Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA

We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn in E. coli DH5alpha reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E. coli or V. vulnificus. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence of V. vulnificus mice.     

Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus

A gene (empV) encoding the extracellular metalloprotease of Vibrio vulnificus CKM-1 has been cloned and sequenced. When the empV gene was expressed in minicells, a unique peptide of approx. 46 kDa was identified. Protease activity staining experiments also indicated a similar M_r for the protease. The empV gene product (EmpV) is secreted into the periplasm of Escherichia coli, but not out of it. The crude enzyme prepared from the periplasmic fraction of recombinant E. coli was inhibited by a metalloprotease inhibitor and Zn²⁺ is essential for its protease activity. Nucleotide sequence analysis predicted a single open reading frame (ORF) of 1818 bp encoding a 606 amino acid (aa) polypeptide, with a potential 24 aa signal peptide followed by a long `pro' sequence consisting of 172 aa. The N-terminal 20 aa sequence for the elastolytic protease (EepV), purified from the culture supernatant of V. vulnificus ATCC 29307, completely identified the beginning of the predicted mature protein within the deduced aa sequence except for 1 aa residue difference. The estimated pI and molecular weight of the predicted mature protein were 5.86 and 44.3 kDa, respectively, which are nearly identical to those of V. vulnificus L-180 extracellular neutral metalloprotease (EnmV) and of strain ATCC 29307 EepV. The estimated molecular weight also closely matches that determined by SDS-PAGE analysis of the minicells and by protease activity staining. The deduced aa sequence of EmpV showed high homology to V. anguillarum metalloprotease (EmpA), V. cholerae HA/protease (HprC), and V. proteolyticus neutral protease (NprP), particularly with respect to active-site residues, zinc-binding residues, and cysteine residues.     

Subcellular localisation of lipoproteins of <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> by the identification of outer membrane vesicles components

Vibrio vulnificus, a Gram-negative halophilic bacterium, is an opportunistic human pathogen that is responsible for the majority of seafood-associated deaths worldwide. Lipoproteins are important components of the bacterial cell envelope and have been shown to be involved in a wide variety of cellular processes. Little is known about the localisation or transport mechanism of lipoproteins in V. vulnificus. To assess the localisation of lipoproteins in V. vulnificus, we tested two established techniques for the rapid separation of membrane-associated proteins: detergent extraction with Sarkosyl and outer membrane vesicles (OMVs) preparation. The results showed that Sarkosyl extraction was not useful for the separation of lipoproteins from the different membranes of V. vulnificus. On the other hand, we confirmed that OMVs produced by V. vulnificus contained lipoproteins from the outer but not the inner membrane. Analysis of the OMVs components confirmed the localisation of several well-known lipoproteins to membranes that were different from expected, based on their predicted functions. Using this technique, we found that Asp at position +2 of mature lipoproteins can function as an inner membrane retention signal in V. vulnificus. Interestingly, the Escherichia coli “+2 rule” does not apply to the V. vulnificus lipoprotein IlpA (G2D) mutant, as a Ser to Asp mutation at position +2 of IlpA did not affect its outer membrane localisation. Furthermore, an IlpA tether-mRFP chimeric lipoprotein and its G2D mutant also behaved like IlpA. Together, these results suggest that the sorting rule of lipoproteins in V. vulnificus might be different from that in E. coli.     

RpoS-Dependent Stress Response and Exoenzyme Production in Vibrio vulnificus

Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection. Like other opportunistic pathogens, V. vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels. In order to begin to understand the ability of V. vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens. An rpoS mutant of V. vulnificus strain C7184o was constructed by homologous recombination. The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions. The most striking difference was a high sensitivity of the mutant to hydrogen peroxide. Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type. Additionally, the motility of the rpoS mutant was severely diminished. Overall, these studies suggest that rpoS in V. vulnificus is important for adaptation to environmental changes and may have a role in virulence.     

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.     

