Genome analysis of the coral bleaching pathogen <Emphasis Type="Italic">Vibrio shiloi</Emphasis>

The past few decades have seen a world-wide increase in coral diseases, yet little is known about coral pathogens. In this study, techniques commonly used in pathogenomic research were applied to the coral pathogen Vibrio shiloi in order to identify genetic elements involved in its virulence. Suppressive subtractive hybridization was used to compare the gene content of V. shiloi to that of a closely related but non-pathogenic bacterium, Vibrio mediterranei, resulting in identification of several putative virulence factors and of three novel genomic islands. The entire genome of V. shiloi was further screened for genes related to previously characterized steps in infection: adhesion, superoxide dismutase production and toxin production. Exposure of pure cultures of V. shiloi to crushed coral tissues strongly affected the expression of seven genes encoding pili, zona occludins toxin (Zot) and a superoxide dismutase. Analysis of eight V. shiloi strains isolated in the last decade shows a shift of the natural population from strains carrying all three genomic islands to strains carrying none of them. This shift occurred following appearance of resistance in the coral Oculina patagonica to infection by V. shiloi. The relevance of these findings to the bleaching disease caused by V. shiloi is discussed.     

The coral bleaching Vibrio shiloi Kushmaro et al. 2001 is a later synonym of Vibrio mediterranei Pujalte and Garay 1986

The coral bleaching Vibrio shiloi LMG 19703T was characterized by means of Fluorescent Amplified Fragment Length Polymorphism (FAFLP), DNA-DNA hybridisation, mol% G+C content, fatty acids methyl ester (FAME) analysis and phenotypical tests. Numerical analysis of the FAFLP band patterns indicated that the type strain of V. shiloi in fact belongs to the species V. mediterranei. The type strains of both species shared 77% DNA similarity, as determined by DNA-DNA hybridisation experiments at stringent conditions. Moreover, V. shiloi and V. mediterranei showed almost identical fatty acid composition and phenotypical features. Collectively, the genotypic and phenotypic data presented in this study suggest that V. shiloi Kushmaro et al. 2001 should be considered a later synonym of V. mediterranei Pujalte and Garay 1986. The involvement of V. mediterranei in coral bleaching was unknown until now.     

Superoxide dismutase is a virulence factor produced by the coral bleaching pathogen Vibrio shiloi

Coral bleaching is a disease that threatens coral reefs throughout the world. The disease is correlated with higher-than-normal seawater temperatures. Data have been reported showing that bleaching of the coral Oculina patagonica during the summer in the Mediterranean Sea is the result of an infection with Vibrio shiloi. The summer temperatures induce the expression of virulence factors in the pathogen. We report here that V. shiloi produces an extracellular superoxide dismutase (SOD) at 30 degrees C, but not at 16 degrees C. An SOD(-) mutant was avirulent. The mutant adhered to corals, penetrated into coral cells, multiplied intracellularly for a short time, and then died. These data support the hypothesis that SOD protects the intracellular V. shiloi from oxidative stress caused by the high concentration of oxygen produced by intracellular zooxanthellae photosynthesis.     

The marine fireworm Hermodice carunculata is a winter reservoir and spring-summer vector for the coral-bleaching pathogen Vibrio shiloi

Vibrio shiloi, the causative agent of bleaching of the coral Oculina patagonica in the Mediterranean Sea, is present in all bleached O. patagonica corals in the summer (25-30 degrees C), but can be not detected in the coral during the winter (16-20 degrees C). Furthermore, the pathogen can not survive in O. patagonica at temperatures below 20 degrees C. Using fluorescence in situ hybridization (FISH) with a V. shiloi-specific oligonucleotide probe, we found that the marine fireworm Hermodice caranculata is a winter reservoir for V. shiloi. Worms taken directly from the sea during the winter contained approximately 10(8) V. shiloi per worm by FISH analysis. However, colony-forming units (cfu) revealed only 4.1-18.3 x 10(4) V. shiloi per worm, indicating that approximately 99.9% of them were in the viable-but-not-culturable (VBNC) state. When worms were infected with V. shiloi, most of the bacteria adhered to the worm within 24 h and then penetrated into epidermal cells. By 48 h, less than 10(-4) of the intact V. shiloi in the worm gave rise to colonies, suggesting that they differentiated inside the worm into the VBNC state. When worms infected with V. shiloi were placed in aquaria containing O. patagonica, all of the corals showed small patches of bleached tissue in 7-10 days and total bleaching in 17 days. This is the first report of a reservoir and vector for a coral disease.     

