Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease.

Considered one of the most devastating coral disease outbreaks in history, stony coral tissue loss disease (SCTLD) is currently spreading throughout Florida's coral reefs and the greater Caribbean. SCTLD affects at least two dozen different coral species and has been implicated in extensive losses of coral cover. Here we show Pseudoalteromonas sp. strain McH1-7 has broad-spectrum antibacterial activity against SCTLD-associated bacterial isolates. Chemical analyses indicated McH1-7 produces at least two potential antibacterials, korormicin and tetrabromopyrrole, while genomic analysis identified the genes potentially encoding an L-amino acid oxidase and multiple antibacterial metalloproteases (pseudoalterins). During laboratory trials, McH1-7 arrested or slowed disease progression on 68.2% of diseased Montastraea cavernosa fragments treated (n = 22), and it prevented disease transmission by 100% (n = 12). McH1-7 is the most chemically characterized coral probiotic that is an effective prophylactic and direct treatment for the destructive SCTLD as well as a potential alternative to antibiotic use.

Comparison of Vibrio coralliilyticus virulence in Pacific oyster larvae and corals.

The bacterium Vibrio coralliilyticus has been implicated in mass mortalities of corals and shellfish larvae. However, using corals for manipulative infection experiments can be logistically difficult compared to other model organisms, so we aimed to establish oyster larvae infections as a proxy model. Therefore, this study assessed the virulence of six wild-type V. coralliilyticus strains, and mutants of one strain with deletions of known virulence factors, between Pacific oyster larvae (Crassostrea gigas) and Hawaiian rice coral (Montipora capitata) infection systems. The wild-type strains tested displayed variable virulence in each system, but virulence levels between hosts were not necessarily comparable. Strains RE98 and OCN008 maintained a medium to high level of virulence across hosts and appeared to be more generalist pathogens. Strain H1, in contrast, was avirulent towards coral but displayed a medium level of virulence towards oyster larvae. Interestingly, the BAA-450 type strain had a medium level of virulence towards coral and was the least virulent to oyster larvae. A comparison of known virulence factors determined that the flagellum, motility or chemotaxis, all of which play a significant role in coral infections, were not crucial for oyster infections with strain OCN008. A genomic comparison of the newly sequenced strain H1 with the other strains tested identified 16 genes potentially specific to coral pathogens that were absent in H1. This is both the first comparison of various V. coralliilyticus strains across infection systems and the first investigation of a strain that is non-virulent to coral. Our results indicate that the virulence of V. coralliilyticus strains in coral is not necessarily indicative of virulence in oyster larvae, and that the set of genes tested are not required for virulence in both model systems. This study increases our understanding of the virulence between V. coralliilyticus strains and helps assess their potential threat to marine environments and shellfish industries.

Disease Diagnostics and Potential Coinfections by Vibrio coralliilyticus During an Ongoing Coral Disease Outbreak in Florida.

A deadly coral disease outbreak has been devastating the Florida Reef Tract since 2014. This disease, stony coral tissue loss disease (SCTLD), affects at least 22 coral species causing the progressive destruction of tissue. The etiological agents responsible for SCTLD are unidentified, but pathogenic bacteria are suspected. Virulence screens of 400 isolates identified four potentially pathogenic strains of Vibrio spp. subsequently identified as V. coralliilyticus. Strains of this species are known coral pathogens; however, cultures were unable to consistently elicit tissue loss, suggesting an opportunistic role. Using an improved immunoassay, the VcpA RapidTest, a toxic zinc-metalloprotease produced by V. coralliilyticus was detected on 22.3% of diseased Montastraea cavernosa (n = 67) and 23.5% of diseased Orbicella faveolata (n = 24). VcpA+ corals had significantly higher mortality rates and faster disease progression. For VcpA- fragments, 21.6% and 33.3% of M. cavernosa and O. faveolata, respectively, died within 21 d of observation, while 100% of similarly sized VcpA+ fragments of both species died during the same period. Further physiological and genomic analysis found no apparent differences between the Atlantic V. coralliilyticus strains cultured here and pathogens from the Indo-Pacific but highlighted the diversity among strains and their immense genetic potential. In all, V. coralliilyticus may be causing coinfections that exacerbate existing SCTLD lesions, which could contribute to the intraspecific differences observed between colonies. This study describes potential coinfections contributing to SCTLD virulence as well as diagnostic tools capable of tracking the pathogen involved, which are important contributions to the management and understanding of SCTLD.