The role of quorum sensing and the effect of environmental conditions on biofilm formation by strains of Vibrio vulnificus

Abstract

It has been suggested that Vibrio vulnificus attaches to plankton and algae and is found in large numbers in the environment. Factors affecting attachment, biofilm formation and morphology of V. vulnificus have not been thoroughly investigated. This study evaluated the role of quorum sensing (QS) and environmental conditions on biofilm development of V. vulnificus. It was found that biofilm development by V. vulnificus was affected by nutrient and glucose concentration, but not by NaCl concentration or temperature under the conditions used here. Moreover, biofilm development of a QS mutant strain proceeded rapidly and sloughing occurred earlier than for the isogenic parent strain. There was a significant loss of viability for the QS mutant biofilm early in development. Hence, it is hypothesised that factors regulated by the QS system play a role in proper biofilm development and maintenance of V. vulnificus. Furthermore, it is shown that biofilm development varied among isolates.     

The Periplasmic, Group III Catalase of Vibrio fischeri Is Required for Normal Symbiotic Competence and Is Induced Both by Oxidative Stress and by Approach to Stationary Phase

The catalase gene, katA, of the sepiolid squid symbiont Vibrio fischeri has been cloned and sequenced. The predicted amino acid sequence of KatA has a high degree of similarity to the recently defined group III catalases, including those found in Haemophilus influenzae, Bacteroides fragilis, and Proteus mirabilis. Upstream of the predicted start codon of katA is a sequence that closely matches the consensus sequence for promoters regulated in Escherichia coli by the alternative sigma factor encoded by rpoS. Further, the level of expression of the cloned katA gene in an E. coli rpoS mutant is much lower than in wild-type E. coli. Catalase activity is induced three- to fourfold both as growing V. fischeri cells approach stationary phase and upon the addition of a small amount of hydrogen peroxide during logarithmic growth. The catalase activity was localized in the periplasm of wild-type V. fischeri cells, where its role could be to detoxify hydrogen peroxide coming from the external environment. No significant catalase activity could be detected in a katA null mutant strain, demonstrating that KatA is the predominately expressed catalase in V. fischeri and indicating that V. fischeri carries only a single catalase gene. The catalase mutant was defective in its ability to competitively colonize the light organs of juvenile squids in coinoculation experiments with the parent strain, suggesting that the catalase enzyme plays an important role in the symbiosis between V. fischeri and its squid host.     

Characterization and Virulence of Hemolysin III from <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the word. A gene (hlyIII) encoding a hemolysin was cloned and sequenced from V. vulnificus. Nucleotide sequence analysis predicted an open reading frame of 642 bp encoding a 214 amino acid polypeptide that showed 48% sequence identity to the hemolysin III of Bacillus cereus. When HlyIII of V. vulnificus was expressed in Escherichia coli, crude extracts exhibited hemolytic activity similar to that of hemolysin III from Bacillus cereus. A hlyIII isogenic mutant was constructed via insertional inactivation and showed an attenuated virulence compared with the wild-type strain when this mutant was administered intraperitoneally in mice.     

DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site

The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA. The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 A resolution. Vvn has a novel mixed alpha/beta topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm. The overall structure of Vvn shows no similarity to other endonucleases; however, a known 'betabetaalpha-metal' motif is identified in the central cleft region. The crystal structure of the mutant Vvn-DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by approximately 20 degrees. Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence-independent recognition of DNA and RNA. Based on the enzyme-substrate and enzyme-product structures observed in the mutant Vvn-DNA crystals, a catalytic mechanism is proposed. This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA.     

Ecology of Vibrio vulnificus in Estuarine Waters of Eastern North Carolina

While several studies on the ecology of Vibrio vulnificus in Gulf Coast environments have been reported, there is little information on the distribution of this pathogen in East Coast waters. Thus, we conducted a multiyear study on the ecology of V. vulnificus in estuarine waters of the eastern United States, employing extensive multiple regression analyses to reveal the major environmental factors controlling the presence of this pathogen, and of Vibrio spp., in these environments. Monthly field samplings were conducted between July 2000 and April 2002 at six different estuarine sites along the eastern coast of North Carolina. At each site, water samples were taken and nine physicochemical parameters were measured. V. vulnificus isolates, along with estuarine bacteria, Vibrio spp., Escherichia coli organisms, and total coliforms, were enumerated in samples from each site by using selective media. During the last 6 months of the study, sediment samples were also analyzed for the presence of vibrios, including V. vulnificus. Isolates were confirmed as V. vulnificus by using hemolysin gene PCR or colony hybridization. V. vulnificus was isolated only when water temperatures were between 15 and 27°C, and its presence correlated with water temperature and dissolved oxygen and vibrio levels. Levels of V. vulnificus in sediments were low, and no evidence for an overwintering in this environment was found. Multiple regression analysis indicated that vibrio levels were controlled primarily by temperature, turbidity, and levels of dissolved oxygen, estuarine bacteria, and coliforms. Water temperature accounted for most of the variability in the concentrations of both V. vulnificus (47%) and Vibrio spp. (48%).     

