Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases

Abstract

Free-living protozoa affect the survival and virulence evolution of pathogens in the environment. In this study, we explored the fate of <i>Aeromonas hydrophila</i> when co-cultured with the bacteriovorous ciliate <i>Tetrahymena thermophila</i> and investigated bacterial gene expression associated with the co-culture. Virulent <i>A. hydrophila</i> strains were found to have ability to evade digestion in the vacuoles of this protozoan. In <i>A. hydrophila</i>, a total of 116 genes were identified as up-regulated following co-culture with <i>T. thermophila</i> by selective capture of transcribed sequences (SCOTS) and comparative dot-blot analysis. A large proportion of these genes (42/116) play a role in metabolism, and some of the genes have previously been characterized as required for bacterial survival and replication within macrophages. Then, we inactivated the genes encoding methionine sulfoxide reductases, <i>msrA</i>, and <i>msrB</i>, in <i>A. hydrophila</i>. Compared to the wild-type, the mutants Δ<i>msrA</i> and Δ<i>msrAB</i> displayed significantly reduced resistance to predation by <i>T. thermophila</i>, and 50% lethal dose (LD₅₀) determinations in zebrafish demonstrated that both mutants were highly attenuated. This study forms a solid foundation for the study of mechanisms and implications of bacterial defenses.