Comparative genomic analysis of Vibrios yields insights into genes associated with virulence towards C. gigas larvae.

BACKGROUND: Vibriosis has been implicated in major losses of larvae at shellfish hatcheries. However, the species of Vibrio responsible for disease in aquaculture settings and their associated virulence genes are often variable or undefined. Knowledge of the specific nature of these factors is essential to developing a better understanding of the environmental and biological conditions that lead to larvae mortality events in hatcheries. We tested the virulence of 51 Vibrio strains towards Pacific Oyster (Crassostreae gigas) larvae and sequenced draft genomes of 42 hatchery-associated vibrios to determine groups of orthologous genes associated with virulence and to determine the phylogenetic relationships among pathogens and non-pathogens of C. gigas larvae. RESULTS: V. coralliilyticus strains were the most prevalent pathogenic isolates. A phylogenetic logistic regression model identified over 500 protein-coding genes correlated with pathogenicity. Many of these genes had straightforward links to disease mechanisms, including predicted hemolysins, proteases, and multiple Type 3 Secretion System genes, while others appear to have possible indirect roles in pathogenesis and may be more important for general survival in the host environment. Multiple metabolism and nutrient acquisition genes were also identified to correlate with pathogenicity, highlighting specific features that may enable pathogen survival within C. gigas larvae. CONCLUSIONS: These findings have important implications on the range of pathogenic Vibrio spp. found in oyster-rearing environments and the genetic determinants of virulence in these populations.

The aerobic respiratory chain of Pseudomonas aeruginosa cultured in artificial urine media: Role of NQR and terminal oxidases.

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that forms part of the normal human microbiota and it is also an opportunistic pathogen, responsible for 30% of all nosocomial urinary tract infections. P. aeruginosa carries a highly branched respiratory chain that allows the colonization of many environments, such as the urinary tract, catheters and other medical devices. P. aeruginosa respiratory chain contains three different NADH dehydrogenases (complex I, NQR and NDH-2), whose physiologic roles have not been elucidated, and up to five terminal oxidases: three cytochrome c oxidases (COx), a cytochrome bo3 oxidase (CYO) and a cyanide-insensitive cytochrome bd-like oxidase (CIO). In this work, we studied the composition of the respiratory chain of P. aeruginosa cells cultured in Luria Broth (LB) and modified artificial urine media (mAUM), to understand the metabolic adaptations of this microorganism to the growth in urine. Our results show that the COx oxidases play major roles in mAUM, while P. aeruginosa relies on CYO when growing in LB medium. Moreover, our data demonstrate that the proton-pumping NQR complex is the main NADH dehydrogenase in both LB and mAUM. This enzyme is resistant to HQNO, an inhibitory molecule produced by P. aeruginosa, and may provide an advantage against the natural antibacterial agents produced by this organism. This work offers a clear picture of the composition of this pathogen's aerobic respiratory chain and the main roles that NQR and terminal oxidases play in urine, which is essential to understand its physiology and could be used to develop new antibiotics against this notorious multidrug-resistant microorganism.

Sodium antiporters of Pseudomonas aeruginosa in challenging conditions: effects on growth, biofilm formation, and swarming motility.

