The shrimp nephrocomplex serves as a major portal of pathogen entry and is involved in the molting process.

Viruses, such as white spot syndrome virus, and bacteria, such as Vibrio species, wreak havoc in shrimp aquaculture [C. M. Escobedo-Bonilla et al., J. Fish. Dis. 31, 1-18 (2008)]. As the main portal of entry for shrimp-related pathogens remain unclear, infectious diseases are difficult to prevent and control. Because the cuticle is a strong pathogen barrier, regions lacking cuticular lining, such as the shrimp's excretory organ, "the antennal gland," are major candidate entry portals [M. Corteel et al., Vet. Microbiol. 137, 209-216 (2009)]. The antennal gland, up until now morphologically underexplored, is studied using several imaging techniques. Using histology-based three-dimensional technology, we demonstrate that the antennal gland resembles a kidney, connected to a urinary bladder with a nephropore (exit opening) and a complex of diverticula, spread throughout the cephalothorax. Micromagnetic resonance imaging of live shrimp not only confirms the histology-based model, but also indicates that the filling of the diverticula is linked to the molting cycle and possibly involved therein. Based on function and complexity, we propose to rename the antennal gland as the "nephrocomplex." By an intrabladder inoculation, we showed high susceptibility of this nephrocomplex to both white spot syndrome virus and Vibrio infection compared to peroral inoculation. An induced drop in salinity allowed the virus to enter the nephrocomplex in a natural way and caused a general infection followed by death; fluorescent beads were used to demonstrate that particles may indeed enter through the nephropore. These findings pave the way for oriented disease control in shrimp.

Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections.

Vibrio species cause infectious diseases in humans and animals, but they can also live as commensals within their host tissues. How Vibrio subverts the host defenses to mount a successful infection remains poorly understood, and this knowledge is critical for predicting and managing disease. Here, we have investigated the cellular and molecular mechanisms underpinning infection and colonization of 2 virulent Vibrio species in an ecologically relevant host model, oyster, to study interactions with marine Vibrio species. All Vibrio strains were recognized by the immune system, but only nonvirulent strains were controlled. We showed that virulent strains were cytotoxic to hemocytes, oyster immune cells. By analyzing host and bacterial transcriptional responses to infection, together with Vibrio gene knock-outs, we discovered that Vibrio crassostreae and Vibrio tasmaniensis use distinct mechanisms to cause hemocyte lysis. Whereas V. crassostreae cytotoxicity is dependent on a direct contact with hemocytes and requires an ancestral gene encoding a protein of unknown function, r5.7, V. tasmaniensis cytotoxicity is dependent on phagocytosis and requires intracellular secretion of T6SS effectors. We conclude that proliferation of commensal vibrios is controlled by the host immune system, preventing systemic infections in oysters, whereas the successful infection of virulent strains relies on Vibrio species-specific molecular determinants that converge to compromise host immune cell function, allowing evasion of the host immune system.

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation.

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

Characteristics of Vibrio vulnificus isolates from clinical and environmental sources.

Researchers have developed multiple methods to characterize clinical and environmental strains of Vibrio vulnificus. The aim of our study was to use four assays to detect virulence factors in strains from infected patients and those from surface waters/sediments/oysters of South Carolina and the Gulf of Mexico. Vibrio vulnificus strains from clinical (n = 81) and environmental (n = 171) sources were tested using three real-time PCR methods designed to detect polymorphisms in the 16S rRNA, vcg and pilF genes and a phenotypic method, the ability to ferment D-mannitol. Although none of the tests correctly categorized all isolates, the differentiation between clinical and environmental isolates was similar for the pilF, vcgC/E and 16S rRNA assays, with sensitivities of 74.1-79.2% and specificities of 77.4-82.7%. The pilF and vcgC/E assays are comparable in efficacy to the widely used 16S rRNA method, while the D-mannitol fermentation test is less discriminatory (sensitivity = 77.8%, specificity = 61.4%). Overall percent agreement for the D-mannitol fermentation method was also lower (66.7%) than overall percent agreement for the 3 molecular assays (78.0%-80.2%). This study demonstrated, using a large, diverse group of Vibrio vulnificus isolates, that three assays could be used to distinguish most clinical vs environmental isolates; however, additional assays are needed to increase accuracy.

Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?

The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3-5 million infections worldwide and 28.800-130.000 deaths per year according to the World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non-O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life-threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases, this review aims to highlight the most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario.

Complete genome analysis of a virulent Vibrio scophthalmi strain VSc190401 isolated from diseased marine fish half-smooth tongue sole, Cynoglossus semilaevis.

