Norepinephrine and dopamine increase motility, biofilm formation, and virulence of <i>Vibrio harveyi</i>

Vibrio harveyi is one of the major pathogens of aquatic organisms, affecting both vertebrates and invertebrates, and causes important losses in the aquaculture industry. In order to develop novel methods to control disease caused by this pathogen, we need to obtain a better understanding of pathogenicity mechanisms. Sensing of catecholamines increases both growth and production of virulence-related factors in pathogens of terrestrial animals and humans. However, at this moment, knowledge on the impact of catecholamines on the virulence of pathogens of aquatic organisms is lacking. In the present study, we report that in V. harveyi, norepinephrine (NE) and dopamine (Dopa) increased growth in serum-supplemented medium, siderophore production, swimming motility, and expression of genes involved in flagellar motility, biofilm formation, and exopolysaccharide production. Consistent with this, pretreatment of V. harveyi with catecholamines prior to inoculation into the rearing water resulted in significantly decreased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. harveyi. Further, NE-induced effects could be neutralized by α-adrenergic antagonists or by the bacterial catecholamine receptor antagonist LED209, but not by β-adrenergic or dopaminergic antagonists. Dopa-induced effects could be neutralized by dopaminergic antagonists or LED209, but not by adrenergic antagonists. Together, our results indicate that catecholamine sensing increases the success of transmission of V. harveyi and that interfering with catecholamine sensing might be an interesting strategy to control vibriosis in aquaculture. We hypothesize that upon tissue and/or hemocyte damage during infection, pathogens come into contact with elevated catecholamine levels, and that this stimulates the expression of virulence factors that are required to colonize a new host

Expression and quorum sensing regulation of type III secretion system genes of <i>Vibrio harveyi</i> during infection of gnotobiotic brine shrimp

Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells

The bacterial storage compound PHB protects <i>Artemia franciscana</i> from pathogenic <i>Vibrio campbellii</i>

Infections caused by luminescent vibrios can cause dramatic losses in aquaculture. These infections are often hard to treat with antibiotics because of the spread of resistant strains and therefore, alternative control strategies are urgently needed. We previously found that the short-chain fatty acid 3-hydroxybutyrate protects Artemia from pathogenic Vibrio campbellii. In this study, we investigated whether the homopolymer of the fatty acid, the well-known bacterial storage compound poly-3-hydroxybutyrate (PHB), could be used to protect the nauplii from the pathogen. A starvation experiment learned us that the addition of 1000mg.l-1 PHB particles (average diameter 30μm) to the culture water of starved Artemia nauplii significantly enhanced their survival. This indicated that the nauplii could obtain energy from the PHB. In order to provide the nauplii from energy, the PHB must have been at least partially degraded into water-soluble products (i.e. 3-hydroxybutyrate monomers and oligomers). Subsequently, an in vivo challenge test was performed with the PHB particles and Artemia nauplii challenged to Vibrio campbellii LMG21363. The addition of the PHB particles (at 100mg.l-1 and 1000mg l-1) to the Artemia culture water together with the pathogen significantly enhanced the survival of the infected nauplii. A complete protection (no significant difference in survival with uninfected nauplii) was observed at the highest concentration. If the PHB particles were added 1 day after the addition of the pathogen, a similar but less pronounced effect was noticed. In a second in vivo challenge test, we investigated the effect of the addition of a PHBaccumulating Brachymonas denitrificans strain on the survival of infected nauplii. The strain, added either untreated or pasteurised at 107CFU ml-1, completely protected the nauplii from the pathogen if it had a high PHB content (32% of the VSS). No protection was observed if the strain had a low PHB content

Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example

The massive (mis)use of antibiotics to control infections in aquaculture has resulted in the development of resistant strains, which have rendered antibiotic treatments ineffective. Moreover, the horizontal transfer of resistance determinants to human pathogens and the presence of antibiotic residues in aquaculture products for human consumption constitute important threats to public health. Therefore, to make the aquaculture industry more sustainable, new strategies to control infections are urgently needed.

Expression of virulence genes in luminescent and nonluminescent isogenic vibrios and virulence towards gnotobiotic brine shrimp (<i>Artemia franciscana</i>)

Aims: This study aimed to evaluate the expression levels of virulence gene regulators (luxR and toxR) and virulence factors (serine protease, metalloprotease and haemolysin) in luminescent and nonluminescent isogenic Vibrio harveyi and Vibrio campbellii.Method and Results: Nonluminescent variants have been reported before to become dominant in cultures of luminescent vibrios when grown under static conditions in the dark. Wild-type V. harveyi BB120, V. campbellii LMG 21363, quorum sensing mutants of V. harveyi BB120 and their previously reported nonluminescent isogenic counterparts were used in this study. The expression level of the virulence genes srp serine protease, vhp metalloprotease and vhh haemolysin, the quorum sensing master regulator gene luxR and the virulence regulator gene toxR in isogenic luminescent and nonluminescent strains were quantified using reverse transcriptase real-time PCR. These experiments revealed that the nonluminescent strains produced lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene (which is known to be regulated by quorum sensing). Finally, challenge tests with gnotobiotic brine shrimp (Artemia franciscana) larvae revealed that the nonluminescent strains are less virulent than their luminescent isogenic counterparts.Conclusion: Nonluminescent variants of V. harveyi and V. campbellii strains produce lower levels of the quorum sensing master regulator gene luxR and the vhp metalloprotease gene and are less virulent to brine shrimp than their isogenic luminescent counterparts.Significance and Impact of the study: These results indicate that adaptation of luminescent vibrios to specific growth conditions that result in a dominant nonluminescent phenotype is accompanied by a decreased adaptation to a host environment because of altered virulence gene regulation

The natural furanone (5<i>Z</i>)-4-bromo-5-(bromomethylene)-3-butyl-2(5<i>H</i>)-furanone disrupts quorum sensing-regulated gene expression in <i>Vibrio harveyi</i> by decreasing the DNA-binding activity of the transcriptional regulator protein luxR

This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)- furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing-regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real-time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxRVh mRNA levels in wild-type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxRVh response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxRVh protein also showed less shifts with furanone-treated LuxRVh, whereas the LuxRVh concentration was found not to be altered by the furanone (as determined by SDS-PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxRVh unable to bind to the promoter sequences of quorum sensing-regulated genes