno (1 isolate), Puck (2 isolates), Chalupy (1 isolate). Our preliminary research study confirmed the presence of potentially pathogenic V. vulnificus in the Gulf of Gdansk in the summer months. Therefore, further monitoring of the presence of Vibrio spp. in the Baltic coast area is necessary.

CONDITIONS OF CLINICAL SYMPTOMS AND LIFE OF VANAME SHRIMP IN PREVENTION OF Vibrio alginolyticus INFECTION USING MIANA LEAF EXTRACT

This study aims to determine the clinical symptoms and survival of white vaname shrimp in the prevention of Vibrio alginolyticus infection using miana leaf extract. The study consisted of 4 treatments and 3 replications. The treatments in this study were 1 treatment and 3 treatments using different concentrations of bacteria, namely 103 CFU/ml, 105 CFU/ml, and 107 CFU/ml. The research was carried out in June-July 2020 at the Hatchery Laboratory of the Faculty of Marine and Fisheries Sciences, Hasanuddin University. Data analysis used descriptive analysis and analysis of variance (ANOVA). The results showed that A (control) shrimp was under normal conditions, treatment B (103 CFU/ml) was red, treated C (105 CFU/ml) was red and had necrosis of the body, and treatment D (107 CFU/ml) was shrimp. reddening, necrosis of tail and body segments. The results of data analysis showed that the use of miana leaf extract had a significant effect (p<0.05) on the use of live vaname shrimp. The highest vaname shrimp survival was treatment A (control) at 100%, treatment B (103 CFU/ml) at 90%, treatment C (105 CFU/ml) at 63.33% and treatment D (107 CFU/ml) by 40%.

Phenylalanine hydroxylase (PAH) plays a positive role during WSSV and Vibrio parahaemolyticus infection in Litopenaeus vannamei

Phenylalanine hydroxylase (PAH) is involved in immune defence reactions by providing the starting material, tyrosine, to synthesise catecholamines and melanin. PAH is an important metabolic enzyme of aromatic amino acids and the rate-limiting enzyme in the hydroxylation of amino acid phenylalanine to tyrosine. In the present study, a PAH gene, LvPAH, was cloned and identified from Litopenaeus vannamei. The open reading frame (ORF) of LvPAH was 1383 bp, encoding a protein of 460 amino acids comprised of an ACT domain and a Biopterin_H domain. LvPAH was constitutively expressed in healthy L. vannamei, with the highest expression levels in the eyestalk and the lowest in the hepatopancreas. Both white spot syndrome virus (WSSV) and Vibrio parahaemolyticus infection upregulated LvPAH expression in hemocytes, hepatopancreas and gills of L. vannamei. Inhibition of LvPAH resulted in a significantly lower survival rate of L. vannamei after WSSV infection than the control group, consistent with the observation that WSSV viral load was significantly higher in LvPAH-silenced L. vannamei. After a V. parahaemolyticus challenge, there was no significant difference between the survival rate of LvPAH-silenced and the control L. vannamei. However, the load of V. parahaemolyticus in LvPAH-silenced L. vannamei was significantly higher than the control population for L. vannamei. The effect of LvPAH on L. vannamei from a neuroendocrinological perspective was assessed by measuring l-DOPA, dopamine (DA) and noradrenaline (NE) levels in the hemocytes after the knockdown of LvPAH. The results showed that phenoloxidase (PO), l-DOPA and DA levels in the hemolymph of LvPAH-silenced L. vannamei were significantly decreased starting from 24hpi. In contrast, the NE levels in the hemolymph of shrimp decreased significantly at first and then increased. The results suggest that LvPAH may play an important role in antiviral and bacterial immunity in L. vannamei.

Inhibitory Effects of Cinnamaldehyde Derivatives on Biofilm Formation and Virulence Factors in Vibrio Species.