Interaction of Nuclease Colicins with Membranes: Insertion Depth Correlates with Bilayer Perturbation

Background

Protein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases) or RNA (RNases) hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7) and one ribosomal RNase domain (E3) using a biomembrane model system.

Principal Results

We demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed.

Conclusions/Significance

Our results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.     

Effects of Physicochemical Factors and Bacterial Colony Morphotype on Association of Vibrio vulnificus with Hemocytes of Crassostrea virginica

Vibrio vulnificus is a naturally occurring marine bacterium that causes invasive disease of immunocompromised humans following the consumption of raw oysters. It is a component of the natural microbiota of Gulf Coast estuaries and has been found to inhabit tissues of oysters, Crassostrea virginica (Gmelin 1791). The interaction of V. vulnificus with oyster host defenses has not been reported in detail. We examined the interaction of V. vulnificus with phagocytic oyster hemocytes as a function of time, temperature, bacterial concentration, pretreatment with hemolymph, and V. vulnificus translucent and opaque colony morphotypes. Within these experimental parameters, the results showed that the association of V. vulnificus with hemocytes increased with time, temperature, and initial V. vulnificus/hemocyte ratio. Pretreatment of V. vulnificus with serum or an increased serum concentration did not enhance V. vulnificus-hemocyte associations, a result suggesting the absence of opsonic activity. More than 50% of hemocytes bound the translucent, avirulent morphotype, whereas 10 to 20% were associated with the opaque, virulent form, a result indicating that the degree of encapsulation was related to resistance to phagocytosis, as previously described for mammalian phagocytes. Understanding these cellular interactions may, in part, explain the persistence of V. vulnificus in oyster tissues and the ecology of V. vulnificus in estuarine environments.     

Sediment and Vegetation as Reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.     

Occurrence of Vibrio vulnificus Biotypes in Danish Marine Environments

During the unusually warm summer in Denmark in 1994, 11 clinical cases of Vibrio vulnificus infection were reported. These reports initiated an investigation of the occurrence of V. vulnificus biotypes in Danish marine environments. Samples of coastal water, sediment, shellfish, and wild fish were analyzed by preenrichment in alkaline peptone water amended with polymyxin B (2.0 × 10⁴ U/liter) followed by streaking onto modified cellobiose-polymyxin B-colistin agar. V. vulnificus-like colonies were tested with a V. vulnificus-specific DNA probe. Low densities of V. vulnificus were detected in water (0.8 to 19 CFU/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) from July until mid-November. The presence of V. vulnificus was strongly correlated with water temperature. However, we isolated V. vulnificus from water from a mussel farm at a lower temperature than previously reported (7°C). In 1 of the 13 locations studied, V. vulnificus was found in mussels in 7 of 17 samples analyzed; this is the first report of V. vulnificus in European shellfish. V. vulnificus was also isolated from gills, intestinal contents, and mucus from wild fish. Although biotyping of 706 V. vulnificus strains isolated during our investigations revealed that the majority of the strains (99.6%) belonged to biotype 1, biotype 2 was detected in seawater at a low frequency (0.4%). Our findings provide further evidence that seawater can serve as a reservoir and might facilitate spread of V. vulnificus biotype 2 to eels, with subsequent spread to persons handling eels. In conclusion, our data demonstrate that V. vulnificus is ubiquitous in a temperate marine environment and that V. vulnificus biotype 2 is not strictly confined to eels.     

