The Probiotic Bacterium Phaeobacter inhibens Downregulates 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 downregulate the transcription of major virulence factors, metalloprotease genes, in V. coralliilyticus cultures. The effects of the S4Sm culture supernatant on the transcription of three genes involved in protease activity, namely, vcpA, vcpB, and vcpR (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by reverse transcriptase quantitative PCR (qRT-PCR). The expression of vcpB and vcpR were reduced to 36% and 6.6%, respectively, compared to that in an untreated control. We constructed a V. coralliilyticus green fluorescent protein (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 (IC50s) against V. coralliilyticus protease activity were 0.26 M, 3.7 M, and 2.9 M, respectively. Our qRT-PCR data demonstrated that exposures to the individual AHLs reduced the transcription of vcpR and vcpB. Combinations of the three AHLs (any two or all three AHLs) on V. coralliilyticus produced additive effects on 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, the 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 Vibrio 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 alphaproteobacterium 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 by using a multipronged array of behaviors that limit the ability of pathogens to become established and cause infection