Motility and flagellin gene expression in the fish pathogen Vibrio salmonicida: effects of salinity and temperature

The success of several Vibrio species, including Vibrio cholerae, Vibrio anguillarum and Vibrio fischeri in colonizing their symbiont, or causing infection is linked to flagella-based motility. It is during early colonization or the initial phase of infection that motility appears to be critical. In this study we used Vibrio salmonicida, a psychrophilic and moderate halophilic bacterium that causes cold-water vibriosis in seawater-farmed Atlantic salmon (Salmo salar), to study motility and expression of flagellins under salt conditions mimicking the initial and later phases of an infection. Our results, which are based on motility in semi-solid agar, membrane protein proteomics, quantitation of flagellin gene expression, challenge infection of fish, and microscopy, show that V. salmonicida is highly motile, expresses elevated levels of flagellins, and typically contains several polar flagella under salt conditions that are seawater-like. In contrast, V. salmonicida cells are non-motile and express significantly lower levels of flagellins under physiological-like salt conditions.     

Curing of a plasmid is correlated with an attenuation of virulence in the marine fish pathogen Vibrio anguillarum

The transposon A sequence Tn1 containing the ampicillin resistance determinants was transposed from RP4 to a plasmid of the marine fish pathogen Vibrio anguillarum. Curing experiments in which plasmid loss was determined by analysis of the segregation of the ampicillin resistance phenotype showed the association of virulence with the specific V. anguillarum plasmid class.     

Vibrio anguillarum evades the immune response of the bony fish sea bass (Dicentrarchus labrax L.) through the inhibition of leukocyte respiratory burst and down-regulation of apoptotic caspases

The mechanisms of the cellular immune response involved in the protection of fish against infection by the pathogenic bacterium Vibrio anguillarum are largely unknown. In the present study, sea bass specimens were injected with live or formalin-killed V. anguillarum and the respiratory burst of leukocytes was measured. The infection of fish resulted in a strong inhibition of the respiratory burst, in contrast with the slight increase in respiratory burst of leukocytes from fish injected with dead bacteria. In addition, we observed a concomitant down-regulation of p22(phox) and p40(phox), two components of the NADPH oxidase, in the leukocytes from infected fish. To investigate whether these differences may be the result of a dysregulation of cytokines expression in infected fish, we cloned several sea bass cytokines, including interleukin-6 (IL-6), IL-8 and three CC chemokines, and performed a detailed expression study with these and other cytokines. Surprisingly, cytokine expression was fairly similar in leukocytes from both live and formalin-killed V. anguillarum-challenged fish, the response being even higher and longer lasting in infected fish. Furthermore, the expression of two key apoptotic caspases, caspase-3 and -9, was down-regulated in leukocytes from infected fish, but remained unaltered in fish injected with formalin-killed bacteria. These results suggest that the virulence mechanisms of V. anguillarum in sea bass involve the inhibition of leukocyte respiratory burst and apoptosis, and thereby providing a safe haven for growth.     

Outer membrane proteins induced under conditions of iron limitation in the marine fish pathogen Vibrio anguillarum 775.

Cells of the marine fish pathogen Vibrio anguillarum 775 harboring a plasmid associated with virulence can grow unaffected in the presence of iron-binding compounds such as transferrin. In contrast, the growth of isogenic plasmidless derivatives is inhibited by the presence of iron chelators. Radioactive from (55Fe3+) uptake experiments indicate that this plasmid-linked ability of V. anguillarum cells to grow under conditions of iron limitation is indeed due to a more rapid and efficient iron uptake mediated by the virulence plasmid. In addition, V. anguillarum cells growing under iron limitation show at least two novel outer membrane proteins. One of them, a 86,000-dalton protein we called OM2, is inducible only in those cells in which the virulence plasmid is present.     

Evidence for plasmid contribution to the virulence of fish pathogen Vibrio anguillarum.

Analysis of the plasmid deoxyribonucleic acid complement of high- and low- virulent strains of the fish pathogen Vibrio anguillarum showed a correlation between enhanced virulence and the presence of a 50-megadalton plasmid class. All 50-megadalton plasmids isolated from different high-virulent V. anguillarum strains were homologous as judged by the analysis of plasmid deoxyribonucleic acid-deoxyribonucleic acid hybridization. The 50-megadalton plasmid class did not have polynucleotide sequences in common with plasmids of different incompatibility groups.     

