Project description:Tenacibaculum maritimum, the etiological agent of tenacibaculosis in marine fish, constitutively secretes extracellular products (ECPs), which play a main role in virulence. However, their protein content has not been yet comprehensively studied. In this study a collection of 64 T. maritimum strains belonging to the four serotypes described so far (O1 to O4) was used to analyse the prevalence of extracellular proteolytic and lipolytic activities related to virulence. Results showed the existence of a large heterogeneity of the enzymatic capacity among serotype O4 isolates. Most notably, the ECPs of T. maritimum SP9.1 belonging to serotype O4 contain a large amount of outer membrane vesicles (OMVs), which were characterized by electron microscopy and purified by successive steps of filtration and centrifugation. Total protein content of ECPs, and the proteins associated to OMVs and soluble fraction of the ECPs (S-ECPs) were identified by nLC-TIMS-QTOF. A total of 641 proteins were identified in ECPs including some virulence related factors, which were mainly found in one of the fractions, either in OMVs or S-ECPs. Outer membrane proteins such as TonB-dependent transporters and the T9SS-related proteins PorP, PorT and SprA appeared to be mainly associated with OMVs. By contrast, putative virulence factors such as sialidase SiaA, chondroitinase CslA, sphingomyelinase Sph, ceramidase Cer and collagenase Col were found only in the S-ECPs. These findings demonstrate that T. maritimum release, through surface blebbing, outer membrane vesicles that are enriched specifically in TonB-dependent siderophore transporters and proteins of the type IX secretion system. Interestingly, our results also showed that OMVs could play a role in virulence by promoting surface adhesion, biofilm formation, and maximizing cytotoxic effects of the ECPs. The T. maritimum secretome analysis provides insights into the extracellular products function and can constitute the basis for future studies aimed to elucidate the role of OMVs in the pathogenesis of fish tenacibaculosis.
Project description:Mouthrot, caused by Tenacibaculum maritimum is a significant disease of farmed Atlantic salmon, Salmo salar on the West Coast of North America. Smolts recently transferred into saltwater are the most susceptible and affected fish die with little internal or external clinical signs other than the characteristic small (usually < 5 mm) yellow plaques that are present inside the mouth. The mechanism by which these smolts die is unknown. This study investigated the microscopic pathology (histology and scanning electron microscopy) of bath infected smolts with Western Canadian T. maritimum isolates TmarCan15-1, TmarCan16-1 and TmarCan16-5 and compared the findings to what is seen in a natural outbreak of mouthrot. A real-time RT-PCR assay based on the outer membrane protein A specific for T. maritimum was designed and used to investigate the tissue tropism of the bacteria. The results from this showed that T. maritimum is detectable internally by real-time RT-PCR. This combined with the fact that the bacteria can be isolated from the kidney suggests that T. maritimum becomes systemic. The pathology in the infected smolts is primarily mouth lesions, including damaged tissues surrounding the teeth; the disease is similar to periodontal disease in mammals. The pathological changes are focal, severe, and occur very rapidly with little associated inflammation. Skin lesions are more common in experimentally infected smolts than in natural outbreaks, but this could be an artefact of the challenge dose, handling and tank used during the experiments.
Project description:Tenacibaculum maritimum is a devastating bacterial pathogen of wild and farmed marine fish with a broad host range and a worldwide distribution. We report here the complete genome sequence of the T. maritimum type strain NCIMB 2154T. The genome consists of a 3,435,971-base pair circular chromosome with 2,866 predicted protein-coding genes. Genes encoding the biosynthesis of exopolysaccharides, the type IX secretion system, iron uptake systems, adhesins, hemolysins, proteases, and glycoside hydrolases were identified. They are likely involved in the virulence process including immune escape, invasion, colonization, destruction of host tissues, and nutrient scavenging. Among the predicted virulence factors, type IX secretion-mediated and cell-surface exposed proteins were identified including an atypical sialidase, a sphingomyelinase and a chondroitin AC lyase which activities were demonstrated in vitro.
Project description:Tenacibaculum maritimum, the etiological agent of tenacibaculosis in marine fish, constitutively secretes extracellular products (ECPs) in which protein content has not been yet comprehensively studied. In this work, the prevalence of extracellular proteolytic and lipolytic activities related to virulence was analyzed in 64 T. maritimum strains belonging to the O1-O4 serotypes. The results showed the existence of a great intra-specific heterogeneity in the enzymatic capacity, particularly within serotype O4. Thus, the secretome of a strain belonging to this serotype was characterized by analyzing the protein content of ECPs and the possible production of outer membrane vesicles (OMVs). Notably, the ECPs of T. maritimum SP9.1 contain a large amount of OMVs that were characterized by electron microscopy and purified. Thus, ECPs were divided into soluble (S-ECPs) and insoluble fractions (OMVs), and their protein content was analyzed by a high-throughput proteomic approach. A total of 641 proteins were identified in ECPs including some virulence-related factors, which were mainly found in one of the fractions, either OMVs or S-ECPs. Outer membrane proteins such as TonB-dependent siderophore transporters and the type IX secretion system (T9SS)-related proteins PorP, PorT, and SprA appeared to be mainly associated with OMVs. By contrast, putative virulence factors such as sialidase SiaA, chondroitinase CslA, sphingomyelinase Sph, ceramidase Cer, and collagenase Col were found only in the S-ECPs. These findings clearly demonstrate that T. maritimum releases, through surface blebbing, OMVs specifically enriched in TonB-dependent transporters and T9SS proteins. Interestingly, in vitro and in vivo assays also showed that OMVs could play a key role in virulence by promoting surface adhesion and biofilm formation and maximizing the cytotoxic effects of the ECPs. The characterization of T. maritimum secretome provides insights into ECP function and can constitute the basis for future studies aimed to elucidate the full role of OMVs in the pathogenesis of fish tenacibaculosis.
Project description:BackgroundEphippidae fish are characterized by a discoid shape with a very small visceral cavity. Among them Platax orbicularis has a high economic potential due to its flesh quality and flesh to carcass ratio. Nonetheless, the development of its aquaculture is limited by high mortality rates, especially due to Tenacibaculum maritimum infection, occurring one to three weeks after the transfer of fishes from bio-secure land-based aquaculture system to the lagoon cages for growth. Among the lines of defense against this microbial infection, the gastrointestinal tract (GIT) is less studied. The knowledge about the morphofunctional anatomy of this organ in P. orbicularis is still scarce. Therefore, the aims of this study are to characterize the GIT in non-infected P. orbicularis juveniles to then investigate the impact of T. maritimum on this multifunctional organ.MethodsIn the first place, the morpho-anatomy of the GIT in non-infected individuals was characterized using various histological techniques. Then, infected individuals, experimentally challenged by T. maritimum were analysed and compared to the previously established GIT reference.ResultsThe overlapped shape of the GIT of P. orbicularis is probably due to its constrained compaction in a narrow visceral cavity. Firstly, the GIT was divided into 10 sections, from the esophagus to the rectum. For each section, the structure of the walls was characterized, with a focus on mucus secretions and the presence of the Na+/K+ ATPase pump. An identification key allowing the characterization of the GIT sections using in toto histology is given. Secondly, individuals challenged with T. maritimum exhibited differences in mucus type and proportion and, modifications in the mucosal and muscle layers. These changes could induce an imbalance in the trade-off between the GIT functions which may be in favour of protection and immunity to the disadvantage of nutrition capacities.