BACKGROUND: Pseudomonas aeruginosa is a bacterial pathogen that can cause grave and sometimes chronic infections in patients with weakened immune systems and cystic fibrosis. It is expected that sodium/proton transporters in the cellular membrane are crucial for the organism's survival and growth under certain conditions, since many cellular processes rely on the maintenance of Na+ and H+ transmembrane gradients. RESULTS: This study focused on the role of the primary and secondary proton and/or sodium pumps Mrp, Nuo, NhaB, NhaP, and NQR for growth, biofilm formation, and swarming motility in P. aeruginosa. Using mutants with gene deletions, we investigated the impact of each sodium pump's absence on the overall growth, biofilm formation, motility, and weak acid tolerance of the organism. We found that the absence of some, but not all, of the sodium pumps have a deleterious effect on the different phenotypes of P. aeruginosa. CONCLUSION: The absence of the Mrp sodium/proton antiporter was clearly important in the organism's ability to survive and function in environments of higher pH and sodium concentrations, while the absence of Complex I, which is encoded by the nuo genes, had some consistent impact on the organism's growth regardless of the pH and sodium concentration of the environment.

Insight into the resilience and susceptibility of marine bacteria to T6SS attack by Vibrio cholerae and Vibrio coralliilyticus.

The type VI secretion system (T6SS) is a nanomachine capable of killing adjacent microbial cells in a contact-dependent manner. Due to limited studies, relatively little is known about the range of marine bacteria that are susceptible to T6SS attack. Here, 15 diverse marine bacterial isolates from the phyla Bacteroidetes and Ɣ-Proteobacteria were challenged against the marine bacterium and human pathogen, Vibrio cholerae, which has a well described T6SS. V. cholerae killed several of the tested Ɣ-Proteobacteria, including members of the orders Vibrionales, Alteromonadales, Oceanospirillales, and Pseudomonadales. In contrast, V. cholerae co-existed with multiple Bacteroidetes and Ɣ-Proteobacteria isolates, but was killed by Vibrio coralliilyticus. Follow-up experiments revealed that five V. coralliilyticus strains, including known coral and shellfish pathogens survived the T6SS challenge and killed V. cholerae. By using predicted protein comparisons and mutagenesis, we conclude that V. coralliilyticus protected itself in the challenge by using its own T6SS to kill V. cholerae. This study provides valuable insight into the resilience and susceptibility of marine bacteria to the V. cholerae T6SS, and provides the first evidence for a functional T6SS in V. coralliilyticus, both of which have implications for human and ocean health.

Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae.

The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation.IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

Microbial Community Shifts Associated With the Ongoing Stony Coral Tissue Loss Disease Outbreak on the Florida Reef Tract.

As many as 22 of the 45 coral species on the Florida Reef Tract are currently affected by stony coral tissue loss disease (SCTLD). The ongoing disease outbreak was first observed in 2014 in Southeast Florida near Miami and as of early 2019 has been documented from the northernmost reaches of the reef tract in Martin County down to Key West. We examined the microbiota associated with disease lesions and apparently healthy tissue on diseased colonies of Montastraea cavernosa, Orbicella faveolata, Diploria labyrinthiformis, and Dichocoenia stokesii. Analysis of differentially abundant taxa between disease lesions and apparently healthy tissue identified five unique amplicon sequence variants enriched in the diseased tissue in three of the coral species (all except O. faveolata), namely an unclassified genus of Flavobacteriales and sequences identified as Fusibacter (Clostridiales), Planktotalea (Rhodobacterales), Algicola (Alteromonadales), and Vibrio (Vibrionales). In addition, several groups of likely opportunistic or saprophytic colonizers such as Epsilonbacteraeota, Patescibacteria, Clostridiales, Bacteroidetes, and Rhodobacterales were also enriched in SCTLD disease lesions. This work represents the first microbiological characterization of SCTLD, as an initial step toward identifying the potential pathogen(s) responsible for SCTLD.

Metabolomics of Vibrio cholerae.

Metabolomics is an "omics" approach to quantitatively measure a large set of metabolites. In this chapter, we describe an example method for performing liquid chromatography coupled to mass spectrometry (LC-MS)-based untargeted metabolomics on a cell extract from Vibrio cholerae.

