Vibrio Campbellii Research Articles

Indole-3-acetic acid increases the survival of brine shrimp challenged with vibrios belonging to the Harveyi clade.

Vibrios belonging to the Harveyi clade (including closely related species such as Vibrio campbellii, Vibrio harveyi and Vibrio parahaemolyticus) are important pathogens of aquatic organisms. In this study, we investigated the use of indole-3-acetic acid to control disease caused by Harveyi clade vibrios. Indole-3-acetic acid, which can be produced by various seaweeds and microalgae, was added to the rearing water of brine shrimp larvae challenged with 12 different Harveyi clade Vibrio strains. Indole-3-acetic acid significantly decreased the virulence of 10 of the strains without any effect on their growth. The latter is important as it will minimize the selective pressure for resistance development. The survival rate of brine shrimp larvae increased from 1.2-fold to 4.8-fold upon treatment with 400 μM indole-3-acetic acid. Additionally, indole-3-acetic acid significantly decreased the swimming motility in 10 of the strains and biofilm formation in eight of the strains. The mRNA levels of the pirA and pirB toxin genes were decreased to 46% and 42% by indole-3-acetic acid in the AHPND-causing strain V.parahaemolyticus M0904. Hence, our data demonstrate that indole-3-acetic acid has the potential to be an effective virulence inhibitor to control infections in aquaculture.

Phytochemical Research and Evaluation of Tarragon (Artemisia dracunculus L.) as a Food Additive

In this study, tarragon (Artemisia dracunculus L.) the amount of antioxidants and phenolic substances of water-based extracts in various concentrations (25g/L, 50g/L, 100g/L) with the aroma components of the plant was determined. Various food pathogens (Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Listeria monocytogenes ATCC 43251) and bacterial strains ((Gram-negative (Vibrio harveyi (KF443058), Vibrio vulnificus (KF443056), Aeromonas veronii (KF443053), Vibrio anguillarum (NR 029254.1) and Vibrio campbellii (MH231447.1), Vibrio rotiferianus (NR 042081.1), Vibrio ponticus (NR 029032.1), Psychrobacter marincola (NR 025458.1), Pseudoalteromonas prydzensis (NR 044803.1), Pseudoalteromonas mariniglutinosa (NR 028992.1) and Gram-positive (Bacillus thuringiensis (NR 043403.1)) obtained from naturally infected Dicentrarchus labrax fish were determined by the disk diffusion method on their antimicrobial properties. As a result of the study, antioxidant values were found to be 88.5% at maximum concentrations of 10%, while the total phenolic substance content was determined between 3.75-5.06 mg GAE/g values. The main component of the tarragon plant was terpinyl acetate (23.16%), followed by α-terpineol (20.08%), anethole-(Z) (8.93%), limonene (5.20%), spathulenol (4.47%), ısoeugenol (3.73%), valeric acid (3.40%), eucalyptol (3.26%). No antimicrobial activity was determined on the test microorganisms used in the study.

ML364 exerts the broad-spectrum antivirulence effect by interfering with the bacterial quorum sensing system.

Antivirulence strategy has been developed as a nontraditional therapy which would engender a lower evolutionary pressure toward the development of antimicrobial resistance. However, the majority of the antivirulence agents currently in development could not meet clinical needs due to their narrow antibacterial spectrum and limited indications. Therefore, our main purpose is to develop broad-spectrum antivirulence agents that could target on both Gram-positive and Gram-negative pathogens. We discovered ML364, a novel scaffold compound, could inhibit the productions of both pyocyanin of Pseudomonas aeruginosa and staphyloxanthin of Staphylococcus aureus. Further transcriptome sequencing and enrichment analysis showed that the quorum sensing (QS) system of pathogens was mainly disrupted by ML364 treatment. To date, autoinducer-2 (AI-2) of the QS system is the only non-species-specific signaling molecule that responsible for the cross-talk between Gram-negative and Gram-positive species. And further investigation showed that ML364 treatment could significantly inhibit the sensing of AI-2 or its nonborated form DPD signaling in Vibrio campbellii MM32 and attenuate the biofilm formation across multi-species pathogens including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus. The results of molecular docking and MM/GBSA free energy prediction showed that ML364 might have higher affinity with the receptors of DPD/AI-2, when compared with DPD molecule. Finally, the in vivo study showed that ML364 could significantly improve the survival rates of systemically infected mice and attenuate bacterial loads in the organs of mice. Overall, ML364 might interfere with AI-2 quorum sensing system to exert broad-spectrum antivirulence effect both in vitro and in vivo.

Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195–0.39 μM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

The prevalence of potential pathogens in ballast water and sediments of oceangoing vessels and implications for management

Ballast water and sediments can serve as prominent vectors for the widespread dispersal of pathogens between geographically distant areas. However, information regarding the diversity and distribution of the bacterial pathogens in ballast water and sediments is highly limited. In this study, using high-throughput sequencing and quantitative PCR, we investigated the composition and abundance of potential pathogens, and their associations with indicator microorganisms. We accordingly detected 48 potential bacterial pathogens in the assessed ballast water and sediments, among which there were significant differences in the compositions and abundances of pathogenic bacterial communities characterizing ballast water and sediments. Rhodococcus erythropolis, Bacteroides vulgatus, and Vibrio campbellii were identified as predominant pathogens in ballast water, whereas Pseudomonas stutzeri, Mycobacterium paragordonae, and Bacillus anthracis predominated in ballast sediments. Bacteroidetes, Vibrio alginolyticus, Vibrio parahaemolyticus, and Escherichia coli were generally detected with median values of 8.54 × 103−1.22 × 107 gene copies (GC)/100 mL and 1.16 × 107−3.97 × 109 GC/100 g in ballast water and sediments, respectively. Notably, the concentrations of Shigella sp., Staphylococcus aureus, and V. alginolyticus were significantly higher in ballast sediments than in the water. In addition, our findings tend to confirm that the indicator species specified by the International Maritime Organization (IMO) might underestimate the pathogen risk in the ballast water and sediments, as these bacteria were unable to predict some potential pathogens assessed in this study. In summary, this study provides a comprehensive insight into the spectrum of the potential pathogens that transferred by ship ballast tanks and emphasizes the need for the implementation of IMO convention on ballast sediment management.

Indole analogues decreasing the virulence of Vibrio campbellii towards brine shrimp larvae.

Indole signalling has been proposed as a potential target for the development of novel virulence inhibitors to control bacterial infections. However, the major structural features of indole analogues that govern antivirulence activity remain unexplored. Therefore, we investigated the impact of 26 indole analogues on indole-regulated virulence phenotypes in Vibrio campbellii and on the virulence of the bacterium in a gnotobiotic brine shrimp model. The results demonstrated that 10 indole analogues significantly increased the fluorescence of indole reporter strain Vibrio cholerae S9149, 21 of them decreased the swimming motility of V. campbellii, and 13 of them significantly decreased the biofilm formation of V. campbellii. Further, we found that 1-methylindole, indene, 2,3-benzofuran, thianaphthene, indole-3-acetonitrile, methyl indole-3-carboxylate, 3-methylindole, and indole-2-carboxaldehyde exhibited a significant protective effect on brine shrimp larvae against V. campbellii infection, resulting in survival rates of challenged brine shrimp above 80%. The highest survival of shrimp larvae (98%) was obtained with indole-3-acetonitrile, even at a relatively low concentration of 20 μM. Importantly, the indole analogues did not affect bacterial growth, both in vitro and in vivo. These results indicate the potential of indole analogues in applications aiming at the protection of shrimp from vibriosis.