Increased production of the siderophore anguibactin mediated by pJM1-like plasmids in Vibrio anguillarum.

The virulence of the fish pathogen Vibrio anguillarum 775 is mediated by the pJM1 plasmid-specified iron uptake system which is expressed under conditions of iron limitation. Other V. anguillarum strains isolated from various geographical locations harbor plasmids that are highly related to pJM1 and that are also associated with the high-virulence phenotype of these strains. In this work, we found that a pJM1-like plasmid, pJHC1, from one of these virulent strains encoded an iron uptake system that resulted in an increased level of production of the siderophore anguibactin. The gene(s) responsible for increased anguibactin production was included within the iron uptake region of plasmid pJHC1. The cloned iron uptake regions of pJHC1 and pJM1 possessed identical restriction endonuclease maps, suggesting that the DNA region encoding those genes in pJHC1 may have diverged subtly from that in pJM1. Analysis of the iron uptake system from other V. anguillarum strains carrying pJM1-like plasmids demonstrated that strains originating from diseased fish from the Atlantic coast carry plasmids encoding an increased-siderophore-production phenotype, while strains isolated from Pacific Ocean locations behaved as the 775 strain.     

Streptococcus-zebrafish model of bacterial pathogenesis

Due to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish, Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 10(3) CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that of S. iniae in farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogen Streptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease.     

Vibrio salmonicida pathogenesis analyzed by experimental challenge of Atlantic salmon (Salmo salar)

Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the Gram-negative bacterium Vibrio (Aliivibrio) salmonicida. To study the pathogenesis of this marine pathogen, Atlantic salmon was experimentally infected by immersion challenge with wild type V. salmonicida and the bacterial distribution in different organs was investigated at different time points. V. salmonicida was identified in the blood as early as 2 h after challenge demonstrating a rapid establishment of bacteremia without an initial period of colonization of the host. Two days after immersion challenge, only a few V. salmonicida were identified in the intestines, but the amount increased with time. In prolonged CV cases, V. salmonicida was the dominating bacterium of the gut microbiota causing a release of the pathogen to the water. We hypothesize that V. salmonicida uses the blood volume for proliferation during the infection of the fish and the salmonid intestine as a reservoir that favors survival and transmission. In addition, a motility-deficient V. salmonicida strain led us to investigate the impact of motility in the CV pathogenesis by comparing the virulence properties of the mutant with the wild type LFI1238 strain in both i.p. and immersion challenge experiments. V. salmonicida was shown to be highly dependent on motility to gain access to the fish host. After invasion, motility was no longer required for virulence, but the absence of normal flagellation delayed the disease development.     

Isolation and characterization of rainbow trout C-reactive protein

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout (Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.     

Molecular characterization of interleukin-6 in the gilthead seabream (Sparus aurata)

A cDNA clone, designated sbIL-6 (seabream interleukin-6), was obtained from a cDNA library of enriched immune-stimulated sequences from gilthead seabream. The deduced sbIL-6 protein corresponds to a 225-amino acid protein with a putative 24-amino acid signal peptide, four conserved alpha helices and one N-linked glycosylation site. At the amino acid level sbIL-6 shares 23-26% identity with mammalian IL-6 sequences and 30-51% identity with other fish IL-6 sequences. The structure of the sbIL-6 gene consisted of 5 exons and 4 introns, spanning 2.4 kb. Healthy fish expressed sbIL-6 in white muscle, skin, spleen, anterior intestine and stomach, while no expression was detected in brain, gill, head kidney, posterior intestine and adipose tissue. A significant up-regulation of sbIL-6 expression was observed after lipopolysaccharide (LPS), Vibrio anguillarum DNA (VaDNA) and peptidoglycan treatment in cultured seabream head kidney leukocytes. Using purified immune cells, sbIL-6 expression was induced similarly in macrophages and acidophilic granulocytes by VaDNA but LPS was more effective in inducing sbIL-6 expression in acidophilic granulocytes than in macrophages. Furthermore, in vivo infection of seabream with live V. anguillarum caused significant increases in sbIL-6 mRNA expression in the thymus, peritoneal exudate, head kidney and gills. In summary, our study provides further evidence for the existence of distinct IL-6 genes in lower vertebrates and for the strong induction of their expression by immune stimuli, supporting the notion of a potentially important role for this cytokine in fish.     

