Sodium Ascorbate as a Quorum-Sensing Inhibitor Leads to Decreased Virulence in Vibrio campbellii.
ABSTRACT: 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:The halophilic aquatic bacterium Vibrio campbellii is an important aquatic pathogen, capable of causing vibriosis in shrimp and fish resulting in significant economic losses. In a previous work, essential oils (EOs) extracts from Melaleuca alternifolia, Litsea citrata, and Eucalyptus citriodora were found to inhibit the growth of V. campbellii in vitro. This study aimed to determine in vivo EOs’ potential protective effect towards gnotobiotic brine shrimp Artemia franciscana, challenged with V. campbellii. The study showed that brine shrimp larvae supplemented with EOs of M. alternifolia (0.0008%) and L. citrata (0.002%) displayed significantly increased survival against V. campbellii. The results indicated that supplementation of these EOs increased the expression of immune-related genes (either in the presence or absence of the pathogen), probably contributing to enhanced protection. Furthermore, in vitro studies indicated that some EOs modulated the expression of virulence factors including swimming motility, biofilm formation, and gelatinase and lipase activity, while flow cytometry data and regrowth assay indicated that these EOs do not exhibit antimicrobial activity as V. campbellii grew at the tested concentrations [M. alternifolia (0.0008%) and L. citrata (0.002%)]. Our findings suggest that EOs extracted from M. alternifolia and L. citrata, can modulate virulence factor production and immunological responses and might hence become part of an intervention strategy to control vibriosis in a fish or shrimp aquaculture setting, a hypothesis that needs to be validated in the future.
Project description:Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority.
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: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 spp. are the most common pathogens for animals reared in aquaculture. Vibrio campbellii, which is often involved in shrimp, fish and mollusks diseases, is widely distributed in the marine environment worldwide, but our knowledge about its pathogenesis and antimicrobial resistance is very limited. The existence of this knowledge gap is at least partially because that V. campbellii was originally classified as Vibrio harveyi, and the detailed information of its comparative genome analysis to other Vibrio spp. is currently lacking. In this study, the complete genome of a V. campbellii predominant strain, LMB29, was determined by MiSeq in conjunction with PacBio SMRT sequencing. This genome consists of two circular DNA chromosomes and four megaplasmids. Comparative genome analysis indicates that LMB29 shares a 96.66% similarity (average nucleotide identity) with the V. campbellii ATCC strain BAA-1116 based on a 75% AF (average fraction) calculations, and its functional profile is very similar to V. campbellii E1 and V. campbellii CAIM115. Both type III secretion system (T3SS) and type VI secretion system (T6SS), along with the tlh gene which encodes a thermolabile hemolysin, are present in LMB29 which may contribute to the bacterial pathogenesis. The virulence of this strain was experimental confirmed by performing a LDH assay on a fish cell infection model, and cell death was observed as early as within 3 h post infection. Thirty-seven antimicrobial resistance genes (>45% identity) were predicted in LMB29 which includes a novel rifampicin ADP ribosyltransferase, arr-9, in plasmid pLMB157. The gene arr-9 was predicted on a genomic island with horizontal transferable potentials which may facilitate the rifampicin resistance dissemination. Future researches are needed to explore the pathogenesis of V. campbellii LMB29, but the availability of this genome sequence will certainly aid as a basis for further analysis.
Project description:<i>Vibrio campbellii</i> is an emerging aquaculture pathogen that causes luminous vibriosis in farmed shrimp. Although prophages in various aquaculture pathogens have been widely reported, there is still limited knowledge regarding prophages in the genome of pathogenic <i>V. campbellii</i>. Here, we describe the full-genome sequence of a prophage named HY01, induced from the emerging shrimp pathogen <i>V. campbellii</i> HY01. The phage HY01 was induced by mitomycin C and was morphologically characterized as long tailed phage. <i>V. campbellii</i> phage HY01 is composed of 41,772 bp of dsDNA with a G+C content of 47.45%. A total of 60 open reading frames (ORFs) were identified, of which 31 could be predicted for their biological functions. Twenty seven out of 31 predicted protein coding regions were matched with several encoded proteins of various <i>Enterobacteriaceae</i>, <i>Pseudomonadaceae</i>, <i>Vibrionaceae</i>, and other phages of Gram-negative bacteria. Interestingly, the comparative genome analysis revealed that the phage HY01 was only distantly related to Vibrio phage Va_PF430-3_p42 of fish pathogen <i>V. anguillarum</i> but differed in genomic size and gene organization. The phylogenetic tree placed the phage together with <i>Siphoviridae</i> family. Additionally, a survey of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) spacers revealed two matching sequences between phage HY01 genome and viral spacer sequence of <i>Vibrio</i> spp. The spacer results combined with the synteny results suggest that the evolution of <i>V. campbellii</i> phage HY01 is driven by the horizontal genetic exchange between bacterial families belonging to the class of Gammaproteobacteria.
Project description:Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e-12), and significantly reduced protease activity (p = 1.6e-07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing-protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.
Project description:The compound poly-ß-hydroxybutyrate (PHB), a polymer of the short chain fatty acid ß-hydroxybutyrate, was shown to protect experimental animals against a variety of bacterial diseases, (including vibriosis in farmed aquatic animals), albeit through undefined mechanisms. Here we aimed at unraveling the underlying mechanism behind the protective effect of PHB against bacterial disease using gnotobiotically-cultured brine shrimp Artemia franciscana and pathogenic Vibrio campbellii as host-pathogen model. The gnotobiotic model system is crucial for such studies because it eliminates any possible microbial interference (naturally present in any type of aquatic environment) in these mechanistic studies and furthermore facilitates the interpretation of the results in terms of a cause effect relationship. We showed clear evidences indicating that PHB conferred protection to Artemia host against V. campbellii by a mechanism of inducing heat shock protein (Hsp) 70. Additionally, our results also showed that this salutary effect of PHB was associated with the generation of protective innate immune responses, especially the prophenoloxidase and transglutaminase immune systems - phenomena possibly mediated by PHB-induced Hsp70. From overall results, we conclude that PHB induces Hsp70 and this induced Hsp70 might contribute in part to the protection of Artemia against pathogenic V. campbellii.
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: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).