Edward J. Spinard

H-NS Is a Negative Regulator of the Two Hemolysin/Cytotoxin Gene Clusters in Vibrio anguillarum

ABSTRACT Hemolysins produced by Vibrio anguillarum have been implicated in the development of hemorrhagic septicemia during vibriosis, a fatal fish disease. Previously, two hemolysin gene clusters responsible for the hemolysis and cytotoxicity of V. anguillarum were identified: the vah1-plp gene cluster and the rtxACHBDE gene cluster. In this study, we identified the hns gene, which encodes the H-NS protein and acts as a negative regulator of both gene clusters. The V. anguillarum H-NS protein shares strong homology with other bacterial H-NS proteins. An hns mutant exhibited increased hemolytic activity and cytotoxicity compared to the wild-type strain. Complementation of the hns mutation restored hemolytic activity and cytotoxicity levels to nearly wild-type levels. Furthermore, expression of rtxA, rtxH, rtxB, vah1, and plp increased in the hns mutant and decreased in the hns-complemented mutant strain compared to expression in the wild-type strain. Additionally, experiments using DNase I showed that purified recombinant H-NS protected multiple sites in the promoter regions of both gene clusters. The hns mutant also exhibited significantly attenuated virulence against rainbow trout. Complementation of the hns mutation restored virulence to wild-type levels, suggesting that H-NS regulates many genes that affect fitness and virulence. Previously, we showed that HlyU is a positive regulator of expression for both gene clusters. In this study, we demonstrate that upregulation by hlyU is hns dependent, suggesting that H-NS acts to repress or silence both gene clusters and HlyU acts to relieve that repression or silencing.

Following the infection process of vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species

Vibriosis represents the main bottleneck for the larval production process in shellfish aquaculture. While the signs of this disease in bivalve larvae are well known, the infection process by pathogenic Vibrio spp. during episodes of vibriosis has not been elucidated. To investigate the infection process in bivalves, the pathogens of larvae as V. tubiashii subsp. europaensis, V. neptunius and V. bivalvicida were tagged with green fluorescent protein (GFP). Larvae of Manila clam (Ruditapes philippinarum) were inoculated with the GFP-labeled pathogens in different infection assays and monitored by microscopy. Manila clam larvae infected by distinct GFP-tagged Vibrio spp. in different challenges showed the same progression in the infection process, defining three infection stages. GFP-tagged Vibrio spp. were filtered by the larvae through the vellum and entered in the digestive system through the esophagus and stomach and colonized the digestive gland and particularly the intestine, where they proliferated during the first 2h of contact (Stage I), suggesting a chemotactic response. Then, GFP-tagged Vibrio spp. expanded rapidly to the surrounding organs in the body cavity from the dorsal to ventral region (Stage II; 6-8h), colonizing the larvae completely at the peak of infection (Stage III) (14-24h). Results demonstrated for the first time that the vibriosis is asymptomatic in Manila clam larvae during the early infection stages. Thus, the early colonization and the rapid proliferation of Vibrio pathogens within the body cavity supported the sudden and fatal effect of the vibriosis, since the larvae exhibited the first signs of disease when the infection process is advanced. As a first step in the elucidation of the potential mechanisms of bacterial pathogenesis in bivalve larvae the enzymatic activities of the extracellular products released from the wild type V. neptunius, V. tubiashii subsp. europaensis and V. bivalvicida were determined and their cytotoxicity was demonstrated in fish and homeothermic cell lines for the first time. That activity was lost after heat treatment.

Draft Genome Sequence of Aliiroseovarius crassostreae CV919-312, the Causative Agent of Roseovarius Oyster Disease (Formerly Juvenile Oyster Disease).

ABSTRACT Aliiroseovarius crassostreae CV919-312 is a marine alphaproteobacterium and the causative agent of Roseovarius oyster disease. We announce here the draft genome sequence of A. crassostreae CV919-312 and identify potential virulence genes involved in pathogenicity.

Reclassification of the larval pathogen for marine bivalves Vibrio tubiashii subsp. europaeus as Vibrio europaeus sp. nov.

