Vibrio Coralliilyticus Research Articles

Outcomes of Infections of Sea Anemone Aiptasia pallida with Vibrio spp. Pathogenic to Corals

Incidents of coral disease are on the rise. However, in the absence of a surrogate animal host, understanding of the interactions between coral pathogens and their hosts remains relatively limited, compared to other pathosystems of similar global importance. A tropical sea anemone, Aiptasia pallida, has been investigated as a surrogate model to study certain aspects of coral biology. Therefore, to test whether the utility of this surrogate model can be extended to study coral diseases, in the present study, we tested its susceptibility to common coral pathogens (Vibrio coralliilyticus and Vibrio shiloi) as well as polymicrobial consortia recovered from the Caribbean Yellow Band Disease (CYBD) lesions. A. pallida was susceptible to each of the tested pathogens. A. pallida responded to the pathogens with darkening of the tissues (associated with an increased melanization) and retraction of tentacles, followed by complete disintegration of polyp tissues. Loss of zooxanthellae was not observed; however, the disease progression pattern is consistent with the behavior of necrotizing pathogens. Virulence of some coral pathogens in Aiptasia was paralleled with their glycosidase activities.

The specifically enhanced cellular immune responses in Pacific oyster (Crassostrea gigas) against secondary challenge with Vibrio splendidus

The increasing experimental evidences suggest that there are some forms of specific acquired immunity in invertebrates, but the underlying mechanism is not fully understood. In the present study, Pacific oyster (Crassostrea gigas) stimulated primarily by heat-killed Vibrio splendidus displayed stronger immune responses at cellular and molecular levels when they encountered the secondary challenge of live V. splendidus. The total hemocyte counts (THC) increased significantly after the primary stimulation of heat-killed V. splendidus, and it increased even higher (p<0.01) and reached the peak earlier (at 6h) after the secondary challenge with live V. splendidus compared with that of the primary stimulation. The number of new generated circulating hemocytes increased dramatically (p<0.01) at 6h after the pre-stimulated oysters received the secondary stimulation with live V. splendidus, and the phagocytic rate was also enhanced significantly (p<0.01) at 12h after the secondary stimulation. Meanwhile, the enhanced phagocytosis of hemocytes was highly specific for V. splendidus and they could distinguish Vibrio anguillarum, Vibrio coralliilyticus, Yarrowia lipolytica, and Micrococcus luteus efficiently. In addition, the mRNA expression of 12 candidate genes related to phagocytosis and hematopoiesis were also monitored, and the expression levels of CgIntegrin, CgPI3K (phosphatidylinositol 3-kinase), CgRho J, CgMAPKK (mitogen-activated protein kinase kinase), CgRab32, CgNADPH (nicotinamide adenine dinucleotide phosphate) oxidase, CgRunx1 and CgBMP7 (bone morphogenetic protein 7) in the hemocytes of pre-stimulated oysters after the secondary stimulation of V. splendidus were higher (p<0.01) than that after the primary stimulation, but there was no statistically significant changes for the genes of CgPKC (protein kinase C), CgMyosin, CgActin, and CgGATA 3. These results collectively suggested that the primary stimulation of V. splendidus led to immune priming in oyster with specifically enhanced phagocytosis and rapidly promoted regeneration of circulating hemocytes when the primed oysters encountered the secondary challenge with V. splendidus.