Antimicrobial properties of resident coral mucus bacteria of Oculina patagonica

The inhibitory properties of the microbial community of the coral mucus from the Mediterranean coral Oculina patagonica were examined. Out of 156 different colony morphotypes that were isolated from the coral mucus, nine inhibited the growth of Vibrio shiloi , a species previously shown to be a pathogen of this coral. An isolate identified as Pseudoalteromonas sp. was the strongest inhibitor of V. shiloi . Several isolates, especially one identified as Roseobacter sp., also showed a broad spectrum of action against the coral pathogens Vibrio coralliilyticus and Thallassomonas loyana , plus nine other selected Gram-positive and Gram-negative bacteria. Inoculation of a previously established biofilm of the Roseobacter strain with V. shiloi led to a 5-log reduction in the viable count of the pathogen within 3 h, while inoculation of a Pseudoalteromonas biofilm led to complete loss of viability of V. shiloi after 3 h. These results support the concept of a probiotic effect on microbial communities associated with the coral holobiont.     

Inhibition of photosynthesis and bleaching of zooxanthellae by the coral pathogen Vibrio shiloi

Vibrio shiloi is the causative agent of bleaching (loss of endosymbiotic zooxanthellae) of the coral Oculina patagonica in the Mediterranean Sea. To obtain information on the mechanism of bleaching, we examined the effect of secreted material (AK1-S) produced by V. shiloi on zooxanthellae isolated from corals. AK1-S caused a rapid inhibition of photosynthesis of the algae, as measured with a Mini-PAM fluorometer. The inhibition of photosynthesis was caused by (i) ammonia produced during the growth of V. shiloi on protein-containing media and (ii) a non-dialysable heat-resistant factor. This latter material did not inhibit photosynthesis of the algae by itself but, when added to different concentrations of NH₄Cl, enhanced the inhibition approximately two- to threefold. Ammonia and the enhancer were effective to different degrees on zooxanthellae isolated from four species of coral examined. In addition to the rapid inhibition of photosynthesis, AK1-S caused bleaching (loss of pigmentation) and lysis of zooxanthellae. Bleaching was more rapid than lysis, reaching a peak (25% bleached algae) after 6 h. The factors in AK1-S responsible for bleaching and lysis were different from those responsible for the inhibition of photosynthesis, because they were heat sensitive, non-dialysable and active in the dark. Thus, the coral pathogen V. shiloi produces an array of extracellular materials that can inhibit photosynthesis, bleach and lyse zooxanthellae.     

Differential effects of temperature and starvation on induction of the viable-but-nonculturable state in the coral pathogens Vibrio shiloi and Vibrio tasmaniensis

We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20 degrees C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17 degrees C), the VBNC state was not induced by incubation at 4 degrees C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30 degrees C), was induced into the VBNC state by incubation at 4 degrees C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20 degrees C, and V. shiloi became VBNC after incubation for 126 days at 20 degrees C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.     

Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica

Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a beta-D-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29 degrees C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloi antibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.     

Coral Disease Diagnostics: What's between a Plague and a Band?

Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.     

Coral disease diagnostics: what's between a plague and a band?

