Immunity Profiles of Wild-Type and Recombinant Shiga-Like Toxin-Encoding Bacteriophages and Characterization of Novel Double Lysogens

Abstract

The pathogenicity of Shiga-like toxin (stx)-producing Escherichia coli (STEC), notably serotype O157, the causative agent of hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura, is based partly on the presence of genes (stx 1 and/or stx 2 ) that are known to be carried on temperate lambdoid bacteriophages. Stx phages were isolated from different STEC strains and found to have genome sizes in the range of 48 to 62 kb and to carry either stx 1 or stx 2 genes. Restriction fragment length polymorphism patterns and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles were relatively uninformative, but the phages could be differentiated according to their immunity profiles. Furthermore, these were sufficiently sensitive to enable the identification and differentiation of two different phages, both carrying the genes for Stx2 and originating from the same STEC host strain. The immunity profiles of the different Stx phages did not conform to the model established for bacteriophage lambda, in that the pattern of individual Stx phage infection of various lysogens was neither expected nor predicted. Unexpected differences were also observed among Stx phages in their relative lytic productivity within a single host. Two antibiotic resistance markers were used to tag a recombinant phage in which the stx genes were inactivated, enabling the first reported observation of the simultaneous infection of a single host with two genetically identical Stx phages. The data demonstrate that, although Stx phages are members of the lambdoid family, their replication and infection control strategies are not necessarily identical to the archetypical bacteriophage , and this could be responsible for the widespread occurrence of stx genes across a diverse range of E. coli serotypes.

Cumulative effect of prophage burden on Shiga toxin production in Escherichia coli

“…Theoretically, this ability has clear implications for the severity of Stx-mediated disease symptoms, commensurate with an increased number of toxin gene copies and increased toxin load, but previously this had not been directly and unequivocally demonstrated. Different stx genes, associated with different prophages or prophage remnants, are regularly found in combination within a single host (Allison et al, 2003;Bielaszewska et al, 2007;Eklund et al, 2002;Muniesa et al, 2003;Zheng et al, 2008), and virulence profiles comprising two stx2 genes are frequently associated with HUS and bloody diarrhoea (Banatvala et al, 1996;Eklund et al, 2002;Elliott et al, 2001;Tozzi et al, 2003;Woodward et al, 2002). Furthermore, the presence of two stx operons can lead to increased in vitro toxin expression (Bielaszewska et al, 2006;Cornick et al, 2002;Eklund et al, 2002;Muniesa et al, 2003), though this observation is not without exceptions (Serra-Moreno et al, 2008), nor does it necessarily equate to more severe clinical disease (Bielaszewska et al, 2006;Cornick et al, 2002).…”

“…host cell (Allison et al, 2003;Fogg et al, 2007;SerraMoreno et al, 2008). Theoretically, this ability has clear implications for the severity of Stx-mediated disease symptoms, commensurate with an increased number of toxin gene copies and increased toxin load, but previously this had not been directly and unequivocally demonstrated.…”

“…Double lysogens were created by infecting the above-mentioned single lysogen carrying pStxCDF (OD 600 0.5) with the isogenic bacteriophage w24 B : : Cat at an m.o.i. of 0.1, as previously described (Allison et al, 2003).…”

“…1a) was created as described previously (Fogg et al, 2011), except that pCDF-Duet (Novagen) was used in place of pACYC. pStxCDF was transformed into competent MC1061 W24 B : : Kan lysogen cells (Allison et al, 2003) and naïve E. coli MC1061 (James et al, 2001) control cells. Single W24 B lysogens are preferentially integrated at a single location in E. coli MC1061, 59 of intS, and the strain used here has been shown by Southern hybridization to harbour only one prophage (Fogg et al, 2007).…”

“…These similarities include the expression of an orthologous global repressor protein, CI, that is expected to effectively shut off all gene expression from the integrated prophage and provide immunity to superinfection from incoming homoimmune phages. The assumption that Stx phages conform to this immunity model was confounded when multiple lysogens of a differentially labelled, isogenic lambdoid Stx2-encoding phage (w24 B ) were created (Allison et al, 2003). Furthermore, it was proved that at least three isogenic prophages could be harboured simultaneously within a single host at distinct integration sites (Fogg et al, 2007), an eventuality not reproducible with bacteriophage lambda (Fogg et al, 2010).…”