Vibrio parahaemolyticus is considered one of the most relevant pathogenic marine bacteria with a range of virulence factors to establish food-related gastrointestinal infections in humans. Cinnamaldehyde (CNMA) and some of its derivatives have antimicrobial and antivirulence activities against several bacterial pathogens. This study examined the inhibitory effects of CNMA and its derivatives on biofilm formation and the virulence factors in Vibrio species, particularly V. parahaemolyticus. CNMA and ten of its derivatives were initially screened against V. parahaemolyticus biofilm formation, and their effects on the production of virulence factors and gene expression were studied. Among the CNMA derivatives tested, 4-nitrocinnamaldehyde, 4-chlorocinnamaldehyde, and 4-bromocinnamaldehyde displayed antibacterial and antivirulence activities, while the backbone CNMA had weak effects. The derivatives could prevent the adhesion of V. parahaemolyticus to surfaces by the dose-dependent inhibition of cell surface hydrophobicity, fimbriae production, and flagella-mediated swimming and swarming phenotypes. They also decreased the protease secretion required for virulence and indole production, which could act as an important signal molecule. The expression of QS and biofilm-related genes (aphA, cpsA, luxS, and opaR), virulence genes (fliA, tdh, and vopS), and membrane integrity genes (fadL, and nusA) were downregulated in V. parahaemolyticus by these three CNMA analogs. Interestingly, they eliminated V. parahaemolyticus and reduced the background flora from the squid surface. In addition, they exhibited similar antimicrobial and antibiofilm activities against Vibrio harveyi. This study identified CNMA derivatives as potential broad-spectrum antimicrobial agents to treat biofilm-mediated Vibrio infections and for surface disinfection in food processing facilities.

Physiological and antioxidant response of Litopenaeus vannamei against Vibrio parahaemolyticus infection after feeding supplemented diets containing Dunaliella sp. flour and β-glucans

The presence of pathogen agents in shrimp farming is the main obstacle for successful aquaculture. Vibrio species are naturally part of water because they play an important role as opportunistic bacteria. Vibrio parahaemolyticus was identified as the causative agent of the Early Mortality Syndrome in 2009, causing the loss of shrimp farming worldwide. Dunaliella sp. flour has been tested against Vibrio infection proving to be an effective prophylactic method that decreases mortality and improves physiological and immune response in Litopenaeus vannamei. Juvenile shrimp were exposed to 2% Dunaliella sp. flour and commercial 1.1% β -glucan diet provided every other day for 15 days and a posterior infection with V. parahaemolyticus (1 × 106 CFU/mL). To evaluate shrimp stress status, some parameters as glucose, lactate, cholesterol, triglycerides, relative superoxide dismutase (SOD) gene expression and circulating hemocytes were analyzed in hemolymph at zero and seven days before infection and at 0, 24, and 48 h post-infection. L. vannamei fed with Dunaliella sp. showed 93% and β -glucan 87% survival, compared with 79% in the infected control group. Additionally, Dunaliella sp. improved hemocyte and lipid concentrations compared to β -glucan while both immunostimulants showed an increase in SOD response against bacteria. The addition of 2% Dunaliella sp. every other day in L. vannamei diet enhanced stress response against V. parahaemolyticus infection.

FADD regulates antibacterial immune responses via the immune deficiency signaling pathway in the Chinese mitten crab

In invertebrates, innate immune responses are the only defense against invading pathogens. The immune deficiency (IMD) signaling pathway protects invertebrates from bacterial infection by secreting antimicrobial peptides (AMPs). Fas-associated protein with death domain (FADD) activates AMPs and triggers apoptosis. However, FADD's function in crustaceans is unclear. Herein, the full-length FADD cDNA (EsFADD) was cloned from the Chinese mitten crab, Eriocheir sinensis. Vibrio parahaemolyticus infection upregulated EsFADD expression markedly. Knockdown of EsFADD in hemocytes suppressed the cytoplasm-to-nucleus translocation of transcription factor Relish under V. parahaemolyticus stimulation, which in turn reduced the expression of several AMPs. In vivo, silencing of EsFADD rendered crabs susceptible to bacterial infection and impaired their bacterial clearance. The results suggest that EsFADD is indispensable in IMD signal transduction in E. sinensis. In contrast to Drosophila, EsFADD barely promoted apoptosis. Our findings revealed the evolutionary conservation of FADD in crustaceans and provided insights into IMD signaling in invertebrates.

