Differences in the Shiga Toxin (Stx) 2a Phage Regulatory Switch Region Influence Stx2 Localization and Virulence of Stx-Producing Escherichia coli in Mice

Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne illness globally, and infection with serotype O157:H7 is associated with increased risk of hospitalization and death in the U.S. The Stxs are encoded on a temperate bacteriophage (stx-phage), and phage induction leads to Stx expression; subtype Stx2a in particular is associated with more severe disease. Our earlier studies showed significant levels of RecA-independent Stx2 production by STEC O157:H7 strain JH2010 (stx(2a)stx(2c)), even though activated RecA is the canonical trigger for stx-phage induction. This study aimed to further compare and contrast RecA-independent toxin production in Stx2-producing clinical isolates. Deletion of recA in JH2010 resulted in higher in vitro supernatant cytotoxicity compared to that from JH2016ΔrecA, and the addition of the chelator ethylenediaminetetraacetic acid (EDTA) and various metal cations to the growth medium exacerbated the difference in cytotoxicity exhibited by the two deletion strains. Both the wild-type and ΔrecA deletion strains exhibited differential cytotoxicity in the feces of infected, streptomycin (Str)-treated mice. Comparison of the stx(2a)-phage predicted protein sequences from JH2010 and JH2016 revealed low amino acid identity of key phage regulatory proteins that are involved in RecA-mediated stx-phage induction. Additionally, other STEC isolates containing JH2010-like and JH2016-like stx(2a)-phage sequences led to similar Stx2 localization, as demonstrated by JH2010ΔrecA and JH2016ΔrecA, respectively. Deletion of the stx(2a)-phage regulatory region in the wild-type strains prevented the differential localization of Stx2 into the culture supernatant, a finding that suggests that the stx(2a)-phage regulatory region is involved in the differential ΔrecA phenotypes exhibited by the two strains. We hypothesize that the amino acid differences between the JH2010 and JH2016 phage repressor proteins (CIs) lead to structural differences that are responsible for differential interaction with RecA. Overall, we discovered that non-homologous stx(2a)-phage regulatory proteins differentially influence RecA-independent, and possibly RecA-dependent, Stx2 production. These findings emphasize the importance of studying non-homologous regulatory elements among stx(2)-phages and their influence on Stx2 production and virulence of STEC isolates.