Zebrafish as a useful model for zoonotic Vibrio parahaemolyticus pathogenicity in fish and human

Vibrio parahaemolyticus is an important aquatic zoonotic pathogen worldwide that causes vibriosis in many marine fish, and sepsis, gastroenteritis and wound infection in humans. However, the pathogenesis of different sources of V. parahaemolyticus is not fully understood. Here, we examined the pathogenicity and histopathology of fish (V. parahaemolyticus 1.2164) and human (V. parahaemolyticus 17) strains in a zebrafish (Danio rerio). We found that different infection routes resulted in different mortality in zebrafish. Moreover, death due to V. parahaemolyticus 1.2164 infection occurred quicker than that caused by V. parahaemolyticus 17 infection. Hematoxylin-eosin staining of liver, kidney and intestine sections showed histological lesions in all three organs after infection with either strain. V. parahaemolyticus 1.2164 caused more severe damage than V. parahaemolyticus 17. In particular, V. parahaemolyticus 1.2164 treatment induced more serious hydropic degeneration and venous sinus necrosis in the liver than V. parahaemolyticus 17 treatment. The expression levels of three proinflammatory cytokines, interleukin 1β (il1β), interferon phi 1 (ifnϕ1) and tumor necrosis factor α (tnfα), as determined by quantitative real-time PCR, were upregulated in all examined tissues of infected fish. Notably, the peak levels of tnfα were significantly higher than those of il1β and ifnϕ1, suggesting, together with pathological results, that tnfα and il1β play an important role in acute sepsis. High amounts of tnfα may be related to acute liver necrosis, while ifnϕ1 may respond to V. parahaemolyticus and play an antibacterial role for chronically infected adult zebrafish. Taken together, our results suggest that the zebrafish model of V. parahaemolyticus infection is useful for studying strain differences in V. parahaemolyticus pathogenesis.     

Transcriptomic profiling revealed the signatures of intestinal barrier alteration and pathogen entry in turbot (Scophthalmus maximus) following Vibrio anguillarum challenge

The mucosal immune system serves as the frontline barriers of host defense against pathogen infection, especially for the fishes, which are living in the pathogen rich aquatic environment. The intestine constitutes the largest surface body area in constantly contact with the external pathogens, and plays a vital role in the immune defense against inflammation and pathogen infection. Previous studies have revealed that fish intestine might serves as the portal of entry for Vibrio anguillarum. To characterize the immune actors and their associated immune activities in turbot intestine barrier during bacterial infection, here we examined the gene expression profiles of turbot intestine at three time points following experimental infection with V. anguillarum utilizing RNA-seq technology. A total of 122 million reads were assembled into 183,101 contigs with an average length of 1151 bp and the N50 size of 2302 bp. Analysis of differential gene expression between control and infected samples at 1 h, 4 h, and 12 h revealed 2079 significantly expressed genes. Enrichment and pathway analysis of the differentially expressed genes showed the centrality of the pathogen attachment and recognition, antioxidant/apoptosis, mucus barrier modification and immune activation/inflammation in the pathogen entry and host immune responses. The present study reported the novel gene expression patterns in turbot mucosal immunity, which were overlooked in previous studies. Our results can help to understand the mechanisms of turbot host defense, and may also provide foundation to identify the biomarkers for future selection of disease-resistant broodstock and evaluation of disease prevention and treatment options.     

The toxR Gene of Vibrio (Listonella) anguillarum Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model

To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two beta-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s).     

Fish soluble Toll-like receptor 5 (TLR5S) is an acute-phase protein with integral flagellin-recognition activity