Temperature-Dependent Inhibition of Opportunistic Vibrio Pathogens by Native Coral Commensal Bacteria

Bacteria living within the surface mucus layer of corals compete for nutrients and space. A number of stresses affect the outcome of this competition. The interactions between native microorganisms and opportunistic pathogens largely determine the coral holobiont's overall health and fitness. In this study, we tested the hypothesis that commensal bacteria isolated from the mucus layer of a healthy elkhorn coral, Acropora palmata, are capable of inhibition of opportunistic pathogens, Vibrio shiloi AK1 and Vibrio coralliilyticus. These vibrios are known to cause disease in corals and their virulence is temperature dependent. Elevated temperature (30 °C) increased the cell numbers of one commensal and both Vibrio pathogens in monocultures. We further tested the hypothesis that elevated temperature favors pathogenic organisms by simultaneously increasing the fitness of vibrios and decreasing the fitness of commensals by measuring growth of each species within a co-culture over the course of 1 week. In competition experiments between vibrios and commensals, the proportion of Vibrio spp. increased significantly under elevated temperature. We finished by investigating several temperature-dependent mechanisms that could influence co-culture differences via changes in competitive fitness. The ability of Vibrio spp. to utilize glycoproteins found in A. palmata mucus increased or remained stable when exposed to elevated temperature, while commensals' tended to decrease utilization. In both vibrios and commensals, protease activity increased at 30 °C, while chiA expression increased under elevated temperatures for Vibrio spp. These results provide insight into potential mechanisms through which elevated temperature may select for pathogenic bacterial dominance and lead to disease or a decrease in coral fitness.

Autolysis in Vibrio tubiashii and Vibrio coralliilyticus

Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams, and shellfish hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis outbreaks since 2006. Like V. tubiashii, Vibrio coralliilyticus has recently been described as an oyster pathogen in addition to its role in coral disease. Here, we describe an autolysis phenotype in V. tubiashii and its close relative V. coralliilyticus and characterize the effects of environmental conditions on this phenotype. We also explored whether the survivors of autolysis were resistant to the phenotype and if material from the autolysed culture would either regrow or have a population of viable cells. Ultimately, this work contributes to the larger understanding of bacterial population dynamics as it relates to aquaculture pathogens.

A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals

Diseases are an emerging threat to ocean ecosystems. Coral reefs, in particular, are experiencing a worldwide decline because of disease and bleaching, which have been exacerbated by rising seawater temperatures. Yet, the ecological mechanisms behind most coral diseases remain unidentified. Here, we demonstrate that a coral pathogen, Vibrio coralliilyticus, uses chemotaxis and chemokinesis to target the mucus of its coral host, Pocillopora damicornis. A primary driver of this response is the host metabolite dimethylsulfoniopropionate (DMSP), a key element in the global sulfur cycle and a potent foraging cue throughout the marine food web. Coral mucus is rich in DMSP, and we found that DMSP alone elicits chemotactic responses of comparable intensity to whole mucus. Furthermore, in heat-stressed coral fragments, DMSP concentrations increased fivefold and the pathogen's chemotactic response was correspondingly enhanced. Intriguingly, despite being a rich source of carbon and sulfur, DMSP is not metabolized by the pathogen, suggesting that it is used purely as an infochemical for host location. These results reveal a new role for DMSP in coral disease, demonstrate the importance of chemical signaling and swimming behavior in the recruitment of pathogens to corals and highlight the impact of increased seawater temperatures on disease pathways.

An Improved Detection and Quantification Method for the Coral Pathogen Vibrio coralliilyticus

DNA- and RNA-based PCR and reverse-transcription real-time PCR assays were developed for diagnostic detection of the vcpA zinc-metalloprotease implicated in the virulence of the coral pathogen Vibrio coralliilyticus. Both PCR methods were highly specific for V. coralliilyticus and failed to amplify strains of closely-related Vibrio species. The assays correctly detected all globally occurring V. coralliilyticus isolates including a newly-described isolate [TAV24] infecting gorgonians in the Mediterranean Sea and highlighted those isolates that had been potentially misidentified, in particular V. tubiashii strains ATCC 19105 and RE22, historically described as important oyster pathogens. The real-time assay is sensitive, detecting 10 gene copies and the relationships between gene copy number and cycle threshold (CT) were highly linear (R2≥99.7). The real-time assay was also not affected by interference from non-target DNA. These assays are useful for rapid detection of V. coralliilyticus and monitoring of virulence levels in environmental samples, allowing for implementation of timely management steps to limit and possibly prevent losses due to V. coralliilyticus infection, as well as furthering investigations of factors affecting pathogenesis of this important marine pathogen.