The Orientalis clade has a relevant significance for bivalve aquaculture since it includes the pathogens Vibrio bivalvicida, Vibrio tubiashii subsp. tubiashii and Vibrio tubiashii subsp. europaeus. However, the previous taxonomic description of the subspecies of V. tubiashii shows some incongruities that should be emended. In the genomic age, the comparison between genome assemblies is the key to clarify the taxonomic position of both subspecies. With this purpose, we have tested the ability of multilocus sequence analysis based on eight housekeeping gene sequences (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA and topA), different in silico genome-to-genome comparisons, chemotaxonomic features and phenotypic traits to reclassify the subspecies V. tubiashii subsp. europaeus within the Orientalis clade. This polyphasic approach clearly demonstrated that this subspecies is phylogenetically and phenotypically distinct from V. tubiashii and should be elevated to the rank of species as Vibrio europaeus sp. nov. This reclassification allows us to update the Orientalis clade (V. bivalvicida,V. brasiliensis, V. crosai, V. hepatarius, V. orientalis, V. sinaloensis, V. tubiashii and V. europaeus sp. nov.) and reconstruct a better phylogeny of the genus Vibrio. An emended description of V. tubiashii is provided. Finally, the proposed novel species is represented by emergent bivalve pathogens [type strain PP-638T (=CECT 8136T=DSM 27349T), PP2-843 and 07/118 T2] responsible for high mortalities in Spanish and French hatcheries.

Draft Genome Sequence of the Marine Pathogen Vibrio coralliilyticus RE22.

ABSTRACT Vibrio coralliilyticus RE22 is a causative agent of vibriosis in larval bivalves. We report here the draft genome sequence of V. coralliilyticus RE22 and describe additional virulence factors that may provide insight into its mechanism of pathogenicity.

Isocitrate dehydrogenase mutation in Vibrio anguillarum results in virulence attenuation and immunoprotection in rainbow trout ( Oncorhynchus mykiss)

BackgroundVibrio anguillarum is an extracellular bacterial pathogen that is a causative agent of vibriosis in finfish and crustaceans with mortality rates ranging from 30% to 100%. Mutations in central metabolism (glycolysis and the TCA cycle) of intracellular pathogens often result in attenuated virulence due to depletion of required metabolic intermediates; however, it was not known whether mutations in central metabolism would affect virulence in an extracellular pathogen such as V. anguillarum.ResultsSeven central metabolism mutants were created and characterized with regard to growth in minimal and complex media, expression of virulence genes, and virulence in juvenile rainbow trout (Oncorhynchus mykiss). Only the isocitrate dehydrogenase (icd) mutant was attenuated in virulence against rainbow trout challenged by either intraperitoneal injection or immersion. Further, the icd mutant was shown to be immunoprotective against wild type V. anguillarum infection. There was no significant decrease in the expression of the three hemolysin genes detected by qRT-PCR. Additionally, only the icd mutant exhibited a significantly decreased growth yield in complex media. Growth yield was directly related to the abundance of glutamate. A strain with a restored wild type icd gene was created and shown to restore growth to a wild type cell density in complex media and pathogenicity in rainbow trout.ConclusionsThe data strongly suggest that a decreased growth yield, resulting from the inability to synthesize α-ketoglutarate, caused the attenuation despite normal levels of expression of virulence genes. Therefore, the ability of an extracellular pathogen to cause disease is dependent upon the availability of host-supplied nutrients for growth. Additionally, a live vaccine strain could be created from an icd deletion strain.

Draft Genome Sequence of the Emerging Bivalve Pathogen Vibrio tubiashii subsp. europaeus

ABSTRACT Vibrio tubiashii subsp. europaeus is a bivalve pathogen isolated during episodes of mortality affecting larval cultures in different shellfish hatcheries. Here, we announce the draft genome sequence of the type strain PP-638 and describe potential virulence factors, which may provide insight into the mechanism of pathogenicity.

Draft Genome Sequence of the New Pathogen for Bivalve Larvae Vibrio bivalvicida.

ABSTRACT Vibrio bivalvicida is a novel pathogen of bivalve larvae responsible for recent vibriosis outbreaks affecting shellfish hatcheries. Here, we announce the draft genome sequence of V. bivalvicida 605T and describe potential virulence factors.

Draft Genome Sequence of the Shellfish Larval Probiotic Bacillus pumilus RI06-95.

ABSTRACT Bacillus pumilus RI06-95 is a marine bacterium isolated in Narragansett, Rhode Island, which has shown probiotic activity against marine pathogens in larval shellfish. We report the genome of B. pumilus RI06-95, which provides insight into the microbes probiotic ability and may be used in future studies of the probiotic mechanism.

Draft Genome Sequence of Bowmanella denitrificans JL63, a Bacterium Isolated from Whiteleg Shrimp (Litopenaeus vannamei) That Can Inhibit the Growth of Vibrio parahaemolyticus

ABSTRACT Bowmanella denitrificans strain JL63 was isolated from a whiteleg shrimp (Litopenaeus vannamei) and was determined to have antibacterial activity against an acute hepatopancreatic necrosis disease (AHPND) strain of Vibrio parahaemolyticus. Here, we report the draft genome sequence of this strain and identify genes that are potentially involved in its antibacterial activity.