Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell & Molecular Biology

First Advisor

David R. Nelson

Abstract

Infections by pathogenic marine bacteria are a major problem for both the shellfish and finfish aquaculture industries, causing severe disease and high mortality, which seriously affect aquaculture production and cause significant economic loss. Marine pathogens like Vibrio tubiashii and Roseovarius crassostreae frequently cause disease in a variety of shellfish. With the understanding that the use of antibiotics in large-scale aquaculture leads to the development and transfer of antibiotic resistance, investigation of probiotic approaches for the prevention of infectious disease has become important. In manuscript I, screening of bacterial isolates from Rhode Island marine organisms and environment using agar-based assay methods for detection of antimicrobial activity against oyster pathogens led to the isolation of candidate probiotic bacteria Phaeobacter gallaeciensis S4. P. gallaeciensis S4 is a gram-negative -Proteobacteria within the Roseobacter clade. Pretreatment of larval and juvenile oysters for 24 h with 104 CFU / mL of P. gallaeciensis S4 protected larval oysters against mortality resulting from challenge with R. crassostreae and V. tubiashii. Probiotic isolates had no negative impact on oyster survival. These results suggest the potential of marine bacterial isolate P. gallaeciensis S4 to serve as probiotic bacterium to control the infection and disease by bacterial pathogens in the culture of Crassostrea virginica. The probiotic bacterium P. gallaeciensis S4, isolated from the inner shell surface of a healthy oyster, secretes the antibiotic tropodithietic acid (TDA), is an excellent biofilm former, and increases oyster larvae survival when challenged with bacterialpathogens. In manuscript II, we investigated the specific roles of TDA secretion and biofilm formation in the probiotic activity of S4Sm (a spontaneous streptomycin-resistant mutant of the parental S4). For this purpose, mutations in clpX (ATP-dependent ATPase) and exoP (an exopolysaccharide biosynthesis gene) were created by insertional mutagenesis using homologous recombination. Mutation of clpX resulted in the loss of TDA production, no decline in biofilm formation, and loss of the ability of S4Sm to inhibit the growth of Vibrio tubiashii and Vibrio anguillarum in vitro. Mutation of exoP resulted in a ~70% decline in biofilm formation, no decline in TDA production, and delayed inhibitory activity towards Vibrio pathogens in vitro. Both clpX and exoP mutants exhibited reduced ability to protect oyster larvae from death when challenged by Vibrio tubiashii. Complementation of the clpX and exoP mutations restored the wild type phenotypes. We also found that pre-colonization by S4Sm was critical for this bacterium to inhibit pathogen colonization and growth. Our observations suggest that probiotic activity by S4Sm involves contributions from both biofilm formation and the production of the antibiotic TDA. In manuscript III, we found that culture supernatant of S4Sm down-regulates protease activity in V. tubiashii cultures. The effects of S4Sm culture supernatant on the transcription of several genes involved in protease activity, including vtpA, vtpB, and vtpR (encoding metalloproteases A and B and their transcriptional regulator, respectively), were examined by qRT-PCR. Expression of vtpB and vtpR were reduced to 35.9% and 6.6%, respectively, compared to an untreated control. In contrast, expression of vtpA was not affected. A V. tubiashii GFP-reporter strain was constructed to detect the inhibitory compounds. Three molecules responsible for V. tubiashii protease inhibition activity were isolated from S4Sm supernatant and identified as N-acyl homoserine lactones (AHLs): 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 (50%) inhibitory concentrations (IC) against V. tubiashii protease activity are 0.264 μM, 3.713 μM and 2.882 μM, respectively. Our qRT-PCR data demonstrated that exposure to the individual AHL reduced transcription of vtpR and vtpB, but not vtpA. Treatment with a combination of three AHLs (any two AHLs or all three AHLs) on V. tubiashii showed that there were additive effects among these three AHL molecules upon protease inhibition activity. These AHL compounds may act by disrupting the quorum-sensing pathway that activates protease transcription of V. tubiashii.

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