Small RNAs carried by Outer Membrane Vesicles produced by hlyF-positive Shiga toxin- producing Escherichia coli represent a complex mechanism to regulate the infection process

Introduction Shiga toxin (Stx)–producing Escherichia coli (STEC) harboring virulence determinants typical 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 pR444_A-like plasmids carry hlyF, a gene associated with an augmented production of Outer Membrane Vesicles (OMV). OMVs produced by E. coli strains have been shown to deliver toxins and small RNAs, but information on the latter genetic component is still scanty. Materials and Methods We investigated the small RNAs 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 small RNAs. Specific Real Time PCR assays were deployed to detect the small RNAs within OMVs. Results The EM and NTA characterization confirmed the presence of bilayered-particles and showed that the introduction of sequential centrifugations in OMVs’ purification resulted in a cleaner preparation obtained at 200.000 x g, suitable for downstream small RNA investigations. We were able to identify the sequences of 27 putative small RNAs (shorter than 200 nucleotides) present in the genome of hlyF-positive STEC strains and absent in that of a non-pathogenic E. coli K12 strain, analyzed for comparison. For 12 small RNAs it was possible to develop specific Real Time PCR assays. 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 small RNAs, some of which encompass 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. Discussion and Conclusions 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. Furthermore, our findings suggest that the small RNA STnc_100 may exert a role in the regulation of Stx production putatively mediated by OMVs delivery.