Antibacterial Activity of Bacillus Strains against Acute Hepatopancreatic Necrosis Disease-Causing Vibrio campbellii in Pacific White Leg Shrimp

Acute hepatopancreatic necrosis disease (AHPND) is a bacterial disease caused by Vibrioparahaemolyticus. Currently, various Vibrio strains, including V. campbellii, V. owensii, and V. harveyi, have been reported as causative pathogens. Thus, controlling AHPND to maintain high production in shrimp aquaculture is difficult. We evaluated the antimicrobial activity of five Bacillus strains (B1, B3, B5, B7, and B8)—isolated from seawater in Jeju, South Korea—against 12 Vibrio strains (10 AHPND strains and 2 non-AHPND strains). All tested Bacillus strains inhibited the growth of at least one of the tested Vibrio strains in the dot-spot method. Among them, B1 and B3, the most effective Bacillus strains against the Vibrio strains, particularly against AHPND-causing V. campbellii (VcAHPND), were further used in a challenge test. After 48–60 h of VcAHPND immersion, a significantly higher survival rate was observed in the B1-treated group (100%) than in the non-Bacillus-treated group (64.3%). Based on the qPCR analysis of AHPND, the cycle threshold values were 31.63 ± 0.2 (B1-treated group) and 38.04 ± 0.58 (B3-treated group), versus 28.70 ± 0.42 in the control group. Genome sequencing and phylogenetic analysis revealed that B1 and B3 were classified as B. velezensis. The 16S rRNA sequences and complete genome sequences of B1 and B3 were deposited in GenBank.

Halogenated Indoles Decrease the Virulence of Vibrio campbellii in a Gnotobiotic Brine Shrimp Model.

ABSTRACTIndole signaling is viewed as a potential target for antivirulence therapy against antibiotic-resistant pathogens because of its link with the production of virulence factors. This study examined the antimicrobial and antivirulence properties of 44 indoles toward Vibrio campbellii. Based on the results, 17 halogenated indole analogues were selected, as they significantly improved the survival of brine shrimp larvae challenged with V. campbellii. Specifically, 6-bromoindole, 7-bromoindole, 4-fluoroindole, 5-iodoindole, and 7-iodoindole showed a high protective effect, improving the survival of brine shrimp to over 80% even at a low concentration of 10 μM. To explore the impact of selected indole analogues on bacterial virulence phenotypes, swimming motility, biofilm formation, protease activity, and hemolytic activity of V. campbellii were determined. The results showed that all of the 17 selected indole analogues decreased swimming motility at both 10 μM and 100 μM. Most of the indole analogues decreased biofilm formation at a concentration of 100 μM. In contrast, only a slightly decreased protease activity and no effect on hemolytic activity were observed at both concentrations. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system, and the results demonstrate the potential of these compounds in applications aiming at the protection of shrimp from vibriosis, a major disease in aquaculture.IMPORTANCE Bacterial diseases are a major problem in the aquaculture industry. In order to counter this problem, farmers have been using antibiotics, and this has led to the evolution and spread of antibiotic resistance. In order for the aquaculture industry to further grow in a sustainable way, novel and sustainable methods to control diseases are needed. We previously reported that indole signaling is a valid target for the development of novel therapies to control disease caused by Vibrio campbellii and related bacteria, which are among the major bacterial pathogens in aquaculture. In the present study, we identified indole analogues that are more potent in protecting brine shrimp (a model organism for shrimp) from V. campbellii. To our knowledge, this is the first study of the structure-activity relation of halogenated indole analogues with respect to virulence inhibition of a pathogenic bacterium in an in vivo host model system.

The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy

Biotin (vitamin B7) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world’s oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*106 molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.

