Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae.
ABSTRACT: Quorum sensing, bacterial cell-to-cell communication, has been linked to the virulence of pathogenic bacteria. Indeed, in vitro experiments have shown that many bacterial pathogens regulate the expression of virulence genes by this cell-to-cell communication process. Moreover, signal molecules have been detected in samples retrieved from infected hosts and quorum sensing disruption has been reported to result in reduced virulence in different host-pathogen systems. However, data on in vivo quorum sensing activity of pathogens during infection of a host are currently lacking. We previously reported that quorum sensing regulates the virulence of Vibrio harveyi in a standardised model system with gnotobiotic brine shrimp (Artemia franciscana) larvae. Here, we monitored quorum sensing activity in Vibrio harveyi during infection of the shrimp, using bioluminescence as a read-out. We found that wild-type Vibrio harveyi shows a strong increase in quorum sensing activity early during infection. In this respect, the bacteria behave remarkably similar in different larvae, despite the fact that only half of them survive the infection. Interestingly, when expressed per bacterial cell, Vibrio harveyi showed around 200-fold higher maximal quorum sensing-regulated bioluminescence when associated with larvae than in the culture water. Finally, the in vivo quorum sensing activity of mutants defective in the production of one of the three signal molecules is consistent with their virulence, with no detectable in vivo quorum sensing activity in AI-2- and CAI-1-deficient mutants. These results indicate that AI-2 and CAI-1 are the dominant signals during infection of brine shrimp.
Project description:Vibrio harveyi is amongst the most important bacterial pathogens in aquaculture. Novel methods to control this pathogen are needed since many strains have acquired resistance to antibiotics. We previously showed that quorum sensing-disrupting furanones are able to protect brine shrimp larvae against vibriosis. However, a major problem of these compounds is that they are toxic toward higher organisms and therefore, they are not safe to be used in aquaculture. The synthesis of brominated thiophenones, sulphur analogues of the quorum sensing-disrupting furanones, has recently been reported. In the present study, we report that these compounds block quorum sensing in V. harveyi at concentrations in the low micromolar range. Bioluminescence experiments with V. harveyi quorum sensing mutants and a fluorescence anisotropy assay indicated that the compounds disrupt quorum sensing in this bacterium by decreasing the ability of the quorum sensing master regulator LuxR to bind to its target promoter DNA. In vivo challenge tests with gnotobiotic brine shrimp larvae showed that thiophenone compound TF310, (Z)-4-((5-(bromomethylene)-2-oxo-2,5-dihydrothiophen-3-yl)methoxy)-4-oxobutanoic acid, completely protected the larvae from V. harveyi BB120 when dosed to the culture water at 2.5 µM or more, whereas severe toxicity was only observed at 250 µM. This makes TF310 showing the highest therapeutic index of all quorum sensing-disrupting compounds tested thus far in our brine shrimp model system.
Project description:Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.
Project description: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.
Project description:Disease caused by antibiotic resistant pathogens is becoming a serious problem, both in human and veterinary medicine. The inhibition of quorum sensing, bacterial cell-to-cell communication, is a promising alternative strategy to control disease. In this study, we determined the quorum sensing-disrupting activity of 20 thiophenones towards the quorum sensing model bacterium V. harveyi. In order to exclude false positives, we propose a new parameter (AQSI) to describe specific quorum sensing activity. AQSI is defined as the ratio between inhibition of quorum sensing-regulated activity in a reporter strain and inhibition of the same activity when it is independent of quorum sensing. Calculation of AQSI allowed to exclude five false positives, whereas the six most active thiophenones (TF203, TF307, TF319, TF339, TF342 and TF403) inhibited quorum sensing at 0.25 ?M, with AQSI higher than 10. Further, we determined the protective effect and toxicity of the thiophenones in a highly controlled gnotobiotic model system with brine shrimp larvae. There was a strong positive correlation between the specific quorum sensing-disrupting activity of the thiophenones and the protection of brine shrimp larvae against pathogenic V. harveyi. Four of the most active quorum sensing-disrupting thiophenones (TF 203, TF319, TF339 and TF342) were considered to be promising since they have a therapeutic potential of at least 10.
