Dataset Information

Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli.

ABSTRACT: Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entire ST21 lineage and found a high level of variation in Stx1a phage genomes caused by various mechanisms, including replacement by a different Stx1a phage at the same or different locus. The evolutionary timescale of events changing Stx1a phages in ST21 was also determined. Furthermore, by using an Stx1 quantification system developed in this study, we found notable variations in the efficiency of Stx1 production upon prophage induction, which sharply contrasted with the conserved iron regulated Stx1 production. These variations were associated with the Stx1a phage alteration in some cases but not in other cases; thus, Stx1 production in this STEC lineage was determined by differences not only in Stx1 phages but also in host-encoded factors.

SUBMITTER: Yano B

PROVIDER: S-EPMC10042803 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli.

Yano Bungo B Taniguchi Itsuki I Gotoh Yasuhiro Y Hayashi Tetsuya T Nakamura Keiji K

Scientific reports 20230327 1

Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entir ...[more]

PMID: 36973327

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Project description: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 (stx2astx2c), 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 stx2a-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 stx2a-phage sequences led to similar Stx2 localization, as demonstrated by JH2010ΔrecA and JH2016ΔrecA, respectively. Deletion of the stx2a-phage regulatory region in the wild-type strains prevented the differential localization of Stx2 into the culture supernatant, a finding that suggests that the stx2a-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 stx2a-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 stx2-phages and their influence on Stx2 production and virulence of STEC isolates.

Project description:Shiga toxin (Stx)-producing Escherichia coli (STEC) is a major cause of foodborne diarrheal illness in the United States and globally, and serotype O157:H7 is frequently associated with STEC outbreaks and sporadic cases in the United States. Severe systemic diseases associated with STEC are mediated by Stx types, particularly subtype Stx2a, encoded on inducible bacteriophages. We previously identified two STEC O157:H7 clinical isolates, JH2010 and JH2012, that exhibit a large difference in virulence in a streptomycin (Str)-treated mouse model. In this study, we aimed to identify a genetic basis for the difference in virulence between those strains. Comparison of the stx2a phage sequences showed that JH2012 lacks the lytic genes S and R on the phage genome. We also demonstrated that compared to JH2012 cultures, cultures of JH2010 released more Stx2 into the supernatant and were more sensitive to bacterial lysis during growth with ciprofloxacin (Cip), an inducer of stx phages. We therefore generated an stx2a phage SR deletion mutant strain of JH2010 to determine if those genes were responsible for the high virulence of that strain. We found that deletion of the SR genes from the stx2a phage in JH2010, and another O157:H7 strain, JH2016, resulted in increased cellular retention of Stx2, but there was no difference in virulence compared to the wild-type strains. Our results indicate that the stx2a phage SR genes are involved in Stx2 localization and phage-mediated cell lysis in vitro but that they are not required in wild-type STEC strains for virulence in a mouse model. IMPORTANCE The release of Stx from STEC has been thought to be tied to phage-mediated lysis of the host bacterial cell. In this study, we found that the stx2a phage lytic genes are not required for the virulence of pathogenic O157:H7 clinical isolates in a murine model of STEC infection or for release of Stx2a into the supernatant of bacterial cultures. These results point to an alternate mechanism for Stx2a release from STEC strains.

Dataset Information

Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli.

Publications

Dynamic changes in Shiga toxin (Stx) 1 transducing phage throughout the evolution of O26:H11 Stx-producing Escherichia coli.

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