Recently, reports of coral disease have increased significantly across the world's tropical oceans. Despite increasing efforts to understand the changing incidence of coral disease, very few primary pathogens have been identified, and most studies remain dependent on the external appearance of corals for diagnosis. Given this situation, our current understanding of coral disease and the progression and underlying causes thereof is very limited. In the present study, we use structural and microbial studies to differentiate different forms of black band disease: atypical black band disease and typical black band disease. Atypical black band diseased corals were infected with the black band disease microbial consortium yet did not show any of the typical external signs of black band disease based on macroscopic observations. In previous studies, these examples, here referred to as atypical black band disease, would have not been correctly diagnosed. We also differentiate white syndrome from white diseases on the basis of tissue structure and the presence/absence of microbial associates. White diseases are those with dense bacterial communities associated with lesions of symbiont loss and/or extensive necrosis of tissues, while white syndromes are characteristically bacterium free, with evidence for extensive programmed cell death/apoptosis associated with the lesion and the adjacent tissues. The pathology of coral disease as a whole requires further investigation. This study emphasizes the importance of going beyond the external macroscopic signs of coral disease for accurate disease diagnosis.     

The coral probiotic hypothesis

Emerging diseases have been responsible for the death of about 30% of corals worldwide during the last 30 years. Coral biologists have predicted that by 2050 most of the world's coral reefs will be destroyed. This prediction is based on the assumption that corals can not adapt rapidly enough to environmental stress-related conditions and emerging diseases. Our recent studies of the Vibrio shiloi/Oculina patagonica model system of the coral bleaching disease indicate that corals can indeed adapt rapidly to changing environmental conditions by altering their population of symbiotic bacteria. These studies have led us to propose the Coral Probiotic Hypothesis. This hypothesis posits that a dynamic relationship exists between symbiotic microorganisms and environmental conditions which brings about the selection of the most advantageous coral holobiont. Changing their microbial partners would allow the corals to adapt to changing environmental conditions more rapidly (days to weeks) than via mutation and selection (many years). An important outcome of the Probiotic Hypothesis would be development of resistance of the coral holobiont to diseases. The following evidence supports this hypothesis: (i) Corals contain a large and diverse bacterial population associated with their mucus and tissues; (ii) the coral-associated bacterial population undergoes a rapid change when environmental conditions are altered; and (iii) although lacking an adaptive immune system (no antibodies), corals can develop resistance to pathogens. The Coral Probiotic Hypothesis may help explain the evolutionary success of corals and moderate the predictions of their demise.     

Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.     

Status and progress in coral reef disease research

Recent findings on the ecology, etiology and pathology of coral pathogens, host resistance mechanisms, previously unknown disease/syndromes and the global nature of coral reef diseases have increased our concern about the health and future of coral reef communities. Much of what has been discovered in the past 4 years is presented in this special issue. Among the significant findings, the role that various Vibrio species play in coral disease and health, the composition of the 'normal microbiota' of corals, and the possible role of viruses in the disease process are important additions to our knowledge. New information concerning disease resistance and vectors, variation in pathogen composition for both fungal diseases of gorgonians and black band disease across oceans, environmental effects on disease susceptibility and resistance, and temporal and spatial disease variations among different coral species is presented in a number of papers. While the Caribbean may still be the 'disease hot spot' for coral reefs, it is now clear that diseases of coral reef organisms have become a global threat to coral reefs and a major cause of reef deterioration.     

How Microbial Community Composition Regulates Coral Disease Development

Reef coral cover is in rapid decline worldwide, in part due to bleaching (expulsion of photosynthetic symbionts) and outbreaks of infectious disease. One important factor associated with bleaching and in disease transmission is a shift in the composition of the microbial community in the mucus layer surrounding the coral: the resident microbial community—which is critical to the healthy functioning of the coral holobiont—is replaced by pathogenic microbes, often species of Vibrio. In this paper we develop computational models for microbial community dynamics in the mucus layer in order to understand how the surface microbial community responds to changes in environmental conditions, and under what circumstances it becomes vulnerable to overgrowth by pathogens. Some of our model''s assumptions and parameter values are based on Vibrio spp. as a model system for other established and emerging coral pathogens. We find that the pattern of interactions in the surface microbial community facilitates the existence of alternate stable states, one dominated by antibiotic-producing beneficial microbes and the other pathogen-dominated. A shift to pathogen dominance under transient stressful conditions, such as a brief warming spell, may persist long after environmental conditions have returned to normal. This prediction is consistent with experimental findings that antibiotic properties of Acropora palmata mucus did not return to normal long after temperatures had fallen. Long-term loss of antibiotic activity eliminates a critical component in coral defense against disease, giving pathogens an extended opportunity to infect and spread within the host, elevating the risk of coral bleaching, disease, and mortality.     

