Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological and Environmental Sciences

Specialization

Cell & Molecular Biology

Department

Cell & Molecular Biology

First Advisor

David R. Nelson

Abstract

Infections by pathogenic marine bacteria present a major problem for both the shellfish and finfish aquaculture industries, resulting in severe disease and high mortality. Pathogen infection seriously affect aquaculture production, and cause significant economic loss. Marine pathogens like Vibrio coralliilyticus frequently cause disease in a variety of shellfish. The use of antibiotics in large-scale aquaculture settings leads to the development, and potential transfer, of antibiotic resistance. In order to mitigate this emerging threat, an understanding of pathogenic mechanisms of infection and novel preventative strategies, such as probiotic treatment, is paramount for understanding and preventing future disease.

In manuscript I, “Two Type VI Secretion Systems in Vibrio coralliilyticus RE22Sm exhibit differential target specificity for bacteria prey and oyster larvae”, the antibacterial and anti-eukaryotic roles of the two T6SSs (T6SS1 and T6SS2) against E. coli Sm10 cells and Crassostrea virginica larvae were evaluated. Mutations in hcp and vgrG genes were created and characterized for their effects upon bacterial antagonism and eukaryotic host virulence. Mutations in hcp1 and hcp2 resulted in significantly reduced antagonism against E. coli Sm10, with the hcp2 mutation demonstrating the greater impact. In contrast, mutations in vgrG1 or vgrG2 had little effect on E. coli killing. In eastern oyster larval challenge assays, T6SS1 mutations in either hcp1 or vgrG1 dramatically attenuated virulence against C. virginica larvae. Strains with restored wild type hcp or vgrG genes reestablished T6SS-mediated killing to that of wild type V. coralliilyticus RE22Sm. These findings suggest that the T6SS1 of V. coralliilyticus RE22Sm principally targets eukaryotes and secondarily bacteria, while the T6SS2 targets bacterial to a large extent than larval oysters. Attenuation of pathogenicity was observed in all T6SS mutants, demonstrating the requirement for proper assembly of the T6SS systems to maintain maximal virulence in either system

In manuscript II, “The Role of Quorum Sensing in V. coralliilyticus RE22Sm during oyster infection”, the pathogenic contributions of four quorum sensing (QS) genes were characterized with regard to growth ability, biofilm formation, extracellular zinc metalloprotease activity, T6SS killing ability and virulence in eastern oyster larvae. A dual function histidine kinase/phosphatase (luxN) mutant, a phosphorelay (luxO) mutant, a quorum sensing transcriptional regulator (vcpR) mutant, and an AHL synthase (luxM) mutant were created. Based on their contributions to the QS system, the luxN, vcpR and luxM mutants were designed to mimic a low cell density (LCD) environment, and the luxO mutant to mimic a high cell density (HCD) environment. Growth under shaking conditions was reduced for the luxN, vcpR, and luxM mutants, while growth increased in the luxO mutant strain. Planktonic growth reduced in the luxO and luxM mutant strains, and remained unaltered in luxN and vcpR mutant strains. Biofilm formation was increased in the luxO strain, and remained unaffected in luxN, vcpR, and luxM mutant strains. Extracellular metalloprotease production was significantly reduced in the luxN, vcpR, and luxM strains, and increased in the luxO mutant strain. Contact mediated T6SS killing was significantly attenuated in the luxN strain, partially attenuated in the luxO and vcpR strains, and was unaffected in the luxM mutant. The luxN, vcpR, and luxM mutants were significantly attenuated in their ability to kill larval eastern oysters, while the luxO mutant strain had no effect on virulence. These data suggest an LCD state in RE22Sm attenuates virulence against larval oysters, whereas a HCD state results in wild-type levels of virulence. These data indicate that QS mediated protease activity is a secondary virulence factor in oyster infection, where the RE22Sm T6SS-1 acts as the primary virulence factor.

In manuscript III, “The Role of Quorum Sensing in Phaeobacter inhibens S4Sm and Effects on Probiotic Activity”, the roles of four quorum sensing (QS) genes were evaluated for their effect on N-acyl homoserine lactone (AHL) production, cell growth, biofilm formation, inhibition of the pathogen Vibrio coralliilyticus RE22Sm, resilience against T6SS contact mediated attack, and protective effects on eastern oyster larvae to infection by RE22Sm. Mutations in pgaI (AHL synthase), pgaR (cognate AHL receptor and transcriptional regulator), luxO (phosphorelay protein), and pgaK (transmembrane histidine kinase/phosphate) were generated. Mutation of pgaI or pgaR resulted in overall loss of probiotic activity marked by reduced biofilm production, inability to inhibit growth of Vibrio sp. on agar plates, and increased susceptibility to T6SS mediated attack by V. coralliilyticus RE22Sm. The pretreatment of Eastern oyster larvae with the pgaI or pgaR strains resulted in partial reduction in protection as compared to wild type. The pgaI and pgaR strains were notably deficient in tropodithietic acid (TDA) production, as they lacked the characteristic yellow pigmentation the wild-type strain. Mutation of either luxO or pgaK resulted in increased by AHL production/detection. Targeted exploitation of the QS system by mutagenesis increased the ability of S4Sm to protect larval oysters to challenge with V. coralliilyticus RE22Sm.

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