Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7

Mondal, Shakhinur Islam; Islam, Rakibul; Sawaguchi, Akira; Asadulghani,; Ooka, Tadasuke; Gotoh, Yasuhiro; Kasahara, Yasuhiro; Hayashi, Tetsuya

doi:10.1038/srep39036

Cited by 12 publications

(9 citation statements)

References 77 publications

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“…TEM classified the stx2a -convering phage of E. coli O104:H4 as short-tailed (Beutin et al, 2012 ). Secondly, Mondal et al ( 2016 ) identified nine genes responsible for phage morphogenesis of short-tailed phage designated SP5, and these were found by BLAST to be nearly identical (>99%) on amino acid level to the homologs from phage PA2. Notably, one of these genes, ECs1228, is predicted to encode a phage tail fiber and 100% identical to the PA2 phage homolog on amino acid level.…”

Section: Discussionmentioning

confidence: 99%

Non-pathogenic Escherichia coli Enhance Stx2a Production of E. coli O157:H7 Through Both bamA-Dependent and Independent Mechanisms

et al. 2018

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Intestinal colonization by the foodborne pathogen Escherichia coli O157:H7 leads to serious disease symptoms, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Synthesis of one or more Shiga toxins (Stx) is essential for HUS and HC development. The genes encoding Stx, including Stx2a, are found within a lambdoid prophage integrated in the E. coli O157:H7 chromosome. Enhanced Stx2a expression was reported when specific non-pathogenic E. coli strains were co-cultured with E. coli O157:H7, and it was hypothesized that this phenotype required the non-pathogenic E. coli to be sensitive to stx-converting phage infection. We tested this hypothesis by generating phage resistant non-pathogenic E. coli strains where bamA (an essential gene and Stx phage receptor) was replaced with an ortholog from other species. Such heterologous gene replacement abolished the ability of the laboratory strain E. coli C600 to enhance toxin production when co-cultured with E. coli O157:H7 strain PA2, which belongs to the hypervirulent clade 8. The extracellular loops of BamA (loop 4, 6, 7) were further shown to be important for infection by stx2a-converting phages. However, similar gene replacement in another commensal E. coli, designated 1.1954, revealed a bamA-independent mechanism for toxin amplification. Toxin enhancement by 1.1954 was not the result of phage infection through an alternative receptor (LamB or FadL), lysogen formation by stx2a-converting phages, or the production of a secreted molecule. Collectively, these data suggest that non-pathogenic E. coli can enhance toxin production through at least two mechanisms.

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Section: Discussionmentioning

confidence: 99%

Non-pathogenic Escherichia coli Enhance Stx2a Production of E. coli O157:H7 Through Both bamA-Dependent and Independent Mechanisms

et al. 2018

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“…The location of the host integration site and genome size for each prophage is indicated in parentheses after the phage name. Open reading frames of prophages λ, 933W, and Sp5 [28, 91, 92] are shown in comparison to those of prophage 1720a-02, and are depicted as arrows pointing in the direction of transcription. The site of insertion of the chloramphenicol cassette ( cat ) in phage 1720a-02 is indicated with an open triangle.…”

Section: Supporting Informationmentioning

confidence: 99%

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

et al. 2019

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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.

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“…The main virulence factors of STEC are the phage-encoded cytotoxins called Shiga toxins (Stx family), with several toxin subtypes [3]. Stx phages can be present, either integrated in the STEC chromosome as temperate bacteriophage or as free particles in the environment [4][5][6]. STEC strains are genetically heterogeneous and grouped as serotypes, and the…”

Section: Introductionmentioning

confidence: 99%

Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

et al. 2020

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Molecular methods may reveal the presence of pathogens in samples through the detection of specific target gene(s) associated with microorganisms, but often, the subsequent cultural isolation of the pathogen is not possible. This discrepancy may be related to low concentration of the cells, presence of dead cells, competitive microflora, injured cells and cells in a viable but non-culturable state, free DNA and the presence of free bacteriophages which can carry the target gene causing the PCR-positive/culture-negative results. Shiga-toxigenic Escherichia coli (STEC) was used as a model for studying this phenomenon, based on the phage-encoded cytotoxins genes (Stx family) as the detection target in samples through real-time qPCR. Stx phages can be integrated in the STEC chromosome or can be isolated as free particles in the environment. In this study, a combination of PCR with culturing was used for investigating the presence of the stx1 and stx2 genes in 155 ovine recto-anal junction swab samples (method (a)-PCR). Samples which were PCR-positive and culture-negative were subjected to additional analyses including detection of dead STEC cells (method (b)-PCR-PMA dye assay), presence of Stx phages (method (c)-plaque assays) and inducible integrated phages (method (d)-phage induction). Method (a) showed that even though 121 samples gave a PCR-positive result (78%), only 68 samples yielded a culturable isolate (43.9%). Among the 53 (34.2%) PCR-positive/culture-negative samples, 21 (39.6%) samples were shown to have STEC dead cells only, eight (15.1%) had a combination of dead cells and inducible stx phage, while two samples (3.8%) had a combination of dead cells, inducible phage and free stx phage, and a further two samples had Stx1 free phages only (3.8%). It was thus possible to reduce the samples with no explanation to 20 (37.7% of 53 samples), representing a further step towards an improved understanding of the STEC PCR-positive/culture-negative phenomenon.

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Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7

Cited by 12 publications

References 77 publications

Non-pathogenic Escherichia coli Enhance Stx2a Production of E. coli O157:H7 Through Both bamA-Dependent and Independent Mechanisms

Non-pathogenic Escherichia coli Enhance Stx2a Production of E. coli O157:H7 Through Both bamA-Dependent and Independent Mechanisms

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

Investigation of the Causes of Shigatoxigenic Escherichia coli PCR Positive and Culture Negative Samples

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