Project description:Pathogenic Vibrio species cause diseases in diverse marine animals reared in aquaculture. Since their pathogenesis, persistence, and survival in marine environments are regulated by quorum sensing (QS), QS interference has attracted attention as a means to control these bacteria in aquatic settings. A few QS inhibitors of Vibrio species have been reported, but detailed molecular mechanisms are lacking. Here, we identified a novel, potent, and selective Vibrio QS inhibitor, named QStatin [1-(5-bromothiophene-2-sulfonyl)-1H-pyrazole], which affects Vibrio harveyi LuxR homologues, the well-conserved master transcriptional regulators for QS in Vibrio species. Crystallographic and biochemical analyses showed that QStatin binds tightly to a putative ligand-binding pocket in SmcR, the LuxR homologue in V. vulnificus, and changes the flexibility of the protein, thereby altering its transcription regulatory activity. Transcriptome analysis revealed that QStatin results in SmcR dysfunction, affecting the expression of SmcR regulon required for virulence, motility/chemotaxis, and biofilm dynamics. Notably, QStatin attenuated representative QS-regulated phenotypes in various Vibrio species, including virulence against the brine shrimp (Artemia franciscana). Together, these results provide molecular insights into the mechanism of action of an effective, sustainable QS inhibitor that is less susceptible to resistance than other antimicrobial agents and useful in controlling the virulence of Vibrio species in aquacultures.IMPORTANCE Yields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenic Vibrio infections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis of Vibrio species in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor of V. harveyi LuxR homologues, which are well-conserved master QS regulators in Vibrio species. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue in V. vulnificus, and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in various Vibrio species, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures.
Project description:Vibrio campbellii is one of the major bacterial pathogens for animals reared in aquaculture, affecting both vertebrates and invertebrates, and causes significant economic losses. It is now evident that the expressions of virulence factors in this pathogen are regulated by the density of the bacterial population. This type of regulation, termed quorum sensing (QS), is mediated by extracellular signal molecules called autoinducers. In this study, the impact of sodium ascorbate (NaAs) on the virulence of V. campbellii was investigated under both in vitro and in vivo conditions, to develop a natural anti-infective strategy to contain V. campbellii infection in aquacultured animals. Results showed that NaAs significantly decreased swimming motility, biofilm production, and the production of virulence enzymes, such as lipase, caseinase, phospholipase, and hemolysin in V. campbellii. Consistent with this, pretreatment of V. campbellii with NaAs before inoculation into the rearing water resulted in significantly increased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. campbellii. Furthermore, NaAs could interfere with QS-regulated bioluminescence in V. campbellii, suggesting the QS-inhibitory activity largely determines the protective effect of NaAs toward the brine shrimp. In essence, due to the potent anti-virulence effects observed in in vitro studies and the clinical brine shrimp-V. campbellii infection model, NaAs constitute a promising novel strategy for the control of V. campbellii infections in aquaculture.
Project description:In a process called quorum sensing, bacteria communicate using extracellular signal molecules termed autoinducers. Two parallel quorum-sensing systems have been identified in the marine bacterium Vibrio harveyi. System 1 consists of the LuxM-dependent autoinducer HAI-1 and the HAI-1 sensor, LuxN. System 2 consists of the LuxS-dependent autoinducer AI-2 and the AI-2 detector, LuxPQ. The related bacterium, Vibrio cholerae, a human pathogen, possesses System 2 (LuxS, AI-2, and LuxPQ) but does not have obvious homologues of V. harveyi System 1. Rather, System 1 of V. cholerae is made up of the CqsA-dependent autoinducer CAI-1 and a sensor called CqsS. Using a V. cholerae CAI-1 reporter strain we show that many other marine bacteria, including V. harveyi, produce CAI-1 activity. Genetic analysis of V. harveyi reveals cqsA and cqsS, and phenotypic analysis of V. harveyi cqsA and cqsS mutants shows that these functions comprise a third V. harveyi quorum-sensing system that acts in parallel to Systems 1 and 2. Together these communication systems act as a three-way coincidence detector in the regulation of a variety of genes, including those responsible for bioluminescence, type III secretion, and metalloprotease production.