Chitosan-harvested polyhydroxybutyrate-rich Zobellella denitrificans ZD1 as a multifunction feed for aquaculture

The security of the aquaculture industry is jeopardized by antibiotics overuse and increasing demand for aquaculture feeds, which remain heavily derived from capture fisheries. Biopolymer poly(3-hydroxybutyrate) (PHB) and some microorganisms are promising replacements for antibiotics and aquaculture feed, respectively. Therefore, we investigated the potential of using chitosan-harvested PHB-rich Zobellella denitrificans ZD1 (designated as CP-ZD1 hereafter) as a multifunction feed for brine shrimp Artemia (model species). Both PHB and chitosan and their intermediates (3-hydroxybutyrate and chitosan oligosaccharides) showed antimicrobial properties toward three known aquaculture pathogens (Vibrio campbellii, Aeromonas hydrophila, and Streptococcus agalactiae) and non-pathogens (non-pathogenic Escherichia coli, Bacillus megaterium, and Rhodococcus jostii RHA1). Low doses of 3-hydroxybutyrate and chitosan oligosaccharides mixtures effectively suppressed the growth of the pathogens. When supplied with CP-ZD1, the starved Artemia showed prolonged survival and a healthy gut microbiome, suggesting that CP-ZD1 can serve as a feed/energy source. Up to 80% enhanced survival and immune response was observed in pathogenic V. campbellii-challenged Artemia when fed with CP-ZD1. Overall, the results showed that CP-ZD1 could serve as an efficient biocontrol agent, a feed/energy source, and an immunostimulant in support of sustainable aquaculture.

On the origin of amphi-enterobactin fragments produced by Vibrio campbellii species

Amphi-enterobactin is an amphiphilic siderophore isolated from a variety of microbial Vibrio species. Like enterobactin, amphi-enterobactin is a triscatecholate siderophore; however, it is framed on an expanded tetralactone core comprised of four l-Ser residues, of which one l-Ser is appended by a fatty acid and the remaining l-Ser residues are appended by 2,3-dihydroxybenzoate (DHB). Fragments of amphi-enterobactin composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA have been identified in the supernatant of Vibrio campbellii species. The origin of these fragments has not been determined, although two distinct isomers could exist for 2-Ser-1-DHB-FA and three distinct isomers could exist for 3-Ser-2-DHB-FA. The fragments of amphi-enterobactin could originate from hydrolysis of the amphi-enterobactin macrolactone, or from premature release due to an inefficient biosynthetic pathway. Unique masses in the tandem MS analysis establish that certain fragments isolated from the culture supernatant must originate from hydrolysis of the amphi-enterobactin macrolactone, while others cannot be distinguished from premature release during biosynthesis or hydrolysis of amphi-enterobactin.Graphical abstract

Bioactivity of orange-spotted grouper (Epinephelus coioides) interleukin-6 in innate immunity: Inducing inflammation, antimicrobial peptides, and innate immune molecular gene expression as well as activating phagocytosis of leukocytes and increasing survival under Vibrio and NNV infection

Interleukin-6 (IL-6) is a multifunctional cytokine involved in innate and acquired immunity. In mammals, IL-6 can induce the secretion of acute-phase proteins (APPs) and antimicrobial peptides, activate macrophages and neutrophils, and regulate the expression of inflammatory cytokines in innate immunity, resulting in enhanced resistance to invading pathogens. In teleost fish, the functions of IL-6 are still unclear. In our previous studies using orange-spotted grouper (Epinephelus coioides) as an animal model, we demonstrated that IL-6 induces the Th2 pathway in acquired immunity and enhances antibody production. In the current study, we evaluated in greater detail the mechanisms underlying the functions of IL-6 in innate immunity. In gene expression assays, the expression of fish inflammatory cytokine genes (e.g., ifnγ, ccl4, il1β, tnfα 1, and tnfα 2), antimicrobial peptide genes (e.g., epinecidin 1, hepcidin 1, and hepcidin 2), and genes related to innate immunity (e.g., mx, crp, and complement C3) was higher in fish injected with recombinant IL-6 (rIL-6). The serum from those same fish also presented higher antibacterial activity and higher phagocytosis activity of leukocytes. In a pathogen challenge involving Vibrio campbellii and nervous necrosis virus, fish injected with rIL-6 presented a higher survival rate and lower pathogen count in the body. These results demonstrate that the administration of rIL-6 can activate innate immune responses and enhance resistance against invading bacterial or viral pathogens.