Vibrio coralliilyticus Search Patterns across an Oxygen Gradient

The coral pathogen, Vibrio coralliilyticus shows specific chemotactic search pattern preference for oxic and anoxic conditions, with the newly identified 3-step flick search pattern dominating the patterns used in oxic conditions. We analyzed motile V. coralliilyticus cells for behavioral changes with varying oxygen concentrations to mimic the natural coral environment exhibited during light and dark conditions. Results showed that 3-step flicks were 1.4× (P = 0.006) more likely to occur in oxic conditions than anoxic conditions with mean values of 18 flicks (95% CI = 0.4, n = 53) identified in oxic regions compared to 13 (95% CI = 0.5, n = 38) at anoxic areas. In contrast, run and reverse search patterns were more frequent in anoxic regions with a mean value of 15 (95% CI = 0.7, n = 46), compared to a mean value of 10 (95% CI = 0.8, n = 29) at oxic regions. Straight swimming search patterns remained similar across oxic and anoxic regions with a mean value of 13 (95% CI = 0.7, n = oxic: 13, anoxic: 14). V. coralliilyticus remained motile in oxic and anoxic conditions, however, the 3-step flick search pattern occurred in oxic conditions. This result provides an approach to further investigate the 3-step flick.

Transcriptional activation of c3 and hsp70 as part of the immune response of Acropora millepora to bacterial challenges.

The impact of disease outbreaks on coral physiology represents an increasing concern for the fitness and resilience of reef ecosystems. Predicting the tolerance of corals to disease relies on an understanding of the coral immune response to pathogenic interactions. This study explored the transcriptional response of two putative immune genes (c3 and c-type lectin) and one stress response gene (hsp70) in the reef building coral, Acropora millepora challenged for 48 hours with bacterial strains, Vibrio coralliilyticus and Alteromonas sp. at concentrations of 106 cells ml-1. Coral fragments challenged with V. coralliilyticus appeared healthy while fragments challenged with Alteromonas sp. showed signs of tissue lesions after 48 hr. Coral-associated bacterial community profiles assessed using denaturing gradient gel electrophoresis changed after challenge by both bacterial strains with the Alteromonas sp. treatment demonstrating the greatest community shift. Transcriptional profiles of c3 and hsp70 increased at 24 hours and correlated with disease signs in the Alteromonas sp. treatment. The expression of hsp70 also showed a significant increase in V. coralliilyticus inoculated corals at 24 h suggesting that even in the absence of disease signs, the microbial inoculum activated a stress response in the coral. C-type lectin did not show a response to any of the bacterial treatments. Increase in gene expression of c3 and hsp70 in corals showing signs of disease indicates their potential involvement in immune and stress response to microbial challenges.

16SrDNA Pyrosequencing of the Mediterranean Gorgonian Paramuricea clavata Reveals a Link among Alterations in Bacterial Holobiont Members, Anthropogenic Influence and Disease Outbreaks

