Small-RNAs carried by Outer Membrane Vesicles: a (novel) complex mechanism to regulate the infection process of hlyF-positive Shiga Toxin-producing Escherichia coli strains

Introduction Shiga toxin (Stx)–producing Escherichia coli (STEC) cause severe disease including Haemolytic Uraemic Syndrom. STEC strains showing features of Extraintestinal pathogenic E. coli are emerging as a cause of serious illness. Among these features, hlyF gene is associated with an increased production of Outer Membrane Vesicles (OMV). OMVs produced by E. coli have been shown to deliver toxins and small-RNAs, but information on the latter component is still scanty. Methods We investigated the small-RNAs within the OMVs produced by two hlyF-positive STEC strains of O26:H11 and O80:H2 serotypes. OMVs were purified in two fractions by ultracentrifugation at different speeds and analyzed by SEM, TEM and NTA to define their morphology, size and concentration. The whole genome sequences of the two strains and three additional hlyF-positive STEC strains were analyzed to predict putative small-RNAs. Real-Time PCR assays were deployed to validate, identify and quantify the small-RNAs within OMVs. Results NTA and electron microscopy analysis of STEC strains showed an OMVs population with diameters distribution between 20-180 nm. Of the 28 putative small-RNAs identified in hlyF-positive STEC genomes, ten were identified and quantified in the OMVs produced by the strains analyzed. The small-RNAs termed ALIL, EFASI and STAXI were detected at highest concentration in the OMVs produced by O26:H11 strain, while ASpc02 and STnc490 in those of O80:H2 strain. STnc100 was identified in the OMVs produced by both strains and is encoded downstream the stx2 operon in both strains. Conclusion and funding A regulatory role in DNA replication/integration and in stress-response was hypothesized for the detected small-RNAs, some of which encompass all the domains of life. The genomic localization of STnc100 suggests a regulatory function in Stx production/release. These results suggest that the small-RNAs identified may exert a role in the regulation of Stx production and in the cross-talk with the host during infection, mediated by OMVs delivery.