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Abstract
Vibrio spp. are an autochthonous inhabitant of coastal marine environments where predation by protozoa is a
shaping force leading to the evolution of antiprotozoal mechanisms that may also function as virulence factors
in animal and human hosts. Vibrio cholerae and Vibrio vulnificus were used in this study in order to further
elucidate bacterial adaptations against different model protozoa and the potential role of these antiprotozoal
factors in providing fitness in the environment and in a host.
The transcriptomic profile of established V. cholerae biofilms to predation by the amoeba, Acanthamoeba
castellanii, was investigated. Genes that were significantly differentially expressed between grazed and
ungrazed cultures were identified. Tyrosine metabolic genes were among the down-regulated transcripts in the
grazed population. Homogentisic acid is the main intermediate of the L-tyrosine catabolic pathway, and is
known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant,
disrupted in the gene encoding homogentisate 1, 2 - dioxygenase (hmgA) was more resistant to grazing by A.
castellanii than the wild type.
Grazing resistance of V. vulnificus of different genotypes and places of isolation were evaluated using the
protozoan predators, Tetrahymena pyriformis and A. castellanii, but no significant correlation was found in
relation to grazing resistance. However, an oyster isolate, V. vulnificus Env1, showed significant grazing
resistance and toxicity towards T. pyriformis. The whole genome sequence of Env1 was completed, annotated
and compared to grazing sensitive strains to identify Env1 unique features.
Further studies revealed one of the antiprotozoal mechanisms of Env1 was secreted and iron-dependent. The
transcriptomic profile of V. vulnificus Env1 under iron-replete and -deplete conditions was characterised. A
master virulence regulator, arcA, was up-regulated when iron was readily available and an arcA mutant, showed
a significant decrease in grazing resistance. Therefore, ArcA is a novel global regulator controlling the grazing
resistance of V. vulnificus. In summary, this project revealed new defence systems against protozoan grazing
expressed by V. cholerae and V. vulnificus that also play dual roles in environmental survival and pathogenicity,
accentuating how protozoan grazing drives the evolution of pathogenicity in bacteria in the environment.