Induction of transgenerational innate immune memory against Vibrio infections in a brine shrimp (Artemia franciscana) model

The occurrence of transgenerational innate immune memory in invertebrates possibly offers an opportunity for the creation of offspring with increased resistance against infectious diseases. Shrimps, being invertebrates rely on their innate immune system as the main defence mechanism to combat invading pathogens. Thus, creating transgenerational innate immune memory could be used for disease prevention in shrimp farming. However, the mechanisms behind the induction of transgenerational innate immune memory and the creation of a broad spectrum protective immune memory are still not fully understood. Here, the creation of transgenerational innate immune memory and the mechanisms involved were studied using the brine shrimp (Artemia franciscana) as a model for shrimps. Hereto, a parental generation was primed by exposure to a live or dead Vibrio parahaemolyticus PV1 while another parental generation was primed with live or dead Vibrio campbellii LMG21363. Both Vibrio strains are known to cause diseases in farmed shrimps, resulting in significant economic losses. Axenic larvae of three subsequent generations were exposed either to the homologous or heterologous (viable) Vibrio species and their survival was recorded. In general, progenies from primed F0 parents developed innate immune memory as compared to offspring of non-primed control F0 parents as they were significantly protected against a subsequent Vibrio infection. Protection in the infected offspring was more pronounced when they were infected with the homologous Vibrio strain. Disease resistance was associated with significantly increased transcription of especially peroxinectin (pxn), prophenoloxidase (proPO), lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), high mobility group box 1 (HMGB1) and heat shock protein 90 (hsp90) genes and significant changes in histone H3 and H4 acetylation and m6A RNA methylation. Overall, results might lead to procedures at shrimp brood stock level for parental conditioning and creation of transgenerational innate immune memory in offspring.

Proteome Expression and Survival Strategies of a Proteorhodopsin-Containing Vibrio Strain under Carbon and Nitrogen Limitation.

ABSTRACTPhotoheterotrophy is a widespread mode of microbial metabolism, notably in the oligotrophic surface ocean, where microbes experience chronic nutrient limitation. One especially widespread form of photoheterotrophy is based on proteorhodopsin (PR), which uses light to generate proton motive force that can drive ATP synthesis, flagellar movement, or nutrient uptake. To clarify the physiological benefits conferred by PR under nutrient stress conditions, we quantified protein-level gene expression of Vibrio campbellii CAIM 519 under both carbon and nitrogen limitation and under both light and dark conditions. Using a novel membrane proteomics strategy, we determined that PR expression is higher under C limitation than N limitation but is not light regulated. Despite expression of PR photosystems, V. campbellii does not exhibit any growth or survival advantages in the light and only a few proteins show significant expression differences between light and dark conditions. While protein-level proteorhodopsin expression in V. campbellii is clearly responsive to nutrient limitation, photoheterotrophy does not appear to play a central role in the survival physiology of this organism under these nutrient stress conditions. C limitation and N limitation, however, result in very different survival responses: under N-limited conditions, viability declines, cultivability is lost rapidly, central carbon flux through the Entner-Doudoroff pathway is increased, and ammonium is assimilated via the GS-GOGAT pathway. In contrast, C limitation drives cell dwarfing with maintenance of viability, as well as utilization of the glyoxylate shunt, glutamate dehydrogenase and anaplerotic C fixation, and a stringent response mediated by the Pho regulon.IMPORTANCE Understanding the nutrient stress responses of proteorhodopsin-bearing microbes like Vibrio campbellii yields insights into microbial contributions to nutrient cycling, lifestyles of emerging pathogens in aquatic environments, and protein-level adaptations implemented during times of nutrient limitation. In addition to its broad taxonomic and geographic prevalence, the physiological role of PR is diverse, so we developed a novel proteomics strategy to quantify its expression at the protein level. We found that proteorhodopsin expression levels in this wild-type photoheterotroph under these experimental conditions, while higher under C than under N limitation, do not afford measurable light-driven growth or survival advantages. Additionally, this work links differential protein expression patterns between C- and N-limited cultures to divergent stationary-phase survival phenotypes.