BACKGROUND: Vibrio scophthalmi is an opportunistic bacterial pathogen, which is widely distributed in the marine environment. Earlier studies have suggested that it is a normal microorganism in the turbot gut. However, recent studies have confirmed that this bacterial strain can cause diseases in many different marine animals. Therefore, it is necessary to investigate its whole genome for better understanding its physiological and pathogenic mechanisms. RESULTS: In the present study, we obtained a pathogenic strain of V. scophthalmi from diseased half-smooth tongue sole (Cynoglossus semilaevis) and sequenced its whole genome. Its genome contained two circular chromosomes and two plasmids with a total size of 3,541,838 bp, which harbored 3185 coding genes. Among these genes, 2648, 2298, and 1915 genes could be found through annotation information in COG, Blast2GO, and KEGG databases, respectively. Moreover, 10 genomic islands were predicted to exist in the chromosome I through IslandViewer online system. Comparison analysis in VFDB and PHI databases showed that this strain had 334 potential virulence-related genes and 518 pathogen-host interaction-related genes. Although it contained genes related to four secretion systems of T1SS, T2SS, T4SS, and T6SS, there was only one complete T2SS secretion system. Based on CARD database blast results, 180 drug resistance genes belonging to 27 antibiotic resistance categories were found in the whole genome of such strain. However, there were many differences between the phenotype and genotype of drug resistance. CONCLUSIONS: Based on the whole genome analysis, the pathogenic V. scophthalmi strain contained many types of genes related to pathogenicity and drug resistance. Moreover, it showed inconsistency between phenotype and genotype on drug resistance. These results suggested that the physiological mechanism seemed to be complex.

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.

Skin proteome profiling of tongue sole (Cynoglossus semilaevis) challenged with Vibrio vulnificus.

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvß, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8ß, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish.

Vibrio parahaemolyticus infection impaired intestinal barrier function and nutrient absorption in Litopenaeus vannamei.

The intestine is the primary target of pathogenic microbes during invasion. However, the interaction of Vibrio parahaemolyticus (V. parahaemolyticus) with intestinal epithelial cells and its effects on the intestinal function of Litopenaeus vannamei (L. vannamei) are poorly studied. Therefore, the aim of this study was to investigate the influence of V. parahaemolyticus infection on intestinal barrier function and nutrient absorption in L. vannamei. In the present study, a total of 90 shrimp were randomly divided into two groups including the control group and V. parahaemolyticus infection group (final concentration of 1 × 105 CFU/mL), with three replicates per group. The result showed that compared with the control group, V. parahaemolyticus infection increased (P < 0.05) serum diamine oxidase activity and endotoxin quantification, and down-regulated (P < 0.05) the mRNA levels of intestinal peroxinectin, integrin, midline fasciclin at 48 h and 72 h; V. parahaemolyticus infection decreased (P < 0.05) the mRNA expression of intestinal amino acid transporter (CAT1, EAAT3 and ASCT1) and glucose transporter (SGLT-1, GLUT) at 24 h, 48 h and 72 h, and increased (P < 0.05) serum glucose and amino acid (Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr, Phe, Lys, His and Arg) concentration at 24 h. The results indicated that V. parahaemolyticus infection increased intestinal permeability, inhibited absorption of glucose and amino acid in L. vannamei.

In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis.

The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

The Rho signalling pathway mediates the pathogenicity of AHPND-causing V. parahaemolyticus in shrimp.

An emerging bacterial disease, acute hepatopancreatic necrosis disease (AHPND), is caused by strains of Vibrio parahaemolyticus with an additional AHPND-associated plasmid pVA1 encoding a virulent toxin (Pirvp ) that damages the shrimp's hepatopancreas. Like other species of Vibrio, these virulent strains initially colonise the shrimp's stomach, but it is not yet understood how the bacteria or toxins are subsequently able to cross the epithelial barrier and reach the hepatopancreas. Here, by using transcriptomics and system biology methods, we investigate AHPND-induced changes in the stomach of AHPND-causing V. parahaemolyticus (5HP)-infected shrimp and identify host molecular mechanisms that might explain how the integrity of the stomach barrier is compromised. We found that the expression of 376 unique genes was differentially regulated by AHPND infection. Gene ontology, protein interaction, and gene-to-gene correlation expression interaction analyses indicated that in addition to the immune system, a number of these genes were involved in cytoskeleton regulation by Rho GTPase. The involvement of Rho pathway regulation during AHPND pathogenesis was further supported by experiments showing that while Rho inhibitor pretreatment delayed the infection, pretreatment with Rho activator enhanced the pathogenicity of 5HP, and both the bacteria and toxin were detected sooner in the hepatopancreas. Further, disruption of the stomach epithelial structure was found in both Rho preactivated shrimp and in 5HP-infected shrimp. Taken together, we interpret our results to mean that Rho signalling helps to mediate AHPND pathogenesis in shrimp.

Epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection: a case report and literature review.

