Characterisation of ggcP, a Putative Virulence Protein of Vibrio anguillarum

Abstract

Vibrio anguillarum causes significant economic losses in aquaculture, and although vaccines are available for some species, their protection can vary. A deeper understanding of V. anguillarum virulence may inform more effective mitigation measures, but the full range of mechanisms employed by this pathogen remains unclear. A previous transposon mutagenesis study discovered a gene that, when disrupted, reduced virulence in an insect (greater wax moth, Galleria mellonella) infection model. This gene, termed here ggcP, encodes a protein with a GlyGly CTERM sorting motif, which is characteristic of some membrane-localised extracellular proteases in Vibrio species. This present study aimed to further characterise GgcP protein through a holistic approach, by combining in-silico bioinformatic analyses, phenotypic comparison of transposon mutant and parent strains, and by expressing the protein in recombinant E. coli. Homology-based searches indicated that GgcP contains a signal peptide and a Domain of Unknown Function (DUF) 3466, with high prevalence across Vibrio species and a presence in all publicly available V. anguillarum genomes with high conservation. Predictive analysis suggested GgcP is either outer membrane-associated or secreted, with mass spectrometry confirming its presence in both whole-cell and extracellular fractions. Disruption of ggcP in the transposon mutant led to the downregulation of a major outer membrane protein (OmpU), suggesting that GgcP may contribute to membrane integrity and stress response, and the mutant displayed growth defects and increased sensitivity to membrane-active compounds. Successful recombinant expression of GgcP was achieved, and its antigenicity was confirmed with rabbit anti-V. anguillarum polyclonal antibodies, suggesting GgcP is an immunogenic protein and may be a candidate for a subunit vaccine. The work presented in this thesis enhances our understanding of GgcP, although the exact role of this protein in membrane regulation remains to be determined, the new knowledge accumulated in this study may aid the development of strategies to mitigate the impact of V. anguillarum in aquaculture.