Identification, characterization and infection dynamics of Vibrio strains in phyllosoma of Thenus unimaculatus

ABSTRACT The present study investigates Vibrio outbreak in the hatchery system designed for larval rearing of shovel-nosed lobster Thenus unimaculatus in India. High larval mortalities during the episode led to an almost complete loss of the larval stock during the first 10 days. Microscopic examination revealed heavy fouling of the phyllosoma appendages. Histopathological analysis showed the presence of bacteria in larval tissues. Investigation by scanning electron microscopy revealed the formation of bacterial plaques on appendages and adhesion of bacteria on fine setae in heavily infected larvae. Bacteria isolated from larvae and identified by 16S rRNA sequencing and phylogenetic analysis indicated that the strains were closely related to luminescent Vibrio campbellii and Vibrio harveyi. The virulence potential of Vibrio strains isolated from larvae was evaluated through an experimental infection. The study helps to understand microbial infection and its impact on phyllosoma survival and may allow a more effective health management regime to improve larval survival in the hatchery phase.

Eukaryotic catecholamine hormones influence the chemotactic control of Vibrio campbellii by binding to the coupling protein CheW

SignificanceHost-emitted stress hormones significantly influence the growth and behavior of various bacterial species; however, their cellular targets have so far remained elusive. Here, we used customized probes and quantitative proteomics to identify the target of epinephrine and the α-adrenoceptor agonist phenylephrine in live cells of the aquatic pathogen Vibrio campbellii. Consequently, we have discovered the coupling protein CheW, which is in the center of the chemotaxis signaling network, as a target of both molecules. We not only demonstrate direct ligand binding to CheW but also elucidate how this affects chemotactic control. These findings are pivotal for further research on hormone-specific effects on bacterial behavior.

Expression of AHPND toxin genes (pirAB), quorum sensing master regulator gene (luxR) and transmembrane transcriptional regulator gene (toxR) in Vibrio parahaemolyticus and Vibrio harveyi during infection of Penaeus vannamei (Bonne, 1931)

This study aimed to detect the presence of quorum-sensing (QS) signalling molecules and evaluate the in vivo virulence gene expression in shrimp challenged with acute hepatopancreatic necrosis disease (AHPND) positive Vibrio isolates. Three types of QS signal molecules, namely N-acyl-homoserine lactone (AHL), Autoinducer-2 (AI-2) and Cholerae autoinducer-1-like (CAI-1) molecules were detected in AHPND causing isolates using Agrobacterium tumefaciens KYC55, Vibrio campbellii JMH597 and V. campbellii JAF375 biosensors respectively. The presence of QS genes in all the 12 AHPND positive isolates was further justified by conducting molecular screening of the QS-related genes, luxR and luxS. Challenged groups demonstrated a significant (P < 0.05) increase in the expression levels of the QS master regulator gene luxR compared with the unchallenged group. The expression of pirA-, pirB-, toxR- and luxR genes peaked at 36 h in shrimp challenged with AHPND positive Vibrio parahaemolyticus BpShHep31 and V. harveyi BpShHep24. However, compared to the group challenged with V. parahaemolyticus BpShHep31, the V. harveyi BpShHep24 challenged group demonstrated 45.1-, 126.0-, 19.7- and 10.7- fold higher expression of pirA, pirB, toxR and luxR respectively.

Characterization of toxicity and structure of PirABvc -like proteins that are structurally almost identical to shrimp AHPND-causing PirAB toxin.

