Identification and characterization of putative Aeromonas spp. T3SS effectors

Authors: RANGEL, LUIZ - University Of Connecticut; MARDEN, JEREMIAH - University Of Connecticut; COLSTON, SOPHIE - University Of Connecticut; SETUBAL, JOAO - Universidade De Sao Paulo; GRAF, JOERG - University Of Connecticut; GOGARTEN, JOHANN - University Of Connecticut

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2019
Publication Date: 6/4/2019
Citation: Rangel, L.T., Marden, J., Sestubal, J.C., Graf, J., Gogarten, J.P. 2019. Identification and characterization of putative Aeromonas spp. T3SS effectors. PLoS One. 14(6):e0214035. https://doi.org/10.1371/journal.pone.0214035.
DOI: https://doi.org/10.1371/journal.pone.0214035

Interpretive Summary: Aeromonads can cause a number of diseases in fishes, particularly in salmon and trout. One important mechanism that allows these bacteria to cause disease is to inject toxins into the animal cell using a type three secretion system, T3SS. One strain of bacteria can carry multiple toxins that are secreted by the T3SS and no large survey of possible toxins has been carried out previously. In this study, we developed a bioinformatic pipeline to identify these toxins. Using this approach we screened 105 Aeromonas genomes and identified 21 different potential T3SS toxins. 13 of them had not been described in Aeromonas before. We validated the toxins experimentally by expressing them inside yeast. 15 of the 21 were toxic to yeast cells, confirming that them as toxins. This approach discovered a large number of new toxins in Aeromonas and can be widely applied to other bacteria.

Technical Abstract: 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 many Aeromonas spp., 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 105 Aeromonas strains 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 21 Aeromonas T3SS likely effector groups, of which 8 represented known or characterized effectors, while the remaining 13 had not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, 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 novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.