Vibrio vulnificus is a Gram-negative bacterium that belongs to the Vibrionaceae family. It represents a deadly opportunistic human pathogen which grows in water with the proper temperature and salinity, and is mostly acquired from seafood eating or direct contact. In susceptible individuals, a traumatic infection could be fatal, causing severe wound infection and even septic shock, and may require amputation. Global warming plays an important role in the geographical area expanding of Vibrio disease. The pathogenesis of Vibrio vulnificus-associated sepsis is very complex, including iron intake, cell injury, and adhesion-related protein and virulence regulation. Vibrio vulnificus infection mainly manifests clinical subtypes such as primary sepsis, traumatic infection, and gastroenteritis, with rapid symptom progression and signs of multiple organ dysfunction syndrome (MODS). It is important to assess these pathogenetic mechanisms in order to select more appropriate measures to prevent and treat Vibrio vulnificus infections, including antibiotic usage and surgical intervention. In this work, we report a typical case of successful treatment of necrotizing fasciitis caused by Vibrio vulnificus, and review the epidemiology, pathogenetic mechanism, clinical characteristics, and treatment of Vibrio vulnificus infection.

First report of isolation and complete genome of Vibrio rotiferianus strain SSVR1601 from cage-cultured black rockfish (Sebastes schlegelii) associated with skin ulcer.

Vibrio rotiferianus is an important marine pathogen of various aquatic organisms and can be found widely distributed in the marine environment. To further characterize this pathogen, the pathogenic properties and genome of V. rotiferianus SSVR1601 isolated from Sebastes schlegelii with skin ulcer were analysed. SSVR1601 was shown to be short rod-shaped cell with a single polar flagellum. Different degrees of pathological changes in fish kidney, intestine, gills and liver were observed after SSVR1601 challenge. The SSVR1601 genome consists of two chromosomes and two plasmids with a total of 5,717,113 bp, 42.04%-44.93% GC content, 5,269 predicted CDSs, 134 tRNAs and 40 rRNAs. The common virulence factors including OMPs, haemolysin, flagellin, DNase, entF, algU, tcpI, acfB and rfaD were found in strain SSVR1601. Furthermore, factors responsible for iron uptake (fur, fepC and ccmC) and types II, IV and VI secretion systems were detected, which are likely responsible for the pathogenicity of SSVR1601. The antimicrobial resistance genes, bacA, tet34 and norM, were detected based on Antibiotic Resistance Genes Database. The phylogenetic analysis revealed SSVR1601 to be most closely related to V. rotiferianus strains CAIM577 and B64D1.

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

Using NanoSIMS coupled with microfluidics to visualize the early stages of coral infection by Vibrio coralliilyticus.

BACKGROUND: Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. RESULTS: Three coral fragments were inoculated with 15N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15N-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. CONCLUSIONS: This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Influence of food matrix type on extracellular products of Vibrio parahaemolyticus.

BACKGROUND: Two strains of Vibrio parahaemolyticus (ATCC 17802 and 33847) in shrimp, oyster, freshwater fish, pork, chicken and egg fried rice were evaluated for production of hemolysin and exoenzymes of potential importance to the pathogenicity of this bacterium. RESULTS: The two strains of V. parahaemolyticus produced hemolysin, gelatinase, caseinase, phospholipase, urease, DNase and amylase in selected food matrices. Significantly higher (p < 0.05) hemolytic activity was produced by V. parahaemolyticus in egg fried rice > shrimp > freshwater fish > chicken > oyster > pork. But the exoenzyme activities were not consistent with the hemolytic activity profile, being significantly higher (p < 0.05) in shrimp > freshwater fish > chicken > oyster > pork > egg fried rice. Filtrates of V. parahaemolyticus from shrimp, freshwater fish and chicken given intraperitoneally to adult mice induced marked liver and kidney damage and were highly lethal compared with the filtrates of V. parahaemolyticus from oyster > egg fried rice > pork. CONCLUSION: From in vitro and in vivo tests, it appears that the food matrix type has a significant impact on the activity of extracellular products and the pathogenicity of V. parahaemolyticus. From a food safety aspect, it is important to determine which food matrices can stimulate V. parahaemolyticus to produce additional extracellular factors. This is the first report of non-seafood including freshwater fish and chicken contaminated with V. parahaemolyticus to have been shown to be toxic to mice in vivo.

In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish challenged with GFP tagged Vibrio parahaemolyticus Dahv2.