Mass mortality events of benthic invertebrates in the Mediterranean Sea are becoming an increasing concern with catastrophic effects on the coastal marine environment. Sea surface temperature anomalies leading to physiological stress, starvation and microbial infections were identified as major factors triggering animal mortality. However the highest occurrence of mortality episodes in particular geographic areas and occasionally in low temperature deep environments suggest that other factors play a role as well. We conducted a comparative analysis of bacterial communities associated with the purple gorgonian Paramuricea clavata, one of the most affected species, collected at different geographic locations and depth, showing contrasting levels of anthropogenic disturbance and health status. Using massive parallel 16SrDNA gene pyrosequencing we showed that the bacterial community associated with healthy P. clavata in pristine locations was dominated by a single genus Endozoicomonas within the order Oceanospirillales which represented ∼90% of the overall bacterial community. P. clavata samples collected in human impacted areas and during disease events had higher bacterial diversity and abundance of disease-related bacteria, such as vibrios, than samples collected in pristine locations whilst showed a reduced dominance of Endozoicomonas spp. In contrast, bacterial symbionts exhibited remarkable stability in P. clavata collected both at euphotic and mesophotic depths in pristine locations suggesting that fluctuations in environmental parameters such as temperature have limited effect in structuring the bacterial holobiont. Interestingly the coral pathogen Vibrio coralliilyticus was not found on diseased corals collected during a deep mortality episode suggesting that neither temperature anomalies nor recognized microbial pathogens are solely sufficient to explain for the events. Overall our data suggest that anthropogenic influence may play a significant role in determining the coral health status by affecting the composition of the associated microbial community. Environmental stressful events and microbial infections may thus be superimposed to compromise immunity and trigger mortality outbreaks.

The domain of unknown function DUF1521 exhibits metal ion-inducible autocleavage activity - a novel example from a putative effector protein ofVibrio coralliilyticusATCC BAA-450

Vibrio coralliilyticus ATCC BAA-450 is a pathogen causing coral bleaching at elevated seawater temperatures. Based on the available genome sequence, the strain has a type III secretion system. Within the corresponding gene cluster, VIC_001052 is encoded, which contains a conserved domain of unknown function DUF1521. In this study, we show that the purified domain exhibits autocleavage activity in the presence of several divalent metal ions, for example, calcium and manganese but not with magnesium or zinc. Autocleavage is not affected by temperatures between 0 and 30°C, indicating that seawater temperature is not a critical factor for this activity. The DUF1521 domain and the cleavage site are conserved in several proteins from proteobacteria, suggesting a similar cleavage activity for these proteins.

Bacterial enhancement of bleaching and physiological impacts on the coral Montipora digitata

Bleaching affects corals worldwide. We studied the effects of high seawater temperature and bacterial challenges on bleaching, metabolism, and antioxidant defenses of coral. The coral Montipora digitata and five bacterial strains (Vibrio coralliilyticus, Vibrio harveyi, Paracoccus carotinifaciens, Pseudoalteromonas sp., and Sulfitobacter sp.) were used in an inoculation experiment. The bacteria were isolated from the bleached corals and their surrounding seawater. At normal temperatures, the bacteria did not cause coral bleaching and did not affect metabolism or antioxidant enzyme activities. However, bacterial challenges in addition to high-temperature stress resulted in coral bleaching, with a 70% decrease in zooxanthellae density compared with the control, a 25% decrease in photosynthetic efficiency (Fv/Fm), a 66% decrease in photosynthesis, and a 101% reduction in calcification activity. Tissue necrosis was observed in the most compromised branches. Respiration and activity levels of antioxidant enzymes were not affected. Corals under thermal stress alone showed signs of bleaching, but the changes in zooxanthellae density and metabolic rates were less severe than those under synergistic thermal stress and bacterial challenge. Among the bacteria examined, Sulfitobacter sp. had a greater capacity to enhance and accelerate the bleaching process under thermal stress. The mechanisms by which bacteria affect corals are not yet understood. Direct actions include infection and production of toxic substances by bacteria; indirect effects due to changes in the bacterial community under the influence of added bacteria must also be considered. Our results suggest that bacterial challenges combined with thermal stress can synergistically lead to negative outcomes in corals.