Factors affecting infection of corals and larval oysters by Vibrio coralliilyticus.

The bacterium Vibrio coralliilyticus can threaten vital reef ecosystems by causing disease in a variety of coral genera, and, for some strains, increases in virulence at elevated water temperatures. In addition, strains of V. coralliilyticus (formally identified as V. tubiashii) have been implicated in mass mortalities of shellfish larvae causing significant economic losses to the shellfish industry. Recently, strain BAA-450, a coral pathogen, was demonstrated to be virulent towards larval Pacific oysters (Crassostrea gigas). However, it is unclear whether other coral-associated V. coralliilyticus strains can cause shellfish mortalities and if infections are influenced by temperature. This study compared dose dependence, temperature impact, and gross pathology of four V. coralliilyticus strains (BAA-450, OCN008, OCN014 and RE98) on larval C. gigas raised at 23°C and 27°C, and evaluated whether select virulence factors are required for shellfish infections as they are for corals. All strains were infectious to larval oysters in a dose-dependent manner with OCN014 being the most pathogenic and BAA-450 being the least. At 27°C, higher larval mortalities (p < 0.05) were observed for all V. coralliilyticus strains, ranging from 38.8-93.7%. Gross pathological changes to the velum and cilia occurred in diseased larvae, but there were no distinguishable differences between oysters exposed to different V. coralliilyticus strains or temperatures. Additionally, in OCN008, the predicted transcriptional regulator ToxR and the outer membrane protein OmpU were important for coral and oyster disease, while mannose sensitive hemagglutinin type IV pili were required only for coral infection. This study demonstrated that multiple coral pathogens can infect oyster larvae in a temperature-dependent manner and identified virulence factors required for infection of both hosts.

Influence of Chemotaxis and Swimming Patterns on the Virulence of the Coral Pathogen Vibrio coralliilyticus.

Chemotaxis, the directed movement toward or away from a chemical signal, can be essential to bacterial pathogens for locating hosts or avoiding hostile environments. The coral pathogen Vibrio coralliilyticus chemotaxes toward coral mucus; however, chemotaxis has not been experimentally demonstrated to be important for virulence. To further examine this, in-frame mutations were constructed in genes predicted to be important for V. coralliilyticus chemotaxis. Most Vibrio genomes contain multiple homologs of various chemotaxis-related genes, and two paralogs of each for cheB, cheR, and cheA were identified. Based on single mutant analyses, the paralogs cheB2, cheR2, and cheA1 were essential for chemotaxis in laboratory assays. As predicted, the ΔcheA1 and ΔcheR2 strains had a smooth-swimming pattern, while the ΔcheB2 strain displayed a zigzag pattern when observed under light microscopy. However, these mutants, unlike the parent strain, were unable to chemotax toward the known attractants coral mucus, dimethylsulfoniopropionate, and N-acetyl-d-glucosamine. The ΔcheB2 strain and an aflagellate ΔfliG1 strain were avirulent to coral, while the ΔcheA1 and ΔcheR2 strains were hypervirulent (90 to 100% infection within 14 h on average) compared to the wild-type strain (66% infection within 36 h on average). Additionally, the ΔcheA1 and ΔcheR2 strains appeared to better colonize coral fragments than the wild-type strain. These results suggest that although chemotaxis may be involved with infection (the ΔcheB2 strain was avirulent), a smooth-swimming phenotype is important for bacterial colonization and infection. This study provides valuable insight into understanding V. coralliilyticus pathogenesis and how this pathogen may be transmitted between hosts.IMPORTANCE Corals are responsible for creating the immense structures that are essential to reef ecosystems; unfortunately, pathogens like the bacterium Vibrio coralliilyticus can cause fatal infections of reef-building coral species. However, compared to related human pathogens, the mechanisms by which V. coralliilyticus initiates infections and locates new coral hosts are poorly understood. This study investigated the effects of chemotaxis, the directional swimming in response to chemical signals, and bacterial swimming patterns on infection of the coral Montipora capitata Infection experiments with different mutant strains suggested that a smooth-swimming pattern resulted in hypervirulence. These results demonstrate that the role of chemotaxis in coral infection may not be as straightforward as previously hypothesized and provide valuable insight into V. coralliilyticus pathogenesis.