Project description:Quorum sensing is a process of bacterial cell-cell communication that enables populations of cells to carry out behaviours in unison. Quorum sensing involves detection of the density-dependent accumulation of extracellular signal molecules called autoinducers that elicit population-wide changes in gene expression. In Vibrio species, CqsS is a membrane-bound histidine kinase that acts as the receptor for the CAI-1 autoinducer which is produced by the CqsA synthase. In Vibrio cholerae, CAI-1 is (S)-3-hydroxytridecan-4-one. The C170 residue of V. cholerae CqsS specifies a preference for a ligand with a 10-carbon tail length. However, a phenylalanine is present at this position in Vibrio harveyi CqsS and other homologues, suggesting that a shorter CAI-1-like molecule functions as the signal. To investigate this, we purified the V. harveyi CqsS ligand, and determined that it is (Z)-3-aminoundec-2-en-4-one (Ea-C8-CAI-1) carrying an 8-carbon tail. The V. harveyi CqsA/CqsS system is exquisitely selective for production and detection of this ligand, while the V. cholerae CqsA/CqsS counterparts show relaxed specificity in both production and detection. We isolated CqsS mutants in each species that display reversed specificity for ligands. Our analysis provides insight into how fidelity is maintained in signal transduction systems.
Project description:Many bacteria use extracellular signaling molecules to coordinate group behavior, a process referred to as quorum sensing (QS). However, some QS molecules are hydrophobic in character and are probably unable to diffuse across the bacterial cell envelope. How these molecules are disseminated between bacterial cells within a population is not yet fully understood. Here, we show that the marine pathogen Vibrio harveyi packages the hydrophobic QS molecule CAI-1, a long-chain amino ketone, into outer membrane vesicles. Electron micrographs indicate that outer membrane vesicles of variable size are predominantly produced and released into the surroundings during the stationary phase of V. harveyi, which correlates with the timing of CAI-1-dependent signaling. The large vesicles (diameter, <55 nm) can trigger a QS phenotype in CAI-1-nonproducing V. harveyi and Vibrio cholerae cells. Packaging of CAI-1 into outer membrane vesicles might stabilize the molecule in aqueous environments and facilitate its distribution over distances.IMPORTANCE Formation of membrane vesicles is ubiquitous among bacteria. These vesicles are involved in protein and DNA transfer and offer new approaches for vaccination. Gram-negative bacteria use hydrophobic signaling molecules, among others, for cell-cell communication; however, due to their hydrophobic character, it is unclear how these molecules are disseminated between bacterial cells. Here, we show that the marine pathogen Vibrio harveyi packages one of its QS molecules, the long-chain ketone CAI-1, into outer membrane vesicles (OMVs). Isolated CAI-1-containing vesicles trigger a QS phenotype in CAI-1 nonproducing V. harveyi and also in Vibrio cholerae cells. Packaging of CAI-1 into OMVs not only solubilizes, stabilizes, and concentrates this class of molecules, but facilitate their distribution between bacteria that live in aqueous environments.
Project description:Vibrio campbellii BAA-1116 was used as a Harveyi clade model organism to determine the impact of indole signaling on virulence. Gene expression analysis of V. campbellii grown in LB35 broth with or without 100 μM indole revealed that indole decreased: (1) V. campbellii virulence in shrimp and prawn challenge assays, (2) exopolysaccharide production, and (3) swimming motility. The results also indicated that indole inhibits quorum sensing-regulated bioluminescence and blocks the three-channel quorum sensing system by interfering with quorum sensing signal transduction. Overall design: Five biological replicates of V. campbellii were grown in LB35 broth (24 h, 200 rpm, 28°C) with and without 100 μM indole and total RNA was extracted from 5.0E+8 cells. Messenger RNA was isolated from the total RNA extracts, treated with DNase, labeled with biotin, fragmented and hybridized to V. campbellii BAA-1116 whole genome microarrays (520694F, Affymetrix).