Antagonistic interactions among coral-associated bacteria

Reef-building corals are comprised of close associations between the coral animal, symbiotic zooxanthellae, and a diversity of associated microbes (including Bacteria, Archaea and Fungi). Together, these comprise the coral holobiont – a paradigm that emphasizes the potential contributions of each component to the overall function and health of the coral. Little is known about the ecology of the coral-associated microbial community and its hypothesized role in coral health. We explored bacteria–bacteria antagonism among 67 bacterial isolates from the scleractinian coral Montastrea annularis at two temperatures using Burkholder agar diffusion assays. A majority of isolates exhibited inhibitory activity (69.6% of isolates at 25°C, 52.2% at 31°C), with members of the γ-proteobacteria ( Vibrionales and Alteromonadales ) being especially antagonistic. Elevated temperatures generally reduced levels of antagonism, although the effects were complex. Several potential pathogens were observed in the microbial community of apparently healthy corals, and 11.6% of isolates were able to inhibit the growth of the coral pathogen Vibrio shiloi at 25°C. Overall, this study demonstrates that antagonism could be a structuring force in coral-associated microbial communities and may contribute to pathogenesis as well as disease resistance.     

Integrating microbiological, microsensor, molecular, and physiologic techniques in the study of coral disease pathogenesis

The study of coral diseases requires an integrated approach that includes a combination of field and laboratory methods. By combining and building upon information available from multiple disciplines, within both field and laboratory applications, we have been successful in characterizing a number of coral diseases. To illustrate the utility of the integrative approach two very different coral diseases, black band disease and plague, are discussed in detail. Comparison of our ongoing characterization of each disease demonstrates that, within the integrative approach, different combinations of microbiological, microsensor, molecular, and physiologic techniques are required. The pathobiology of black band disease, which consists of a complicated, synergistic microbial consortium functioning around a dynamic sulfur cycle, is slowly being unraveled using a combination of methods. Our study of plague, on the other hand, has progressed in a very different manner that is controlled by the fact that this disease has, to date, emerged in three forms on reefs of the Florida Keys. The study of plague types I, II, and III will be detailed to illustrate the difficulty of characterizing a disease that rapidly evolves in the natural environment of the reef. Our ongoing study of additional (also very different) coral diseases will be summarized from the perspective of combined methodologies to illustrate the range and magnitude of questions that must be addressed and answered in order to understand coral disease pathogenesis and thus coral disease etiology.     

Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef

The microbial community associated with the reef building coral Pocillopora damicornis located on the Great Barrier Reef was investigated using culture-independent molecular microbial techniques. The microbial communities of three separate coral colonies were assessed using clone library construction alongside restriction fragment length polymorphism and phylogenetic analysis. Diversity was also investigated spatially across six replicate samples within each single coral colony using 16S rDNA and rpoB-DGGE analysis. Clone libraries demonstrated that the majority of retrieved sequences from coral tissue slurry libraries affiliated with gamma-Proteobacteria. This contrasted with clone libraries of seawater and coral mucus, which were dominated by alpha-Proteobacteria. A number of retrieved clone sequences were conserved between coral colonies; a result consistent with previous studies suggesting a specific microbe-coral association. rpoB-DGGE patterns of replicate tissue slurry samples underestimated microbial diversity, but demonstrated that fingerprints were identical within the same coral. These fingerprints were also conserved across coral colonies. The 16S rDNA-DGGE patterns of replicate tissue slurry samples were more complex, although non-metric multidimensional scaling (nMDS) analysis showed groupings of these banding patterns indicating that some bacterial diversity was uniform within a coral colony. Sequence data retrieved from DGGE analysis support clone library data in that the majority of affiliations were within the gamma-Proteobacteria. Many sequences retrieved also affiliated closely with sequences derived from previous studies of microbial diversity of healthy corals in the Caribbean. Clones showing high 16S rDNA sequence identity to both Vibrio shiloi and Vibrio coralliilyticus were retrieved, suggesting that these may be opportunist pathogens. Comparisons of retrieved microbial diversity between two different sampling methods, a syringe extracted coral mucus sample and an airbrushed coral tissue slurry sample were also investigated. Non-metric multidimensional scaling of clone library data highlighted that clone diversity retrieved from a coral mucus library more closely reflected the diversity of surrounding seawater than a corresponding coral tissue clone library.     

