THE IMPACT OF QUORUM SENSING DISRUPTION ON THE VIRULENCE OF AQUATIC PATHOGENS TOWARDS GNOTOBIOTIC ARTEMIA FRANCISCANA

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1 THE IMPACT OF QUORUM SENSING DISRUPTION ON THE VIRULENCE OF AQUATIC PATHOGENS TOWARDS GNOTOBIOTIC ARTEMIA FRANCISCANA TOM DEFOIRDT, PETER BOSSIER & WILLY VERSTRAETE Laboratory of Microbial Ecology and Technology, Ghent University Lab. of Aquaculture & Artemia Reference Center, Ghent University

2 INTRODUCTION ν Infections caused by pathogenic and opportunistic bacteria can cause dramatic losses (up to 100%) in aquaculture Luminescent vibriosis ν Traditional solution: antibiotics and disinfectants Resistance!!! Alternative methods are needed

3 INTRODUCTION ν QUORUM SENSING (QS) = bacterial cell-to-cell communication with signal molecules AHL HAI-1 AI-2 CAI-1 LuxP AhyR LuxN LuxQ LuxS CqsS AhyI IM LuxM LuxU CqsA OM srna s + Hfq LuxO σ 54 Target genes LuxR Target genes

4 INTRODUCTION ν QS has been linked to virulence of pathogenic bacteria ν QS systems of aquatic pathogens regulate virulence factor production: - proteases (A.hydrophila, V.anguillarum) - haemolysin (V.vulnificus) - siderophore, toxin and type III secretion (V.harveyi) ν What about virulence (all virulence factors together)? In vivo experiments

5 CHALLENGE TESTS ν QS mutants of Aeromonas hydrophila, Vibrio anguillarum and Vibrio harveyi have been described in literature ν Challenge tests with gnotobiotic Artemia and WT pathogens vs. QS mutants - Fed autoclaved LVS3 (10 7 CFU/mL) - Addition of strain of interest (10 4 CFU/mL) - Counting survival after 2 days

6 CHALLENGES WITH MUTANTS Control Wildtype HAI-1 synthase HAI-1 receptor AI-2 synthase AI-2 receptor HAI-1 & AI-2 synthase LuxO Challenge with V.harveyi wildtype and QS mutants: AI-2 mutants not virulent * * * Control Washwater AI-2 nonproducer AI-2 nonproducer + washwater Effect of exogenous AI-2 on virulence AI-2 neg. mutant: restoration of virulence Survival (%) Survival (%) *

7 CHALLENGES WITH MUTANTS ν V.harveyi can distinguish between the autoinducers HAI-1 AI-2 CAI-1 LuxP LuxN LuxQ LuxS CqsS LuxM LuxU CqsA srna s + Hfq LuxO σ 54 LuxR VIRULENCE

8 CHALLENGES WITH MUTANTS ν For A. hydrophila and V. anguillarum, wildtypes as well as mutants caused significant mortality No effect of disruption of QS

9 QS DISRUPTING FURANONES ν Halogenated furanones produced by the macroalga Delisea pulchra Br H Br O O ν Shown before to be able to block AHL- and AI-2- mediated QS ν No inhibition of growth of Gram-negative bacteria

10 FURANONE IN VITRO 140 H 120 Br Br Bioluminescence of reporter (%) HAI-1 AI-2 CAI-1 O O 0,01 0, [Furanone] (mg/l)

11 FURANONE IN VITRO ν The furanone acts downstream in the QS signal transduction cascade (LuxR?) ν There is no effect on growth of V. harveyi and V. campbellii 10 1 OD 600 0,1 BB120 BB120 + Fur 50 mg/l PN9801 PN Fur 50 mg/l 0, Time (h)

12 FURANONE IN VIVO 100 Protection at 20 mg/l Survival (%) * * * Fur 0 mg/l Fur 5 mg/l Fur 10 mg/l Fur 20 mg/l Fur 50 mg/l Toxicity furanone 20 * 0 BB120 PN9801

13 FURANONE IN VIVO ν The furanone (20 mg/l) protects Artemia from several different pathogenic V.harveyi and V.campbellii isolates ν Protection is complete for weaker pathogens ν There is still significant mortality in furanone-treated Artemia for virulent pathogens ν No difference in Vibrio concentration in culture water between treated and untreated tubes

14 CONCLUSIONS ν AI-2 mediated QS regulates virulence of V.harveyi towards Artemia ν The QS systems of the A. hydrophila and V. anguillarum strains have no influence on their virulence towards Artemia ν Halogenated furanones have potential in treating infections caused by V.harveyi and related bacteria although compounds with lower toxicity need to be developed

15 PERSPECTIVES Testing other strategies to disrupt AI-2-mediated QS: ν Inhibitors of the LuxS protein (AI-2 synthase) ν Isolation of bacteria that can grow on AI-2 or its precursor as sole source of carbon probiotic

16 THANKS FOR YOUR ATTENTION!!! SPECIAL THANKS TO: IWT & FWO (financial support) Paul Williams, Debra Milton & Bonnie Bassler (QS mutants) Thomas Wood (synthesis of brominated furanone) Roselien Crab (MSc. thesis student)