The Probiotic Bacterium Phaeobacter inhibens Downregulates Virulence Factor Transcription in the Shellfish Pathogen Vibrio coralliilyticus by <i>N</i> -Acyl Homoserine Lactone Production

Abstract

<i>Phaeobacter inhibens</i> S4Sm acts as a probiotic bacterium against the oyster pathogen <i>Vibrio coralliilyticus</i> Here, we report that <i>P. inhibens</i> S4Sm secretes three molecules that downregulate the transcription of major virulence factors, metalloprotease genes, in <i>V. coralliilyticus</i> cultures. The effects of the S4Sm culture supernatant on the transcription of three genes involved in protease activity, namely, <i>vcpA</i>, <i>vcpB</i>, and <i>vcpR</i> (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by reverse transcriptase quantitative PCR (qRT-PCR). The expression of <i>vcpB</i> and <i>vcpR</i> were reduced to 36% and 6.6%, respectively, compared to that in an untreated control. We constructed a <i>V. coralliilyticus</i> green fluorescent protein (GFP) reporter strain to detect the activity of inhibitory compounds. Using a bioassay-guided approach, the molecules responsible for <i>V. coralliilyticus</i> protease inhibition activity were isolated from S4Sm supernatant and identified as three <i>N</i>-acyl homoserine lactones (AHLs). The three AHLs are <i>N</i>-(3-hydroxydecanoyl)-l-homoserine lactone, <i>N</i>-(dodecanoyl-2,5-diene)-l-homoserine lactone, and <i>N</i>-(3-hydroxytetradecanoyl-7-ene)-l-homoserine lactone, and their half maximal inhibitory concentrations (IC₅₀s) against <i>V. coralliilyticus</i> protease activity were 0.26 μM, 3.7 μM, and 2.9 μM, respectively. Our qRT-PCR data demonstrated that exposures to the individual AHLs reduced the transcription of <i>vcpR</i> and <i>vcpB</i> Combinations of the three AHLs (any two or all three AHLs) on <i>V. coralliilyticus</i> 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 <i>V. coralliilyticus</i><b>IMPORTANCE</b> 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, <i>Phaeobacter inhibens</i> S4Sm, which protects oyster larvae from <i>Vibrio coralliilyticus</i> RE22Sm infection. We also demonstrated that both antibiotic secretion and biofilm formation play important roles in S4Sm probiotic activity. Here, we report that <i>P. inhibens</i> S4Sm, an alphaproteobacterium and member of the <i>Roseobacter</i> clade, also secretes secondary metabolites that hijack the quorum sensing ability of <i>V. coralliilyticus</i> 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.