Early termination of the Shiga toxin transcript generates a regulatory small RNA

Abstract

Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

Small RNA Regulation of Virulence in Pathogenic Escherichia coli

“…Sakai ( Tree et al., 2014 ) this region of the Sp5 Stx2 phage was found to be bound by Hfq (termed sRNA EcOnc53). In our recent transcriptome-wide 5’ and 3’ end mapping data (dRNA-seq and Term-seq; Sy et al., 2020 ) we find that EcOnc53 is 67 nt in length ( Figure 1 ). Gruber and Sperandio (2015) also identified EcOnc53 in the Stx phage BP933W, termed sRNA108 in their study.…”

“…Surprisingly, this short transcript is not simply degraded by the cell but is processed by RNase E to produce the stable sRNA termed StxS. StxS is expressed from both lysogenic Stx1 and Stx2a bacteriophages, and represses Shiga toxin 1 production 3-fold under lysogenic conditions by directly binding to stx1B RBS to silence translation ( Sy et al., 2020 ). StxS does not appear to regulate stx2AB (that is only produced during lytic induction), although Stx2a is also likely regulated by as yet unidentified Hfq-dependent sRNAs (discussed below).…”

“…StxS sRNA also activates the stationary phase general stress response regulator RpoS through interactions with an activating seed region in the rpoS 5’ UTR ( Sy et al., 2020 ). StxS binds to rpoS at the same site as the other known rpoS- activating sRNAs ArcZ, DsrA, and RprA ( Lease et al., 1998 ; Majdalani et al., 2001 ; Mandin and Gottesman, 2010 ).…”

“…StxS likely acts through the same mechanism to constitutively activate rpoS , at least partly uncoupling rpoS from post-transcriptional repression in EHEC. Through StxS activation of rpoS translation, it was shown that EHEC is able to increase stationary phase cell density ~20% in nutrient limited minimal media ( Sy et al., 2020 ).…”

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity

“…Interestingly, a continuous transcription activity at phage late promoter p R’ , which is terminated directly downstream at t R’ , generates a short RNA byproduct under lysogenic conditions [ 44 ]. It was demonstrated that this regulatory small RNA represses expression of Stx1 under lysogenic conditions and modulates host fitness [ 45 ].…”