Characterization of the small RNAs carried by Outer Membrane Vesicles produced by hlyFpositive Shiga toxin-producing Escherichia coli

Giorgia Barbieri^1,2*Alessia Stefano¹Agostina Pietrantoni¹FEDERICA FRATINI¹Serena Cavallero²Rosangela Tozzoli¹Paola Chiani¹Arianna Boni¹Stefano Morabito¹Valeria Michelacci¹

• ¹National Institute of Health (ISS), Rome, Italy
• ²Sapienza University of Rome, Rome, Italy

Shiga toxin (Stx)–producing Escherichia coli (STEC) harboring virulence determinants of Extraintestinal pathogenic E. coli (ExPEC) are currently emerging in Europe as a cause of severe disease. Among the ExPEC features identified in STEC, the gene hlyF is associated with an augmented production of Outer Membrane Vesicles (OMV). OMVs produced by E. coli strains deliver toxins and smallRNAs, but information on the latter genetic component is scanty. We investigated the smallRNAs contained in the OMVs produced by two hlyF-positive STEC strains producing Stx2 belonging to O26:H11 and O80:H2 serotypes, isolated from a human case of Haemolytic Uraemic Syndrome (HUS) and from a milk sample, respectively. The OMVs were purified from overnight cultures using two sequential ultracentrifugation steps at 100.000 and 200.000 x g of 2h each. The OMVs collected were analyzed by Nanoparticle Tracking Analysis (NTA) and Electron Microscopy (EM) to define their morphology, size and concentration. The whole genome sequences of the two test strains and three additional hlyF-positive STEC strains were analyzed to identify sequences of smallRNAs. Real Time PCR assays were deployed to detect the smallRNAs within OMVs. The EM and NTA characterization confirmed the presence of spherical particles, and showed that a cleaner preparation was obtained after the second step of ultracentrifugation. We identified the sequences of 27 putative small RNAs present in the genome of hlyF-positive STEC strains and absent in that of a non-pathogenic E. coli. Nine of them were identified and quantified in the OMVs produced by the two test strains. A regulatory role in bacterial DNA replication, integration and in stress-response was hypothesized for the identified smallRNAs, some of which encompassing all the domains of life. STnc_100 was the only small RNA identified in the OMVs produced by both strains. Interestingly, a copy of STnc_100 sequence is harboured downstream the stx2 operon carried by Stx2-encoding phages in both the strains and has a complementarity region for stxB gene, suggesting a modulation in Stx production or release. Our results indicate that OMVs release may exert regulatory functions, putatively influencing the crosstalk with the host and the gut microbiota during the infection process.