Article Figures & Data
Figures
- FIG 1
Neutralizing Stx2 production from E. coli using an engineered temperate phage. (A) As an alternative to bacteriolytic strategies that aim to block pathogenesis by killing bacteria, we propose an alternative approach that aims to reduce expression of the virulence factor. We do so by introducing a phage to lysogenize the targeted bacteria within the mammalian gut and to express a transcriptional repressor of the virulence factor. (B) (i) Genetic schemes of E. coli933W showing the 933W prophage expressing cI to maintain a lysogenic state in which stx2 is not expressed and (ii) induction that causes degradation of the cI protein leading to expression of the lytic genes including cro and stx2. (iii) This leads to cell lysis, releasing phage progeny and Stx2 protein. Expression of a nondegradable cI for the 933W prophage, 933W⋅cIind-, from a genomically integrated engineered temperate phage (antivirulence prophage) can force the 933W prophage to remain lysogenic despite induction and degradation of endogenous cI protein.
- FIG 2
Temperate phage robustly transduces E. coli colonizing the mouse gut. (A) Experimental timeline examining the impact of λBH1 phage on precolonized E. coli in mice. (B) Fecal concentrations of free λBH1 phage and (C) E. coli. (D) Percentage of fecal E. coli lysogenized by λBH1. Symbols represent distinct samples from individual mice (n = 3) with lines or bars representing the geometric mean.
- FIG 3
Hybrid λ phages overcome superinfection exclusion by prophage 933W. (A) Depiction of superinfection exclusion by the 933W prophage that inhibits infection by λ phage but is ineffective against a hybrid phage that contains the immunity region from another lambdoid phage in a λ phage background. (B) Schematic representation of a portion of the λ phage genome containing the λ immunity region and its hybrids containing immunity regions from other lambdoid phages (933W, 21, 434, and P22) in a λ phage background. Efficiencies of plating (EOPs) for λ phage and its hybrids on E. coli933W are shown to the right. (C) Schematic representation of λBH1 phage, which is a λ phage with a kanamycin resistance cassette (Kanr) and 933W⋅cIind- inserted into the nonessential b2 region of the phage genome. λBH2 phage is a product of a phage cross between λBH1 and λimmP22dis resulting in a phage containing Kanr and 933W⋅cIind- genes with a P22 immunity region in a λ phage background. Their respective EOPs against E. coli933W are shown to the right. Symbols represent biological replicates with bars representing the geometric mean.
- FIG 4
Engineered λ phage neutralizes Shiga toxin production from E. coli933W in vitro. (A) E. coli933W was mixed with buffer, λimmP22dis, or λBH2 free phage (MOI of ∼1) at t = 0 from which the concentration of Stx2 was measured after 8 h of in vitro culture under (B) noninduced and (C) induced conditions (0.5 μg/ml of mitomycin C). Significance was calculated by one-way ANOVA with the post hoc Tukey test. (D) Total E. coli933W and (E) the percentage of bacteria lysogenized by λBH2 were measured over 8 h under noninduced and mitomycin C-induced conditions. (F) E. coli933W lysogenized with λBH1 or λBH2 was cultured in vitro and analyzed for Stx2 produced under (G) noninduced or (H) induced conditions. Symbols represent biological replicates with bars or lines representing the geometric mean.
- FIG 5
λBH2 phage lysogenizes E. coli933W in the murine gut and reduces fecal Shiga toxin concentrations. (A) Streptomycin-treated mice precolonized with E. coli933W received one dose of 5 × 108 PFU of λimmP22dis phage or λBH2 phage orally. Mitomycin C was administered three times at 3-h intervals by intraperitoneal injection to induce stx2 expression in the gut. (B) Concentrations of fecal Stx2 after induction with mitomycin C. A one-way ANOVA with post hoc Tukey test was used to compare Stx2 levels between buffer, λimmP22dis, and λBH2 conditions while a two-tailed Wilcoxon test was used to compare Stx2 levels after λBH2 phage treatment between days 3 and 4. (C) Concentrations of total fecal E. coli933W with significance calculated by two-way ANOVA with post hoc Tukey test and (D) percentage of fecal E. coli933W found to be lysogenized by λBH2 phage. (E) Concentration of fecal Stx2 as a function of fraction of fecal E. coli933W lysogenic for λBH2 phage on day 3 and (F) day 4. Solid and dashed lines represent means and 95% confidence intervals of linear regression, respectively. P value describes significance of slope being nonzero. Symbols represent individual mice for buffer (n = 9), λimmP22dis (n = 10), and λBH2 (n = 10) conditions. On days 3 and 4, one λimmP22dis phage-treated mouse and one buffer-treated mouse, respectively, were unable to produce stool for analysis. Bars or lines represent geometric means.
Supplemental Material
FIG S1
Spot testing of λ and λimm933W phage against lawns of their lysogens in E. coli. The zone of lysis (dark circle) on a lawn of E. coli indicates successful phage infection. Both phages are capable of infecting nonlysogens and lysogens of the other phage (i.e., λ phage infecting a λimm933W lysogen and vice versa), indicating that 933W⋅cI and λ⋅cI are not inhibitory to λ phage and 933W phage infection, respectively. Download FIG S1, EPS file, 49.2 MB.
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FIG S2
Genetic map of immunity regions within λ phage and its hybrids. (A) Genetic map of portions of the λ phage genome with brackets indicating the regions within λ that have been replaced with immunity regions from other lambdoid phages. (B) Within λimm933W phage, the genes homologous to 933W (accession no. NC_000924) are labeled in red and those homologous to λ (NC_001416) are labeled in black. Percent identities for genes less than identical are indicated in parentheses. (C) Within λimmP22dis phage, the genes homologous to P22 (AF217253) are labeled in green and those homologous to λ (NC_001416) are labeled in black. Download FIG S2, EPS file, 3.5 MB.
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TABLE S3
DNA sequences in this study. Download Table S3, DOCX file, 0.03 MB.
Copyright © 2020 Hsu et al.
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FIG S3
Genetic maps of λBH1 and λBH2 phage. Download FIG S3, EPS file, 0.8 MB.
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FIG S4
Spot assays of 3 μl of ∼107 PFU/ml of λ, λimm933W, λimm434, and λimmP22dis phages against nonlysogenic E. coli or its λ, λBH1, or λBH2 lysogen. Download FIG S4, PDF file, 0.8 MB.
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FIG S5
Growth curves of lysogens or nonlysogens grown in the absence or presence of mitomycin C. Download FIG S5, PDF file, 0.04 MB.
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TABLE S1
Bacteria used in this study. Download Table S1, DOCX file, 0.01 MB.
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TABLE S2
Phages used in this study. Download Table S2, DOCX file, 0.01 MB.
Copyright © 2020 Hsu et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
