Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection.

Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage stx(2dact). This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that phi stx(2dact) infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (phi stx(2dact)) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (phi stx(2dact)) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not. Author summary Enterohemorrhagic Escherichia coli (EHEC), a food-borne pathogen that produces Shiga toxin, is associated with serious disease outbreaks worldwide, including over 390 food poisoning outbreaks in the U.S. in the last two decades. Humans acquire EHEC by ingesting contaminated food or water, or through contact with animals or their environment. Infection and toxin production may result in localized hemorrhagic colitis, but may progress to life-threatening systemic hemolytic uremic syndrome (HUS), the leading cause of kidney failure in children. Treatment for EHEC or HUS remains elusive, as antibiotics have been shown to exacerbate disease. Shiga toxin genes reside on a dormant bacterial virus present in the EHEC genome, but are expressed when the virus is induced to leave its dormant state and begin to replicate. Extensive virus replication has been thought necessary to produce sufficient toxin to cause disease. Using viral and bacterial mutants in our EHEC disease mouse model, we showed that whereas an inducing signal needed to begin viral replication was essential for lethal disease, virus production was not: sufficient Shiga toxin was produced to cause lethal mouse disease, even without viral replication. Future analyses of EHEC-infected human samples will determine whether this same phenomenon applies, potentially directing intervention strategies.