In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish (Danio rerio) challenged with GFP tagged Vibrio parahaemolyticus Dahv2 was studied. The cell free extract of probiotic B. licheniformis Dahb1 at 100 µg mL-1 showed growth inhibition of V. parahaemolyticus Dahv2 in vitro. B. licheniformis Dahb1 also inhibited the biofilm growth of GFP tagged V. parahaemolyticus Dahv2 at 100 µg mL-1 in vitro. The growth and survival of zebrafish was tested using probiotic B. licheniformis Dahb1. Weight (1.28 g) of zebrafish that received the cell free extract was much higher than in control (1.04 g). The mortality of zebrafish infected with GFP tagged V. parahaemolyticus Dahv2 at 107 Cfu mL-1 (Group IV) was 100%, whereas a complete survival of zebrafish that received the cell free extract of B. licheniformis Dahb1 at 107 Cfu mL-1 (Group VII) was observed after 30 days. The number of GFP tagged V. parahaemolyticus Dahv2 colonies in the intestine and gills significantly reduced after treatment with the cell free extract of B. licheniformis Dahb1. Furthermore, a significant decrease in the fluorescent colonies of GFP tagged V. parahaemolyticus Dahv2 was observed after treatment with the cell free extract of B. licheniformis Dahb1 under confocal laser scanning microscopy (CLSM). In conclusion, the cell free extract of B. licheniformis Dahb1 could prevent Vibrio infection by enhancing the growth and survival of zebrafish.

First isolation of Vibrio tapetis and an atypical strain of Aeromonas salmonicida from skin ulcerations in common dab (Limanda limanda) in the North Sea.

Skin ulcerations rank amongst the most prevalent lesions affecting wild common dab (Limanda limanda) with an increase in prevalence of up to 3.5% in the Belgian part of the North Sea. A complex aetiology of these ulcerations is suspected, and many questions remain on the exact factors contributing to these lesions. To construct the aetiological spectrum of skin ulcerations in flatfish, a one-day monitoring campaign was undertaken in the North Sea. Fifteen fish presented with one or more ulcerations on the pigmented and/or non-pigmented side. Pathological features revealed various stages of ulcerations with loss of epidermal and dermal tissue, inflammatory infiltrates and degeneration of the myofibers bordering the ulceration, albeit in varying degrees. Upon bacteriological examination, pure cultures of Vibrio tapetis were retrieved in high numbers from five fish and of Aeromonas salmonicida in one fish. The V. tapetis isolates showed cross-reactivity with the sera against the representative strain of serotype O2 originating form a carpet-shell clam (Ruditapes descussatus). Moreover, the A. salmonicida isolates displayed a previously undescribed vapA gene sequence (A-layer type) with possible specificity towards common dab. Further research is necessary to pinpoint the exact role of these agents in the development of skin ulcerations in common dab.

Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis.

Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter PgbpA. DNase I protection assays, together with the deletion analyses of PgbpA, demonstrated that IscR binds to two specific sequences centered at -164.5 and -106, and CRP and SmcR bind specifically to the sequences centered at -68 and -45, respectively. Furthermore, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.

Vibrio vulnificus MARTX cytotoxin causes inactivation of phagocytosis-related signaling molecules in macrophages.

BACKGROUND: Vibrio vulnificus is a marine bacterial species that causes opportunistic infections manifested by serious skin lesions and fulminant septicemia in humans. We have previously shown that the multifunctional autoprocessing repeats in toxin (MARTXVv1) of a biotype 1 V. vulnificus strain promotes survival of this organism in the host by preventing it from engulfment by the phagocytes. The purpose of this study was to further explore how MARTXVv1 inhibits phagocytosis of this microorganism by the macrophage. METHODS: We compared between a wild-type V. vulnificus strain and its MARTXVv1-deficient mutant for a variety of phagocytosis-related responses, including morphological change and activation of signaling molecules, they induced in the macrophage. We also characterized a set of MARTXVv1 domain-deletion mutants to define the regions associated with antiphagocytosis activity. RESULTS: The RAW 264.7 cells and mouse peritoneal exudate macrophages underwent cell rounding accompanied by F-actin disorganization in the presence of MARTXVv1. In addition, phosphorylation of some F-actin rearrangement-associated signaling molecules, including Lyn, Fgr and Hck of the Src family kinases (SFKs), focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), phosphoinositide 3-kinase (PI3K) and Akt, but not p38, was decreased. By using specific inhibitors, we found that these kinases were all involved in the phagocytosis of MARTXVv1-deficient mutant in an order of SFKs-FAK/Pyk2-PI3K-Akt. Deletion of the effector domains in the central region of MARTXVv1 could lead to reduced cytotoxicity, depending on the region and size of deletion, but did not affect the antiphagocytosis activity and ability to cause rounding of macrophage. Reduced phosphorylation of Akt was closely associated with inhibition of phagocytosis by the wild-type strain and MARTXVv1 domain-deletion mutants, and expression of the constitutively active Akt, myr-Akt, enhanced the engulfment of these strains by macrophage. CONCLUSIONS: MARTXVv1 could inactivate the SFKs-FAK/Pyk2-PI3K-Akt signaling pathway in the macrophages. This might lead to impaired phagocytosis of the V. vulnificus-infected macrophage. The majority of the central region of MARTXVv1 is not associated with the antiphagocytosis activity.