Phage therapy of coral disease

At present there are no known procedures for preventing or treating infectious diseases of corals. Toward this end, the use of phage therapy has been investigated. Lytic bacteriophages (phages) were isolated for two bacterial pathogens that are responsible for coral diseases, Vibrio coralliilyticus, which is the causative agent of bleaching and tissue lysis of Pocillopora damicornis, and Thalosomonas loyaeana, which causes the white plague-like disease of Favia favus. By using these phages in controlled aquaria experiments, it was demonstrated that each of these diseases could be controlled by the pathogen-specific phage. The data indicate that initially the phages bind to the pathogen in seawater and are then brought to the coral surface where they multiply and lyse the pathogen. The phages remained associated with the coral and could prevent subsequent infections. These data suggest that phage therapy has the potential to control the spread of infectious coral diseases.     

Bacterial Communities Associated with Porites White Patch Syndrome (PWPS) on Three Western Indian Ocean (WIO) Coral Reefs

The scleractinian coral Porites lutea, an important reef-building coral on western Indian Ocean reefs (WIO), is affected by a newly-reported white syndrome (WS) the Porites white patch syndrome (PWPS). Histopathology and culture-independent molecular techniques were used to characterise the microbial communities associated with this emerging disease. Microscopy showed extensive tissue fragmentation generally associated with ovoid basophilic bodies resembling bacterial aggregates. Results of 16S rRNA sequence analysis revealed a high variability between bacterial communities associated with PWPS-infected and healthy tissues in P. lutea, a pattern previously reported in other coral diseases such as black band disease (BBD), white band disease (WBD) and white plague diseases (WPD). Furthermore, substantial variations in bacterial communities were observed at the different sampling locations, suggesting that there is no strong bacterial association in Porites lutea on WIO reefs. Several sequences affiliated with potential pathogens belonging to the Vibrionaceae and Rhodobacteraceae were identified, mainly in PWPS-infected coral tissues. Among them, only two ribotypes affiliated to Shimia marina (NR043300.1) and Vibrio hepatarius (NR025575.1) were consistently found in diseased tissues from the three geographically distant sampling localities. The role of these bacterial species in PWPS needs to be tested experimentally.     

The bacterial ecology of a plague-like disease affecting the Caribbean coral Montastrea annularis

The bacterial communities associated with the Caribbean coral Montastrea annularis showing tissue lesions indicative of a White Plague (WP)-like disease were investigated. Two molecular screening techniques using bacterial 16S rDNA genes were used and demonstrated distinct differences between the bacterial communities of diseased and non-diseased coral tissues, and also in relation to the proximity of tissue lesions on diseased corals. Differences between non-diseased corals and the apparently healthy tissues remote from the tissue lesion in affected corals indicates a 'whole coral' response to a relatively small area of infection with a perturbation in the normal microbial flora occurring prior to the onset of visible signs of disease. These whole organism changes in the microbial flora may serve as a bioindicator of environmental stress and disease. There were striking similarities between the 16S rDNA sequence composition associated with the WP-like disease studied here and that previously reported in association with black band disease (BBD) in coral. Similarities included the presence of a potential pathogen, an alpha-proteobacterium identified as the causal agent of juvenile oyster disease (JOD). The WP-like disease studied here is apparently different to WP Type ii because the bacterial species previously identified as the causal agent of WP Type ii was not detected, although the symptoms of the two diseases are similar.