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PLoS One. 2019 Jun 4;14(6):e0214035. doi: 10.1371/journal.pone.0214035. eCollection 2019.

Identification and characterization of putative Aeromonas spp. T3SS effectors.

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Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, United States of America.
Interunidades em Bioinformática, Universidade de São Paulo, São Paulo, Brasil.
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brasil.
Institute for Systems Genomics, University of Connecticut, Storrs, Connecticut, United States of America.


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 Aeromonas gene families are initially selected using Hidden Markov Model (HMM) profile searches against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. This approach identified 21 Aeromonas T3SS likely effector families, of which 8 represent known or characterized effectors, while the remaining 13 have 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.

Conflict of interest statement

J. Graf is a leech microbiology consultant for the German leech farm Biebertaler Blutegelzucht GmbH, Biebertal, Germany, and the company does not direct or approve J. Graf’s research and publications. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors have declared that no competing interests exist.

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