Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae

Vibrio coralliilyticus is an extracellular bacterial pathogen and a causative agent of vibriosis in larval oysters. Host mortality rates can quickly reach 100% during vibriosis outbreaks in oyster hatcheries. Type VI Secretion Systems (T6SS) are rapidly polymerizing, contact dependent injection apparatus for prey cell intoxication and play important roles in pathogenesis. DNA sequencing of V. coralliilyticus RE22Sm indicated the likely presence of two functional T6SSs with one on each of two chromosomes. Here, we investigated the antibacterial and anti-eukaryotic roles of the two T6SSs (T6SS1 and T6SS2) against E. coli Sm10 cells and Crassostrea virginica larvae, respectively. Mutations in hcp and vgrG genes were created and characterized for their effects upon bacterial antagonism and eukaryotic host virulence. Mutations in hcp1 and hcp2 resulted in significantly reduced antagonism against E. coli Sm10, with the hcp2 mutation demonstrating the greater impact. In contrast, mutations in vgrG1 or vgrG2 had little effect on E. coli killing. In eastern oyster larval challenge assays, T6SS1 mutations in either hcp1 or vgrG1 dramatically attenuated virulence against C. virginica larvae. Strains with restored wild type hcp or vgrG genes reestablished T6SS-mediated killing to that of wild type V. coralliilyticus RE22Sm. These data suggest that the T6SS1 of V. coralliilyticus RE22Sm principally targets eukaryotes and secondarily bacteria, while the T6SS2 primarily targets bacterial cells and secondarily eukaryotes. Attenuation of pathogenicity was observed in all T6SS mutants, demonstrating the requirement for proper assembly of the T6SS systems to maintain maximal virulence. Importance: Vibriosis outbreaks lead to large-scale hatchery losses of oyster larvae (product and seed) where Vibrio sp. associated losses of 80 to 100 percent are not uncommon. Practical and proactive biocontrol measures can be taken to help mitigate larval death by Vibrio sp. by better understanding the underlying mechanisms of virulence in V. coralliilyticus. In this study, we demonstrate the presence of two Type VI Secretion Systems (T6SS) in V. coralliilyticus RE22Sm and interrogate the roles of each T6SS in bacterial antagonism and pathogenesis against a eukaryotic host. Specifically, we show that the loss of T6SS1 function results in the loss of virulence against oyster larvae.

Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization and Remediation of Larval Oyster Mortalities

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortalities in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family and two were Siphoviridae. In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% at 6 days post-challenge with lethal doses of V. coralliilyticus. Larvae survival depended on the oyster species, the quantity of phages and vibrios applied, and the species and strain of Vibrio. A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phage vB_VcorM-GR7B; vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A and 28A). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by > 90% (P < 0.0001) over 6 days with phage treatment compared to untreated controls. Genomic sequencing of phages 7B, 11A and 28A revealed 207,758; 194,800; and 154,046 bp, linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries. IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of the hatcheries and commercial shellfish industry. Shellfish pathogens, like Vibrio coralliilyticus and Vibrio tubiashii, have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species, and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach in stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Chemotactic Response of Vibrio coralliilyticus to mucus from various coral species

<i>Vibrio coralliilyticus</i>, a prominent pathogenic bacteria, is known to cause tissue damage in the coral <i>Pocillopora damicornis</i> and is attracted towards the coral via chemotaxis. However, the potential of <i>V. coralliilyticus</i> to infect most of the other coral hosts via chemotaxis is unknown. The present study used capillary assays to quantify the chemotactic response of <i>V. coralliilyticus</i> to the mucus of four tank-cultivated corals, <i>Cataphyllia jardine</i>, <i>Mussidae</i> sp., <i>Nemenzophyllia turbida </i>and <i>Euphyllia ancora</i> and mucus from three wild corals, <i>Acropora</i> sp., <i>Porites</i> sp. & <i>Montipora</i> sp. The bacteria showed positive chemotactic response to each coral mucus tested, with the highest response recorded to the mucus of <i>Acropora</i> sp and the lowest response to the mucus of <i>Montipora</i> sp. A microfluidic chip was then used to assess the chemotactic preference of <i>V. coralliilyticus </i>to the mucus of the tank cultivated corals. Here too, the bacteria showed positive response with a slightly different ranking order. The strong chemotactic response of <i>V. coralliilyticus</i> towards the mucus tested could indicate a broader host range of <i>V. coralliilyticus</i> and in extension its threat to weakened coral reefs worldwide.