Environmental Controls of Oyster-Pathogenic Vibrio spp. in Oregon Estuaries and a Shellfish Hatchery.

Vibrio spp. have been a persistent concern for coastal bivalve hatcheries, which are vulnerable to environmental pathogens in the seawater used for rearing larvae, yet the biogeochemical drivers of oyster-pathogenic Vibrio spp. in their planktonic state are poorly understood. Here, we present data tracking oyster-pathogenic Vibrio bacteria in Netarts Bay and Yaquina Bay in Oregon, USA, as well as in adjacent coastal waters and a local shellfish hatchery, through the 2015 upwelling season. Vibrio populations were quantified using a culture-independent approach of high-throughput Vibrio-specific 16S rRNA gene sequencing paired with droplet digital PCR, and abundances were analyzed in the context of local biogeochemistry. The most abundant putative pathogen in our samples was Vibrio coralliilyticus Environmental concentrations of total Vibrio spp. and V. coralliilyticus were highest in Netarts Bay sediment samples and higher in seawater from Netarts Bay than from nearshore coastal waters or Yaquina Bay. In Netarts Bay, the highest V. coralliilyticus concentrations were observed during low tide, and abundances increased throughout the summer. We hypothesize that the warm shallow waters in estuarine mudflats facilitate the local growth of the V. coralliilyticus pathogen. Samples from larval oyster tanks in Whiskey Creek Shellfish Hatchery, which uses seawater pumped directly from Netarts Bay, contained significantly lower total Vibrio species concentrations, but roughly similar V. coralliilyticus concentrations, than did the bay water, resulting in a 30-fold increase in the relative abundance of the V. coralliilyticus pathogen in hatchery tanks. This suggests that the V. coralliilyticus pathogen is able to grow or persist under hatchery conditions.IMPORTANCE It has been argued that oyster-pathogenic Vibrio spp. have contributed to recent mortality events in U.S. shellfish hatcheries (R. A. Elston, H. Hasegawa, K. L. Humphrey, I. K. Polyak, and C. Häse, Dis Aquat Organ 82:119-134, 2008, https://doi.org/10.3354/dao01982); however, these events are often sporadic and unpredictable. The success of hatcheries is critically linked to the chemical and biological composition of inflowing seawater resources; thus, it is pertinent to understand the biogeochemical drivers of oyster-pathogenic Vibrio spp. in their planktonic state. Here, we show that Netarts Bay, the location of a local hatchery, is enriched in oyster-pathogenic V. coralliilyticus compared to coastal seawater, and we hypothesize that conditions in tidal flats promote the local growth of this pathogen. Furthermore, V. coralliilyticus appears to persist in seawater pumped into the local hatchery. These results improve our understanding of the ecology and environmental controls of the V. coralliilyticus pathogen and could be used to improve future aquaculture efforts, as multiple stressors impact hatchery success.

Characterizing the Adherence Profiles of Virulent Vibrio parahaemolyticus Isolates.

The human pathogen Vibrio parahaemolyticus is a leading cause of seafood-borne illness in the USA, and infections with V. parahaemolyticus typically result from eating raw or undercooked oysters. V. parahaemolyticus has been shown to be highly resistant to oyster depuration, suggesting that the bacterium possesses specific mechanisms or factors for colonizing oysters and persisting during depuration. In this study, we characterized eight different V. parahaemolyticus strains for differences in resistance to oyster depuration, biofilm formation, and motility. While each strain exhibited distinct phenotypes in the various assays, we determined that biofilm formation on abiotic surfaces, such as glass or plastic, does not directly correlate with bacterial retention in oysters during depuration. However, we did observe that the motility phenotype of a strain appeared to be a better indicator for persistence in the oyster. Further studies examining the molecular mechanisms underlying the observed colonization differences by these and other V. parahaemolyticus strains may provide beneficial insights into what critical factors are required for proficient colonization of the Pacific oyster.