The immune function of a NLR like gene, LvNLRPL1, in the Pacific whiteleg shrimp Litopenaeus vannamei

NOD-like receptors (NLRs) are a kind of pattern recognition receptors, which are vital for detection of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and then trigger downstream immune responses in vertebrates. Although many NLR like genes have been identified in invertebrates in recent years, knowledge about their immune functions is still very limited. In the present study, a NLR like gene, designated as LvNLRPL1, was identified in Litopenaeus vannamei. It was widely expressed in multiple tissues and responsive to the infection of Vibrio parahaemolyticus. Knockdown of LvNLRPL1 could accelerate the proliferation of Vibrio in hepatopancreas and increase the mortality rate of shrimp after Vibrio infection. Meanwhile, knockdown of LvNLRPL1 also up-regulated the expression of Caspase 2, 3 and 5 in hemocytes, which caused apoptosis of more hemocytes. These results indicated that LvNLRPL1 played important immune functions in shrimp during Vibrio infection through regulating the apoptosis of hemocytes in shrimp. To our knowledge, this is the first time to reveal the immune function of a NLR like gene in crustaceans.

Changes in gluconeogenesis pathways and key genes associated with mass mortality in the clam Meretrix petechialis upon Vibrio infection

Mass mortality events frequently occur in marine bivalves growing in natural habitats and artificial culture environments due to pathogen infection. The clam Meretrix petechialis is an important commercial bivalve species, and the clam culture industry has suffered frequent mass mortality events during summer. However, the pathogenesis of the host is still poorly understood. In this study, M. petechialis was challenged with Vibrio parahaemolyticus, and the clams were sampled at different infection phases and physiological statuses. The transcriptome analysis of the clam hepatopancreas in the onset phase of Vibrio challenge showed the significant enrichment of multiple metabolism-related signaling pathways, including peroxisome proliferator-activated receptors (PPARs), adenosine 5′-monophosphate activated protein kinase (AMPK), phosphatidylinositol 3′-kinase (PI3K)-Akt signaling pathways and glycolysis/gluconeogenesis, which revealed the involvement of metabolism-related signaling pathways in host immune defense. Weighted correlation network analysis (WGCNA) revealed that key enzymes involved in gluconeogenesis were enriched in this phase, suggesting that gluconeogenesis may be associated with infection-induced mortality in clams. Examinations of the glucose content, phosphoenolpyruvate carboxykinase (PEPCK) activity and the expression of key genes involved in gluconeogenesis showed that gluconeogenesis was repressed during Vibrio infection. An analysis of the expression of eight key genes in the PI3K-Akt signaling pathway between moribund and surviving individuals implied that gluconeogenesis was related to clam mass mortality. The results might help us to understand the molecular mechanism of death outbreak events and provide potential markers to detect the physiological status of this clam species.

Heatwave-associated Vibrio infections in Germany, 2018 and 2019.

Background Vibrio spp. are aquatic bacteria that prefer warm seawater with moderate salinity. In humans, they can cause gastroenteritis, wound infections, and ear infections. During the summers of 2018 and 2019, unprecedented high sea surface temperatures were recorded in the German Baltic Sea.AimWe aimed to describe the clinical course and microbiological characteristics of Vibrio infections in Germany in 2018 and 2019.MethodsWe performed an observational retrospective multi-centre cohort study of patients diagnosed with domestically-acquired Vibrio infections in Germany in 2018 and 2019. Demographic, clinical, and microbiological data were assessed, and isolates were subjected to whole genome sequencing and antimicrobial susceptibility testing.ResultsOf the 63 patients with Vibrio infections, most contracted the virus between June and September, primarily in the Baltic Sea: 44 (70%) were male and the median age was 65 years (range: 2–93 years). Thirty-eight patients presented with wound infections, 16 with ear infections, six with gastroenteritis, two with pneumonia (after seawater aspiration) and one with primary septicaemia. The majority of infections were attributed to V. cholerae (non–O1/non-O139) (n = 30; 48%) or V. vulnificus (n = 22; 38%). Phylogenetic analyses of 12 available isolates showed clusters of three identical strains of V. vulnificus, which caused wound infections, suggesting that some clonal lines can spread across the Baltic Sea.ConclusionsDuring the summers of 2018 and 2019, severe heatwaves facilitated increased numbers of Vibrio infections in Germany. Since climate change is likely to favour the proliferation of these bacteria, a further increase in Vibrio-associated diseases is expected.

Immune correlates of NF-κB and TNFα promoter DNA methylation in Japanese flounder (Paralichthys olivaceus) muscle and immune parameters change response to vibrio anguillarum infection

Vibrio anguillarum infection can activate NF-κB/TNFα pathway in the immune organs of fish. Fish muscle is also an important immune organ, but the research on its immune function is few. Our aim was to study regulating mechanism of NF-κB and TNFα gene expressions in the muscle of Japanese flounder (Paralichthys olivaceus) which was under Vibrio anguillarum infection (0, 24, 48, 72 and 96h). The results showed that the expressions of NF-κB and TNFα increased significantly at 48h, and there was a significant positive correlation between them. In situ hybridization confirmed the co-existence of NF-κB and TNFα genes in Japanese flounder muscle. Interestingly, the expression of the TNFα gene was regulated by the DNA methylation and its methylation level was negatively correlated with the expression. The lowest methylation level of TNFα occurred at 48h under Vibrio anguillarum infection (P<0.05). And more, when the fragment (-2122∼-730) was deleted on TNFα gene promoter, double luciferase activity was the highest, indicating that fragment (-730-0) was the transcription factor binding region. The site (-78 ~ -69) on the fragment (-730-0) binding NF-κB was mutated, and double luciferase activity decreased significantly. The results confirmed that the site (-78 ~ -69) was indeed an important binding site for NF-κB. In addition, the activity of TNFα in the serum of Japanese flounder changed with the prolongation of vibrio anguillarum infection, and the concentration of other immune factors such as ALP, ALT, AST and LDH also changed in the muscle under vibrio anguillarum infection. They all showed a trend of first increasing and then decreasing. Above studies implied that Japanese flounder responded to Vibrio anguillarum infection at the immune level with the change of its methylation status and the activation of transcription factor. By studying the mechanism of immune pathways, understanding the response to immune stress is great significant to the research of fish breeding for disease resistance.

Salmonella - foodborne pathogen and antimicrobial resistance

Foodborne diseases encompass a wide spectrum of illnesses and are a growing public health problem worldwide. They are caused by consumption of food or water contaminated by pathogenic (disease-causing) microorganisms such as bacteria, viruses and parasites. The contamination of food can occur at any stage in the process from food production to consumption (“farm to fork”) and can result from environmental contamination (water, soil or air). They enter the body through the gastrointestinal tract where the first symptoms often occur like nausea, vomiting, stomach cramps, and diarrhoea. However, symptoms differ among the different types of foodborne diseases and the patient’s immune status. Symptoms can sometimes be severe and some foodborne illnesses can even be fatal. Commonly recognized foodborne infections are: campylobacteriosis, Escherichia coli O157:H7 infection and haemolytic uremic syndrome (HUS), salmonellosis, cryptosporidiosis, listeriosis, giardiasis. norovirus infection, scombroid fish poisoning, shigellosis, toxoplasmosis, Vibrio infection and yersiniosis. One of the top three germs that cause illnesses from food eaten in EU is Salmonella.

Activation of TLR2 heterodimers-mediated NF-κB, MAPK, AKT signaling pathways is responsible for Vibrio alginolyticus triggered inflammatory response in vitro

Vibrio alginolyticus is an important zoonotic marine pathogenic bacterium. Previous studies on the mechanism of innate immune against V. alginolyticus infection have been limited to aquatic animals, however, how V. alginolyticus activates mammalian immune cells has not been fully clarified. Here, ELISA combined RT-qPCR assays were used to detect the secretion and transcription level of pro-inflammatory cytokines and TLRs during V. alginolyticus infection of mice peritoneal macrophages (PMϕs). Western blotting was used to explore the phosphorylation levels of p38, JNK, ERK, AKT and NF-κB protein. Immunofluorescence assay was used to determine the location of NF-κB protein. Inhibition assay was used to study the role of up-regulated TLR in activated signaling pathways and the role of these pathways in the release of pro-inflammatory cytokines. Our data showed that V. alginolyticus can up-regulate the expression levels of IL-1β, IL-6, IL-12 and TNF-α in PMϕs. In addition, V. alginolyticus stimulation activated the phosphorylation of p38, JNK and ERK were TLR2 heterodimers-dependent, whereas inhibitors of SB203580 (p38), SCH772984 (ERK) and SP600125 (JNK) significantly reduced IL-1β, IL-6, IL-12 and TNF-α production. We further revealed that V. alginolyticus activated the signaling pathways of AKT via TLR2 heterodimers. The inhibitor of MK-2206 2HCl (AKT) negatively regulated the IL-1β, IL-6 and TNF-α release levels. Moreover, V. alginolyticus infection of PMϕs resulted in TLR2 heterodimers-mediated activation of NF-κB and induced translocation of phosphorylated NF-κB protein from the cytoplasm into the nucleus via IκBα degradation. V. alginolyticus induced IL-1β, IL-6, IL-12 and TNF-α release were blocked by the specific NF-κB inhibitor, BAY 11–7082. Taken together, our results suggested that activation of the TLR2 heterodimers-mediated downstream signaling pathways NF-κB, MAPK and AKT is responsible for inflammatory response during Vibrio alginolyticus infection in vitro.

Cloning and characterization of a key enzyme in octopaminergic pathway: Tyramine beta-hydroxylase from Litopenaeus vannamei, as expressed during Vibrio alginolytics infection and hypothermal stress

Tyramine beta-hydroxylase (TBH) is needed for the biosynthesis of the octopamine (OA) from tyramine (TA). Both OA and TA act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. In this study, TBH was identified in white shrimp, Litopenaeus vannamei, and further investigation on its potential function was conducted after inducing hypothermal stress and Vibrio alginolyticus infection. TBH of L. vannamei (LvTBH) was comprised 2178 nucleotide residues and contained an open reading frame encoding 408 amino acids, belonging to the Copper type II, ascorbate-dependent monooxygenases, was characterized by two Cu2_monooxygen domains and five glycosylation sites. LvTBH expression was especially abundant in muscle, and mainly in brain and thoracic ganglia of nervous system, eyestalk tissues, epithelium, and stomach, as determined by quantitative real-time PCR. The effects of hypothermal stress showed significant increases in LvTBH at 15, 30 and 60 min in brain and at 30 min in haemocyte, accompanied by an increase in OA level in haemolymph from 15 to 60 min. Significant increases in LvTBH occurred at 15, 30 and 60 min in haemocyte and at 60 min in brain tissue, and was proportional to the OA level of haemolymph under Vibrio alginolyticus infection from 30 to 60 min. Here, we demonstrated that LvTBH is functionally responsible for biogenic amine synthesis, suggesting that the increased release of OA in haemolymph for potential modulation of physiological and immunological responses is the consequence of the upregulated LvTBH gene expression in L. vannamei exposed to hypothermal stress and Vibrio alginolyticus infection.

Identification, antibacterial activities and action mode of two macins from manila clam Venerupis philippinarum

In this study, two macins were identified from clam Venerupis philippinarum (designated as VpMacin-1 and VpMacin-2). They showed 64.71% similarity with each other. The highest mRNA expression of VpMacin-1 and VpMacin-2 was detected in gills and hepatopancreas, respectively, in non-stimulated clams, and their expression could be induced significantly in hemocytes after Vibrio anguillarum infection. Silencing of VpMacin-1 and VpMacin-2 led to 22% and 49% mortality 6 days post infection. Escherichia coli cells were killed by recombinant protein rVpMacin-1 and rVpMacin-2 within 1000 and 400 min, respectively, at a concentration of 1.0 × MIC. Compared with rVpMacin-1, rVpMacin-2 not only showed higher broad-spectrum antimicrobial activities towards Vibrio strains, but possessed stronger abilities to inhibit the formation of bacterial biofilm. Both membrane integrity and electrochemical assay indicated that rVpMacins were capable of causing bacterial membrane permeabilization, especially for rVpMacin-2. Besides, rVpMacin-1 significantly induced both phagocytic (0.1 and 1.0 × MIC, p < 0.05) and chemotactic effects (0.1 × MIC, p < 0.01) of hemocytes, while there was no significant increase for rVpMacin-2. Overall, our results suggested that VpMacin-1 and VpMacin-2 play important roles in host defense against invasive pathogens.