Enterohaemorrhagic E. coli (EHEC) is a significant foodborne pathogen and a leading cause of bloody diarrhea, haemorrhagic colitis and haemolytic uremic syndrome. The acquisition of pathogenicity islands by horizontal gene transfer, such as the Shiga toxin (Stx) is the main cause of the severity. Stx is a virulence factor of EHEC and is encoded on the Stx1Φ and Stx2Φ lambdoid bacteriophages. DNA damage induces the StxΦ lytic cycle and leads to toxin release. The level of toxin expression is variable between clinical isolates and isolates that produce high titres of toxin are more likely to cause severe disease. The genetic factors that contribute to variable toxin expression level is unclear.
Many cryptic phages in the EHEC genome encode small regulatory RNAs (sRNAs) that control gene expression post-transcriptionally. In earlier work, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions in EHEC and found that the sRNA EcOnc10 interacts with the mRNA encoding the toxin-antitoxin (TA) system mazEFG. TA systems have been shown to mediate abortive infection and protect cells from phage attack. In particular, mazEF has been shown to protect E. coli from P1 and T4 phage. We hypothesise that the phage-encoded sRNA EcOnc10 represses mazF expression and functions as an anti-abortive infection module that increases phage replication or sensitivity. CRISPRi knockdown of EcOnc10 reduced lytic induction of the Stx2Φ from EHEC by 10-fold supporting the idea that EcOnc10 promotes phage replication. In ongoing work, we will verify the role of mazEFG in phage defence and identify the function of the EcOnc10-mazF interaction.
EcOnc10 is exclusively encoded on bacteriophage elements and is variably present in 1-5 copies in EHEC isolates. We propose that variation in EcOnc10 copy number may contribute to variability in Shiga toxin production by EHEC isolates. Understanding the molecular basis of variation in Shiga toxin production will allow rapid identification of EHEC isolates that are likely to cause severe disease.