Chloroform-free permeabilization for improved detection of ß-galactosidase activity in Vibrio cholerae.

LacZ (ß-galactosidase) is used to monitor the transcription of genes in reporter strains carrying the lacZ gene under the control of a promotor of interest. This protocol for LacZ activity determinations in Vibrio cholerae following detergent lysis results in 2.5-fold increase of LacZ activities compared to lysis with chloroform.

Physiology of the Vc-NhaP paralogous group of cation-proton antiporters in Vibrio cholerae.

The genome of Vibrio cholerae encodes three cation-proton antiporters of NhaP-type, Vc-NhaP1, 2, and 3. To examine physiological roles of Vc-NhaP antiporters, triple ΔnhaP1ΔnhaP2ΔnhaP3 and single ΔnhaP3 deletion mutants of V. cholerae were constructed and characterized. Vc-NhaP3 was, for the first time, cloned and biochemically characterized. Activity measurements on the inside-out membrane vesicle experimental model defined Vc-NhaP3 as a potassium-specific cation-proton antiporter. While elimination of functional Vc-NhaP3 resulted in only minor growth defect in potassium-rich medium at pH 6.0, the triple Vc-NhaP mutant demonstrated severe growth defects at both low and high [K+] at pH 6.0 and failed to grow at high [K+] in mildly alkaline (pH 8.0 and 8.5) media, as well. Expressed from a plasmid, neither of the Vc-NhaP paralogues was able to complement the severe potassium-sensitive phenotype of the triple deletion mutant completely. Vc-NhaP1 provided much better complementation at acidic pH compared to Vc-NhaP2, despite the fact that Vc-NhaP2 showed much higher antiport activity in sub-bacterial vesicles. In mildly alkaline pH only Vc-NhaP2 complemented the potassium-sensitive phenotype of the triple deletion mutant. Taken together, these data suggest that in vivo all three isoforms operate in concert, contributing to K+ resistance of V. cholerae. We suggest that the Vc-NhaP paralogue group might play a role in passing gastric acid barrier by ingested V. cholerae cells.

Effects of chromosomal deletion of the operon encoding the multiple resistance and pH-related antiporter in Vibrio cholerae.

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae.

UNLABELLED: We searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogen Vibrio cholerae and addressed the mechanism of ROS formation using the dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparing V. cholerae strains with or without active Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR), this respiratory sodium ion redox pump was identified as a producer of ROS in vivo The amount of cytoplasmic ROS detected in V. cholerae cells producing variants of Na(+)-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-type V. cholerae showed increased superoxide production activity (9.8 ± 0.6 µmol superoxide min(-1) mg(-1) membrane protein) compared to membranes from the mutant lacking Na(+)-NQR (0.18 ± 0.01 µmol min(-1) mg(-1)). Overexpression of plasmid-encoded Na(+)-NQR in the nqr deletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 µmol min(-1) mg(-1)). By analyzing a variant of Na(+)-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation in V. cholerae The impact of superoxide formation by the Na(+)-NQR on the virulence of V. cholerae is discussed. IMPORTANCE: In several studies, it was demonstrated that the Na(+)-NQR in V. cholerae affects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na(+)-NQR as the site of superoxide formation in the cytoplasm of V. cholerae Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, like V. cholerae, depend on the Na(+)-NQR as the sole electrogenic NADH dehydrogenase.

Mortalities of Eastern and Pacific oyster Larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii.

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD50) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10(4) to 3.0 × 10(4) CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD50s between 1.2 × 10(4) and 4.0 × 10(4) CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10(4) CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.

Roles of the sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) on vibrio cholerae metabolism, motility and osmotic stress resistance.

The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology.