The probiotic bacterium, Phaeobacter inhibens , down-regulates virulence factor transcription in the shellfish pathogen, Vibrio coralliilyticus , by N -acyl homoserine lactone production.

Phaeobacter inhibens S4Sm acts as a probiotic bacterium against the oyster pathogen, Vibrio coralliilyticus Here we report that P. inhibens S4Sm secretes three molecules that down-regulate transcription of major virulence factors, metalloprotease genes, in V. coralliilyticus cultures. The effects of S4Sm culture supernatant on the transcription of three genes involved in protease activity, vcpA, vcpB , and vcpR (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by qRT-PCR. Expression of vcpB and vcpR were reduced to 36% and 6.6%, respectively, compared to an untreated control. We constructed a V. coralliilyticus GFP-reporter strain to detect the activity of inhibitory compounds. Using a bioassay-guided approach, the molecules responsible for V. coralliilyticus protease inhibition activity were isolated from S4Sm supernatant and identified as three N -acyl homoserine lactones (AHLs). The three AHLs are N -(3-hydroxydecanoyl)-L-homoserine lactone, N -(dodecanoyl-2,5-diene)-L-homoserine lactone and N -(3-hydroxytetradecanoyl-7-ene)-L-homoserine lactone, and their half maximal inhibitory concentrations (IC50 ) against V. coralliilyticus protease activity are 0.26 μM, 3.7 μM and 2.9 μM, respectively. Our qRT-PCR data demonstrated that exposure to the individual AHLs reduced transcription of vcpR and vcpB Combinations of the three AHLs (any two or all three AHLs) on V. coralliilyticus showed additive effects upon protease inhibition activity. These AHL compounds may contribute to the host protective effects of S4Sm by disrupting the quorum-sensing pathway that activates protease transcription of V. coralliilyticus Importance Probiotics represent a promising alternative strategy to control infection and disease caused by marine pathogens of aquaculturally important species. Generally, beneficial effects of probiotics include improved water quality, control of pathogenic bacteria and their virulence, stimulation of the immune system, and improved animal growth. Previously, we isolated a probiotic bacterium, Phaeobacter inhibens S4Sm, which protects oyster larvae from V. coralliilyticus RE22Sm infection. We also demonstrated that both antibiotic secretion and biofilm formation play important roles in S4Sm probiotic activity. Here, we report that P. inhibens S4Sm, an α- Proteobacterium and member of the Roseobacter clade, also secretes secondary metabolites that hijack the quorum sensing ability of V. coralliilyticus RE22Sm, suppressing virulence gene expression. This finding demonstrates that probiotic bacteria can exert their host protection using a multi-pronged array of behaviors that limit the ability of pathogens to become established and cause infection.