Phage therapy treatment of the coral pathogen Vibrio coralliilyticus

Vibrio coralliilyticus is an important coral pathogen demonstrated to cause disease outbreaks worldwide. This study investigated the feasibility of applying bacteriophage therapy to treat the coral pathogen V. coralliilyticus. A specific bacteriophage for V. coralliilyticus strain P1 (LMG23696), referred to here as bacteriophage YC, was isolated from the seawater above corals at Nelly Bay, Magnetic Island, central Great Barrier Reef (GBR), the same location where the bacterium was first isolated. Bacteriophage YC was shown to be a lytic phage belonging to the Myoviridae family, with a rapid replication rate, high burst size, and high affinity to its host. By infecting its host bacterium, bacteriophage YC was able to prevent bacterial-induced photosystem inhibition in pure cultures of Symbiodinium, the photosymbiont partner of coral and a target for virulence factors produced by the bacterial pathogen. Phage therapy experiments using coral juveniles in microtiter plates as a model system revealed that bacteriophage YC was able to prevent V. coralliilyticus-induced photoinactivation and tissue lysis. These results demonstrate that bacteriophage YC has the potential to treat coral disease outbreaks caused by the bacterial pathogen V. coralliilyticus, making it a good candidate for phage therapy treatment of coral disease.

Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system

Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo. However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/vox. Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/vox is a novel powerful and safe instrument in the ‘genetic toolbox’ that can be used alone or in combination with other SSRs in heterologous hosts.

NMR analysis of metabolic responses to extreme conditions of the temperature-dependent coral pathogen Vibrio coralliilyticus

To identify and understand the presence of metabolites responsible for the variation in the metabolic profile of Vibrio coralliilyticus under extreme conditions. Multiple batches of V. coralliilyticus were grown under normal conditions. Four samples in one batch were subjected to extreme conditions via a freeze-thaw cycle during lyophilization. Polar metabolites were extracted using a combination of methanol, water and heat. Nuclear magnetic resonance (NMR)-based metabolic profiles indicated significant differences between the normal and stressed samples. Three compounds identified in the stressed metabolome were maltose, ethanolamine, and the bioplastic-type compound (BTC) 2-butenoic acid, 2-carboxy-1-methylethyl ester. This is the first report of the production of this BTC by V. coralliilyticus. The presence of maltose and ethanolamine indicates a state of acute nutrient limitation; therefore, we hypothesize that the cell's metabolism turned to its own cell wall, or perhaps neighbouring cells, for sources of carbon and nitrogen. The presence of the BTC also supports the acute nutrient limitation idea because of the parallels with polyhydroxyalkanoate (PHA) production in other gram-negative bacteria, including other Vibrio species. Recent metabolomics research on the temperature-dependent coral pathogen V. coralliilyticus has led to the discovery of several compounds produced by the organism as a response to high density, low nutrient conditions. The three metabolites, along with (1) H NMR metabolic fingerprints of the nutrient limited samples, are proposed to serve as metabolic markers for extremely stressful conditions of V. coralliilyticus.

Corals shed bacteria as a potential mechanism of resilience to organic matter enrichment

Understanding the mechanisms of resilience of coral reefs to anthropogenic stressors is a critical step toward mitigating their current global decline. Coral-bacteria associations are fundamental to reef health and disease, but direct observations of these interactions remain largely unexplored. Here, we use novel technology, high-speed laser scanning confocal microscopy on live coral (Pocillopora damicornis), to test the hypothesis that corals exert control over the abundance of their associated bacterial communities by releasing ('shedding') bacteria from their surface, and that this mechanism can counteract bacterial growth stimulated by organic inputs. We also test the hypothesis that the coral pathogen Vibrio coralliilyticus can evade such a defense mechanism. This first report of direct observation with high-speed confocal microscopy of living coral and its associated bacterial community revealed a layer (3.3-146.8 μm thick) on the coral surface where bacteria were concentrated. The results of two independent experiments showed that the bacterial abundance in this layer was not sensitive to enrichment (5 mg l(-1) peptone), and that coral fragments exposed to enrichment released significantly more bacteria from their surfaces than control corals (P<0.01; 35.9±1.4 × 10(5) cells cm(-2) coral versus 1.3±0.5 × 10(5) cells cm(-2) coral). Our results provide direct support to the hypothesis that shedding bacteria may be an important mechanism by which coral-associated bacterial abundances are regulated under organic matter stress. Additionally, the novel ability to watch this ecological behavior in real-time at the microscale opens an unexplored avenue for mechanistic studies of coral-microbe interactions.