PirAB is a binary toxic protein that causes acute hepatopancreatic necrosis disease (AHPND) in shrimp. Their closest homologs, PirAvc -like and PirBvc -like proteins, are encoded by two adjacent genes on a non-pVH plasmid from a Vibrio campbellii strain. Herein, PirABvc -like protein caused neither abnormalities nor death in shrimp postlarvae (Litopenaeus vannamei); furthermore, typical AHPND clinical signs were not observed. PirAvc -like protein corresponds to Cry toxin domain III (ligand-binding domain) and likely binds to N-acetylgalactosamine. The C-terminal and N-terminal of PirBvc -like resemble Cry toxin domain II (receptor-binding domain) and domain I (pore-forming domain), respectively. PirAvc -like and PirBvc -like proteins are structurally similar to PirA and PirB, respectively. Subtle structural differences between PirAvc -like protein and PirA appear to be involved in ligand-binding and binary protein complex formation. The difference in virulence of PirABvc -like and PirAB may result from the specific binding of the protein complex to distinct host receptors. These results shed light on the potential functions and host receptors of PirABvc -like proteins and their relationship with PirAB.

Phloroglucinol shows prophylactic and metaphylactic effects against pathogenic stressors in Macrobrachium larvae

Vibriosis caused by Vibrio campbellii and related species is amongst the major hindrance to the sustainable expansion of giant freshwater prawn Macrobrachium rosenbergii larviculture. Induction of heat shock protein Hsp70 is a natural response of stressed organisms that protect against many insults including vibriosis in aquaculture animals. Therefore, there is a great interest in searching for natural compounds that could induce Hsp70 in animals in a non-invasive manner. Previously, in a series of in vivo studies, we have shown that the phenolic compound phloroglucinol could induce Hsp70 in aquaculture organisms Macrobrachium and Artemia. This led to a significant increase in the resistance of the animals towards subsequent challenges with V. parahemolyticus. As V. parahaemolyticus belongs to the Harveyi clade similar to V. campbellii, our above findings triggered the hypothesis that phloroglucinol is a potential anti-microbial agent that could protect the freshwater prawn against V. campbellii infection. The results presented here provide evidence that the Hsp70-inducing compound phloroglucinol could induce both metaphylactic and prophylactic effects against infection stress mediated by V. campbellii. The wide-spectrum property of the compound to both prevent the occurrence and reduce the spread of V. campbellii infection in prawn larvae without affecting the larval growth makes it a potential natural agent for health management and V. campbellii-mediated disease control in freshwater prawn larvae. Overall results add new information about the functional properties of phloroglucinol and advance our knowledge of this compound as a potential antimicrobial agent for the sustainable production of giant freshwater prawns.

The C2 entity of chitosugars is crucial in molecular selectivity of the Vibrio campbellii chitoporin

The marine bacterium Vibrio campbellii expresses a chitooligosaccharide-specific outer-membrane channel (chitoporin) for the efficient uptake of nutritional chitosugars that are externally produced through enzymic degradation of environmental host shell chitin. However, the principles behind the distinct substrate selectivity of chitoporins are unclear. Here, we employed black lipid membrane (BLM) electrophysiology, which handles the measurement of the flow of ionic current through porins in phospholipid bilayers for the assessment of porin conductivities, to investigate the pH dependency of chitosugar–chitoporin interactions for the bacterium’s natural substrate chitohexaose and its deacetylated form, chitosan hexaose. We show that efficient passage of the N-acetylated chitohexaose through the chitoporin is facilitated by its strong affinity for the pore. In contrast, the deacetylated chitosan hexaose is impermeant; however, protonation of the C2 amino entities of chitosan hexaose allows it to be pulled through the channel in the presence of a transmembrane electric field. We concluded from this the crucial role of C2-substitution as the determining factor for chitoporin entry. A change from N-acetylamino- to amino-substitution effectively abolished the ability of approaching molecules to enter the chitoporin, with deacetylation leading to loss of the distinctive structural features of nanopore opening and pore access of chitosugars. These findings provide further understanding of the multistep pathway of chitin utilization by marine Vibrio bacteria and may guide the development of solid-state or genetically engineered biological nanopores for relevant technological applications.