Influence of Water Temperature and Salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast Oysters (Crassostrea virginica)

This study investigated the temperature and salinity parameters associated with waters and oysters linked to food-borne Vibrio vulnificus infections. V. vulnificus was enumerated in oysters collected at three northern Gulf Coast sites and two Atlantic Coast sites from July 1994 through September 1995. Two of these sites, Black Bay, La., and Apalachicola Bay, Fla., are the source of the majority of the oysters implicated in V. vulnificus cases. Oysters in all Gulf Coast sites exhibited a similar seasonal distribution of V. vulnificus: a consistently large number (median concentration, 2,300 organisms [most probable number] per g of oyster meat) from May through October followed by a gradual reduction during November and December to ≤10 per g, where it remained from January through mid-March, and a sharp increase in late March and April to summer levels. V. vulnificus was undetectable (<3 per g) in oysters from the North and South Carolina sites for most of the year. An exception occurred when a late-summer flood caused a drop in salinity in the North Carolina estuary, apparently causing V. vulnificus numbers to increase briefly to Gulf Coast levels. At Gulf Coast sites, V. vulnificus numbers increased with water temperatures up to 26°C and were constant at higher temperatures. High V. vulnificus levels (>10³ per g) were typically found in oysters from intermediate salinities (5 to 25 ppt). Smaller V. vulnificus numbers (<10² per g) were found at salinities above 28 ppt, typical of Atlantic Coast sites. On 11 occasions oysters were sampled at times and locations near the source of oysters implicated in 13 V. vulnificus cases; the V. vulnificus levels and environmental parameters associated with these samples were consistent with those of other study samples collected from the Gulf Coast from April through November. These findings suggest that the hazard of V. vulnificus infection is not limited to brief periods of unusual abundance of V. vulnificus in Gulf Coast oysters or to environmental conditions that are unusual to Gulf Coast estuaries.     

Intraspecific Diversity of Vibrio vulnificus in Galveston Bay Water and Oysters as Determined by Randomly Amplified Polymorphic DNA PCR

Randomly amplified polymorphic DNA (RAPD) PCR was used to analyze the temporal and spatial intraspecific diversity of 208 Vibrio vulnificus strains isolated from Galveston Bay water and oysters at five different sites between June 2000 and June 2001. V. vulnificus was not detected during the winter months (December through February). The densities of V. vulnificus in water and oysters were positively correlated with water temperature. Cluster analysis of RAPD PCR profiles of the 208 V. vulnificus isolates revealed a high level of intraspecific diversity among the strains. No correlation was found between the intraspecific diversity among the isolates and sampling site or source of isolation. After not being detected during the winter months, the genetic diversity of V. vulnificus strains first isolated in March was 0.9167. Beginning in April, a higher level of intraspecific diversity (0.9933) and a major shift in population structure were observed among V. vulnificus isolates. These results suggest that a great genetic diversity of V. vulnificus strains exists in Galveston Bay water and oysters and that the population structure of this species is linked to changes in environmental conditions, especially temperature.     

Structural and functional insight into sugar-nonspecific nucleases in host defense

In prokaryotes, sugar-nonspecific nucleases that cleave DNA and RNA in a sequence-independent manner take part in host defense, as well as site-specific restriction enzymes. Examples include the periplasmic nuclease Vvn and the secreted nuclease ColE7, which degrade foreign nucleic acid molecules in the host periplasm and in the cytoplasm of foreign cells, respectively. Recently determined crystal structures of Vvn and ColE7 in complex with double-stranded DNA provide structural insight into nonspecific DNA interactions and cleavage by sugar-nonspecific nucleases. Both nucleases bind DNA at the minor groove through a common 'betabetaalpha-metal' endonuclease motif and primarily contact the DNA phosphate backbone, probably to avoid sequence-dependent base recognition. In eukaryotes, several apoptotic endonucleases that are responsible for DNA degradation in programmed cell death also contain a betabetaalpha-metal fold at the active site, suggesting that they may recognize and cleave DNA in a comparable way.     

Functional Analysis of TolC Homologs in Vibrio vulnificus

Gram-negative bacteria use tripartite pumps to transport antibacterial drugs and other toxic compounds across the inner and outer membranes, which are separated by the periplasmic space. The TolC protein is an outer membrane factor that participates in the formation of tripartite efflux pumps. The genome of Vibrio vulnificus encodes two E. coli TolC homologs, TolCV1 and TolCV2. Here, we show that both TolCV1 and TolCV2 are involved in the efflux of antimicrobial agents. Deletion of tolCV1 resulted in increased susceptibility of V. vulnificus to chemical detergents, DNA intercalating agents, and antibiotics including erythromycin, novobiocin, and tetracycline, whereas deletion of tolCV2 rendered V. vulnificus more susceptible to the above mentioned antibiotics only. We also observed that tolCV1 deletion resulted in reduced motility of V. vulnificus. Our results indicate active roles for TolCV1 and TolCV2 in the physiology of V. vulnificus.