From an EST fragment of the rainbow trout that was predicted to contain leucine-rich repeats (LRR), we cloned the whole cDNA and identified a soluble form of TLR5 ortholog (rtTLR5S), which does not exist in the mouse and human. rtTLR5S was about 38% homologous to the extracellular domains of human (hu) and mouse (mo)TLR5, while rtTLR5S showed <25% homologous to those of other human or mouse TLRs. A chimera constructed of rtTLR5S and the intra-cellular TIR of huTLR5 expressed on HeLa cells signaled the presence of flagellin A and C from V. anguillarum, resulting in NF-kappaB activation. The mRNA of rtTLR5S was predominantly detected in the liver. The hepatoma cell line of the rainbow trout RTH149 that responded to flagellin, allowed to up-regulate rtTLR5S in response to V. anguillarum or its purified flagellin within 8 h. rtTLR5S, when co-expressed with membrane huTLR5 in HeLa cells, augmented huTLR5-mediated NF-kappaB activation in response to flagellin. These results, together with the genome information of the pufferfish Fugu (Fugu rubripes), suggest that in fish the soluble TLR5 is an acute-phase protein sensing bacterial infection via recognition of a variety of bacterial flagellins to augment NF-kappaB activation, and may be important for fish to survive from bacterial infection in the water.     

Infection of pronephros cell cultures derived from rainbow trout (Oncorhynchus mykiss,Walbaum) with bacterial fish pathogens: A comparison with whole fish infectivity studies

Primary cell cultures were developed from the pronephros of juvenile rainbow trout (Oncorhynchus mykiss, Walbaum), and used to determine their interaction with bacterial pathogens as compared to whole fish pathogenicity experiments. Overall, there was excellent agreement with Aeromonas hydrophila, A. salmonicida, Citrobacter freundii, Streptococcus iniae, Vibrio anguillarum, V. damsela, V. harveyi, V. ordalii, V. viscosus, V. vulnificus and Yersinia ruckeri, which were harmful to cell cultures and pathogenic to Atlantic salmon (Salmo salar L.) and rainbow trout. Also, a culture of Enterococcus faecium, which is not a recognised fish pathogen, caused mortalities in salmonids and damaged the cell cultures. In contrast, negligible damage to fish and/or cell cultures resulted from challenge with a Carnobacterium sp., Escherichia coli, Lactobacillus sp., V. alginolyticus and commercial formalin- inactivated vaccines for enteric redmouth (= Y. ruckeri) and furunculosis (= A. salmonicida). Use of cell cultures and whole fish experiments revealed that inactivation of V. harveyi for use in vaccines was troublesome, insofar as the addition of formalin and chloroform resulted in harmful preparations. In contrast, cultures of V. harveyi, which were inactivated by heat (100 °C for 1 hour), lysis at pH 9.5 and glutaraldehyde (to 0.5% w/v), were less harmful.     

Activation of trout macrophages and production of CRP after immunization with Vibrio anguillarum

To examine the mechanism of the protection of rainbow trout (Salmo gairdneri) against Vibrio anguillarum in the early stage of immunization, the activation of macrophages and production of C-reactive protein (CRP) were investigated. Fish immunized with formalin-killed bacteria emulsified in Freund's complete adjuvant (FCA) resisted intraperitoneal challenge with living bacteria seven and ten days after immunization. The activation of macrophages was demonstrated by a significant increase of the chemiluminescent (CL) response and phagocytic activity. These fish also showed a significant increase of the CRP level in sera. Fish immunized with V. anguillarum alone or injected with FCA, however, did not resist the challenge. Though FCA itself increased CRP level and the sera enhanced phagocytic activity, increase of CL activity was weak. These results indicated that the increase of CL activity and opsonising effect of CRP on the phagocytosis of specifically activated macrophages concern to host defense in the early stage of infection.     

High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans

Vibrio vulnificus biotype 1, a causative agent of fatal septicemia or wound infection in humans, is known to produce a toxic metalloprotease as an important virulence determinant. V. vulnificus biotype 2 (serovar E), a primary eel pathogen, was found to elaborate an extracellular metalloprotease that was indistinguishable from that of biotype 1. The potential of V. vulnificus biotype 1 for production of the metalloprotease was compared with biotype 2 and other human non-pathogenic Vibrio species (Vibrio anguillarum and Vibrio proteolyticus). When cultivated at 25 degrees C in tryptone-yeast extract broth supplemented with 0.9% NaCl, all bacteria multiplied sufficiently and secreted significant amounts of the metalloprotease. However, at 37 degrees C with 0.9% NaCl, V. anguillarum neither grew nor produced the metalloprotease. In human serum, only V. vulnificus biotype 1 revealed a steady multiplication accompanied with production of the extracellular metalloprotease. This prominent ability of biotype 1 in growth and protease production may contribute to cause serious systemic diseases in humans.