Identification and characterization of putative Aeromonas spp. T3SS effectors

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including manyAeromonasspp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105Aeromonasstrains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate families are initially selected using HMM profiles with minimal similarity scores against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. Using our approach, we identified 21AeromonasT3SS likely effector groups, of which 8 represented known or characterized effectors, while the remaining 13 had not previously been described inAeromonas. We experimentally validated ourin silicofindings by assessing the cytotoxicity of representative effectors inSaccharomyces cerevisiaeBY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novelin silicosearch method within vivoexperimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.