Enterohaemorrhagic Escherichia coli (EHEC) causes severe intestinal disease and kidney damage through release of Shiga toxins (Stx) encoded on lambdoid Stx bacteriophages. The EHEC pathotype has evolved through horizontal-acquisition of virulence genes, many of which were acquired on phages. These phage elements have brought up to 55 novel regulatory small RNAs (sRNAs) into the genome but their function in EHEC pathogenesis is largely unknown.
One EHEC-specific sRNA, termed EcOnc10, is encoded exclusively within cryptic phage elements and present in up to 9 copies in EHEC genomes. EcOnc10 is present in higher copy numbers in genomes that encode Shiga toxin 2, the toxin serotype known to cause more severe human disease, suggesting a function in virulence.
Using sRNA interactome data (RNase E-CLASH), we identified mRNA targets of EcOnc10 and have experimentally verified repression of the nucleoid structuring protein, Hha. Hha regulates over 120 genes including many horizontally-acquired virulence genes, motility, and the type 3 secretion system (T3SS) that is essential for colonisation. The later were confirmed phenotypically. RNA-seq analysis also demonstrated repression of genes involved in nitrate/nitrite metabolism and nitric oxide resistance suggesting that EcOnc10 promotes virulence gene expression while sensitising the cell to nitric oxide.
Strikingly, most EHEC strains carry a deletion within the nitric oxide resistance gene norV, particularly those encoding Stx1. Nitric oxide is used as a source of nitrate for anaerobic growth during gastrointestinal colonisation. Our results suggest that phage-encoded sRNAs further disable nitric oxide resistance in EHEC strains and we speculate that this impacts gastrointestinal colonisation and toxin release.