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

Date Published: January 10, 2019

Publisher: Public Library of Science

Author(s): Sowmya Balasubramanian, Marcia S. Osburne, Haley BrinJones, Albert K. Tai, John M. Leong, Steven R. Blanke.


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 Φstx2dact. 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 Φstx2dact 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 (Φstx2dact) 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 (Φstx2dact) 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.

Partial Text

Shiga toxin-producing Escherichia coli (STEC) is a food-borne zoonotic agent associated with worldwide disease outbreaks that pose a serious public health concern. Enterohemorrhagic Escherichia coli (EHEC), a subset of STEC harboring specific virulence factors that promote a specific mode of colonization of the intestinal epithelium (see below), is acquired by humans by ingestion of contaminated food or water, or through contact with animals or their environment. EHEC serotype O157:H7 is a major source of E. coli food poisoning in the United States, accounting for more than 390 outbreaks in the last two decades [1–5]. EHEC infection usually presents as localized hemorrhagic colitis, and may progress to the life-threatening systemic hemolytic uremic syndrome (HUS), characterized by the triad of hemolytic anemia, thrombocytopenia, and renal failure [5, 6]. HUS is the leading cause of renal failure in children [7].

Commensal organisms have the potential to suppress or enhance phage induction and Stx production. Although a role for induction of stx-encoding prophages in the production of Stx and serious disease during animal infection has been well documented in antibiotic-treated and germ-free mice [40, 41, 69], we used a murine model of EHEC infection that features an intact microbiome.




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