Temperature regulation of virulence factors in the pathogen Vibrio coralliilyticus

Sea surface temperatures (SST) are rising because of global climate change. As a result, pathogenic Vibrio species that infect humans and marine organisms during warmer summer months are of growing concern. Coral reefs, in particular, are already experiencing unprecedented degradation worldwide due in part to infectious disease outbreaks and bleaching episodes that are exacerbated by increasing SST. For example, Vibrio coralliilyticus, a globally distributed bacterium associated with multiple coral diseases, infects corals at temperatures above 27 °C. The mechanisms underlying this temperature-dependent pathogenicity, however, are unknown. In this study, we identify potential virulence mechanisms using whole genome sequencing of V. coralliilyticus ATCC (American Type Culture Collection) BAA-450. Furthermore, we demonstrate direct temperature regulation of numerous virulence factors using proteomic analysis and bioassays. Virulence factors involved in motility, host degradation, secretion, antimicrobial resistance and transcriptional regulation are upregulated at the higher virulent temperature of 27 °C, concurrent with phenotypic changes in motility, antibiotic resistance, hemolysis, cytotoxicity and bioluminescence. These results provide evidence that temperature regulates multiple virulence mechanisms in V. coralliilyticus, independent of abundance. The ecological and biological significance of this temperature-dependent virulence response is reinforced by climate change models that predict tropical SST to consistently exceed 27 °C during the spring, summer and fall seasons. We propose V. coralliilyticus as a model Gram-negative bacterium to study temperature-dependent pathogenicity in Vibrio-related diseases.

Innate Immune Responses of a Scleractinian Coral to Vibriosis

Scleractinian corals are the most basal eumetazoan taxon and provide the biological and physical framework for coral reefs, which are among the most diverse of all ecosystems. Over the past three decades and coincident with climate change, these phototrophic symbiotic organisms have been subject to increasingly frequent and severe diseases, which are now geographically widespread and a major threat to these ecosystems. Although coral immunity has been the subject of increasing study, the available information remains fragmentary, especially with respect to coral antimicrobial responses. In this study, we characterized damicornin from Pocillopora damicornis, the first scleractinian antimicrobial peptide (AMP) to be reported. We found that its precursor has a segmented organization comprising a signal peptide, an acidic proregion, and the C-terminal AMP. The 40-residue AMP is cationic, C-terminally amidated, and characterized by the presence of six cysteine molecules joined by three intramolecular disulfide bridges. Its cysteine array is common to another AMP and toxins from cnidarians; this suggests a common ancestor, as has been proposed for AMPs and toxins from arthropods. Damicornin was active in vitro against Gram-positive bacteria and the fungus Fusarium oxysporum. Damicornin expression was studied using a combination of immunohistochemistry, reverse phase HPLC, and quantitative RT-PCR. Our data show that damicornin is constitutively transcribed in ectodermal granular cells, where it is stored, and further released in response to nonpathogenic immune challenge. Damicornin gene expression was repressed by the coral pathogen Vibrio coralliilyticus. This is the first evidence of AMP gene repression in a host-Vibrio interaction.