AhaP, A Quorum Quenching Acylase from Psychrobacter sp. M9-54-1 That Attenuates Pseudomonas aeruginosa and Vibrio coralliilyticus Virulence

Although Psychrobacter strain M9-54-1 had been previously isolated from the microbiota of holothurians and shown to degrade quorum sensing (QS) signal molecules C6 and C10-homoserine lactone (HSL), little was known about the gene responsible for this activity. In this study, we determined the whole genome sequence of this strain and found that the full 16S rRNA sequence shares 99.78–99.66% identity with Psychrobacter pulmonis CECT 5989T and P. faecalis ISO-46T. M9-54-1, evaluated using the agar well diffusion assay method, showed high quorum quenching (QQ) activity against a wide range of synthetic N-acylhomoserine lactone (AHLs) at 4, 15, and 28 °C. High-performance liquid chromatography-mass-spectrometry (HPLC-MS) confirmed that QQ activity was due to an AHL-acylase. The gene encoding for QQ activity in strain M9-54-1 was identified from its genome sequence whose gene product was named AhaP. Purified AhaP degraded substituted and unsubstituted AHLs from C4- to C14-HSL. Furthermore, heterologous expression of ahaP in the opportunistic pathogen Pseudomonas aeruginosa PAO1 reduced the expression of the QS-controlled gene lecA, encoding for a cytotoxic galactophilic lectin and swarming motility protein. Strain M9-54-1 also reduced brine shrimp mortality caused by Vibrio coralliilyticus VibC-Oc-193, showing potential as a biocontrol agent in aquaculture.

The Antibiotic Andrimid Produced by Vibrio coralliilyticus Increases Expression of Biosynthetic Gene Clusters and Antibiotic Production in Photobacterium galatheae

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266–7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae, carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6–2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications.

Bacteriophage Cocktail for the Prevention of Multiple-Antibiotic-Resistant and Mono-Phage-Resistant Vibrio coralliilyticus Infection in Pacific Oyster (Crassostrea gigas) Larvae

Vibrio coralliilyticus (V. coralliilyticus) is a pathogen that causes mass mortality in marine bivalve hatcheries worldwide. In this study, we used a bacteriophage (phage) cocktail to prevent multiple-antibiotic-resistant (MAR) and phage-resistant (PR) V. coralliilyticus infection in Pacific oyster (Crassostreagigas) larvae. To prevent the occurrence of phage-resistant strains and decrease the effect of mono-phage treatment, we prepared a phage cocktail containing three types of V. coralliilyticus-specific phages and tested its prophylactic efficacy against MAR and PR V. coralliilyticus infection. The results of the cell lysis test showed that the phage cocktail showed an excellent bactericidal effect against the MAR and PR variants in contrast to the experimental group treated with two mono phages (pVco-5 and pVco-7). An in vivo test using Pacific oyster larvae also confirmed the preventive effect against MAR and PR variants. In conclusion, the application of the phage cocktail effectively prevented V. coralliilyticus infection in marine bivalve seedling production. Furthermore, it is expected to reduce damage to the aquaculture industry caused by the occurrence of MAR and PR V. coralliilyticus. Therefore, phage cocktails may be used for the control of various bacterial diseases.

Complete Genome Sequence of Vibrio coralliilyticus OCN008

ABSTRACT Here, we report the complete genome sequence of Vibrio coralliilyticus OCN008, a marine bacterium that infects reef-building coral. Previous sequencing efforts yielded an incomplete sequence (210 contigs). We used Nanopore and Illumina sequencing data to obtain complete sequences of the two circular chromosomes (3.48 and 1.91 Mb) and one megaplasmid (244.69 kb).

Virulence as a Side Effect of Interspecies Interaction in Vibrio Coral Pathogens

ABSTRACT The increase in prevalence and severity of coral disease outbreaks produced by Vibrio pathogens, and related to global warming, has seriously impacted reef-building corals throughout the oceans. The coral Oculina patagonica has been used as a model system to study coral bleaching produced by Vibrio infection. Previous data demonstrated that when two coral pathogens (Vibrio coralliilyticus and Vibrio mediterranei) simultaneously infected the coral O. patagonica, their pathogenicity was greater than when each bacterium was infected separately. Here, to understand the mechanisms underlying this synergistic effect, transcriptomic analyses of monocultures and cocultures as well as experimental infection experiments were performed. Our results revealed that the interaction between the two vibrios under culture conditions overexpressed virulence factor genes (e.g., those encoding siderophores, the type VI secretion system, and toxins, among others). Moreover, under these conditions, vibrios were also more likely to form biofilms or become motile through induction of lateral flagella. All these changes that occur as a physiological response to the presence of a competing species could favor the colonization of the host when they are present in a mixed population. Additionally, during coral experimental infections, we showed that exposure of corals to molecules released during V. coralliilyticus and V. mediterranei coculture induced changes in the coral microbiome that favored damage to coral tissue and increased the production of lyso-platelet activating factor. Therefore, we propose that competition sensing, defined as the physiological response to detection of harm or to the presence of a competing Vibrio species, enhances the ability of Vibrio coral pathogens to invade their host and cause tissue necrosis. IMPORTANCE Vibrio coralliilyticus and Vibrio mediterranei are important coral pathogens capable of inducing serious coral damage, which increases severely when they infect the host simultaneously. This has consequences related to the dispersion of these pathogens among different locations that could enhance deleterious effects on coral reefs. However, the mechanisms underlying this synergistic interaction are unknown. The work described here provides a new perspective on the complex interactions among these two Vibrio coral pathogens, suggesting that coral infection could be a collateral effect of interspecific competition. Major implications of this work are that (i) Vibrio virulence mechanisms are activated in the absence of the host as a response to interspecific competition and (ii) release of molecules by Vibrio coral pathogens produces changes in the coral microbiome that favor the pathogenic potential of the entire Vibrio community. Thus, our results highlight that social cues and competition sensing are crucial determinants of development of coral diseases.