Chitin stimulates production of the antibiotic andrimid in a Vibrio coralliilyticus strain

Vibrio coralliilyticus is a putative coral pathogen in tropical oceans, but also possesses antagonistic traits. We previously reported antibacterial activity in Vibrio coralliilyticus strain S2052 based upon the antibiotic andrimid. The purpose of the present study was to determine whether V. coralliilyticus S2052 produces the antibiotic under conditions mimicking natural habitats of vibrios. S2052 synthesized andrimid with both chitin and macroalgal extracts as sole nutrient source. With chitin, the biosynthesis of metabolites other than andrimid was largely abolished, and the yield of the antibiotic per cell was twofold higher. In cultures with Artemia as live chitin model system, S2052 reached up to 10(8) cells ml(-1) , produced andrimid and showed attachment to the exoskeleton and chitinous exuviae. The metabolic focus on andrimid production with chitin indicates that the antibiotic could serve an ecophysiological function. S2052 was compared with two related V. coralliilyticus strains (LMG20984(T) and LMG10953). Despite overall similar secondary metabolomes, LMG20984(T) and LMG10953 did not produce andrimid, and their optimum biosynthetic temperature was 30 as compared with 25°C for S2052. In addition, S2052 appeared less pathogenic towards Artemia than reported for the type strain. Different physiologies of S2052 and closely related strains indicated that V. coralliilyticus subspecies may be adapted to different niches.

Physiological responses of the scleractinian coral Pocillopora damicornis to bacterial stress from Vibrio coralliilyticus

As the effects of climate change have become increasingly visible over the past three decades, coral reefs have suffered from a number of natural and anthropogenic disturbances that have caused a critical decline in coral populations. Among these disturbances are coral diseases, which have appeared with increasing frequency and severity, often in correlation with increases in water temperature. Although the crucial role played by Vibrio species in coral disease has been widely documented, the scientific community does not yet fully understand the infection process of Vibrio or its impact on coral physiology and immunology. Here, we investigated the physiological and transcriptomic responses of a major reef-building coral, Pocillopora damicornis, when exposed to a specific pathogen (Vibrio coralliilyticus) under virulent (increasing water temperature) and non-virulent (constant low temperature) conditions. The infection process was examined by electron microscopy and quantitative reverse-transcription PCR, and coral health was monitored by visual observations and measurements of zooxanthellar density. The results obtained suggest that coral tissue invasion occurs upon increasing water temperature only. Transcriptomic variations were investigated using a suppression-subtractive-hybridization approach, and the expression levels of six candidate immune-related genes were examined during bacterial exposure. These genes correspond to three lectin-like molecules putatively involved in the recognition of pathogens, two metal-binding proteins putatively involved in antibacterial response and one cystein protease inhibitor. The transcription patterns of these selected genes provide new insights into the responses of coral colonies to virulent versus non-virulent bacteria.

Genomic and proteomic analyses of the coral pathogen Vibrio coralliilyticus reveal a diverse virulence repertoire

Vibrio coralliilyticus has been implicated as an important pathogen of coral species worldwide. In this study, the nearly complete genome of Vibrio coralliilyticus strain P1 (LMG23696) was sequenced and proteases implicated in virulence of the strain were specifically investigated. The genome sequence of P1 (5,513,256 bp in size) consisted of 5222 coding sequences and 58 RNA genes (53 tRNAs and at least 5 rRNAs). Seventeen metalloprotease and effector (vgrG, hlyA and hcp) genes were identified in the genome and expressed proteases were also detected in the secretome of P1. As the VcpA zinc-metalloprotease has been considered an important virulence factor of V. coralliilyticus, a vcpA deletion mutant was constructed to evaluate the effect of this gene in animal pathogenesis. Both wild-type and mutant (ΔvcpA) strains exhibited similar virulence characteristics that resulted in high mortality in Artemia and Drosophila pathogenicity bioassays and strong photosystem II inactivation of the coral dinoflagellate endosymbiont (Symbiodinium). In contrast, the ΔvcpA mutant demonstrated higher hemolytic activity and secreted 18 proteins not secreted by the wild type. These proteins included four types of metalloproteases, a chitinase, a hemolysin-related protein RbmC, the Hcp protein and 12 hypothetical proteins. Overall, the results of this study indicate that V. coralliilyticus strain P1 has a diverse virulence repertoire that possibly enables this bacterium to be an efficient animal pathogen.