Sustained in situ protein production and release in the mammalian gut by an engineered bacteriophage

Abstract

Oral administration of biologic drugs is challenging because of the degradative activity of the upper gastrointestinal tract. Strategies that use engineered microbes to produce biologics in the lower gastrointestinal tract are limited by competition with resident commensal bacteria. Here we demonstrate the engineering of bacteriophage (phage) that infect resident commensals to express heterologous proteins released during cell lysis. Working with the virulent T4 phage, which targets resident, nonpathogenic Escherichia coli, we first identify T4-specific promoters with maximal protein expression and minimal impact on T4 phage titers. We engineer T4 phage to express a serine protease inhibitor of a pro-inflammatory enzyme with increased activity in ulcerative colitis and observe reduced enzyme activity in a mouse model of colitis. We also apply the approach to reduce weight gain and inflammation in mouse models of diet-induced obesity. This work highlights an application of virulent phages in the mammalian gut as engineerable vectors to release therapeutics from resident gut bacteria.

Figure 1.

Phage-induced lysis of bacteria releases intracellular proteins. (A) Scheme of the potential utility of this strategy in the gut, by introducing a recombinant phage to target an endogenous commensal gut bacterium. (B) Biological replicates of batch culture E. coli and wildtype T4 phage demonstrates the presence of phage increases the mean extracellular quantity of sfGFP (n=3).

Figure 2.

Survey of T4 phage promoters for in vitro sfGFP production. (A) During T4 infection, promoters are expressed in a series of stages. Representative promoters were selected from early, middle, and late stages of expression. (B) To survey the protein production from these promoters, they were encoded upstream of sfgfp and inserted into the ac region of the T4 phage genome to produce a small library of recombinant T4 phages. (C) After 12 h of coculture of the T4 library with E. coli, the fluorescence and (D) phage concentration was measured in the supernatant (n=3 biological replicates). Bars represent mean values. Statistical analyses were performed by one-way ANOVA compared to wildtype T4 phage with Dunnett’s multiple comparison test.

Figure 3.

In vivo sfGFP production by engineered phage. (A) Schematic representation of the mouse model. (B) Fecal E. coli and phage concentrations immediately prior to phage administration and at experimental conclusion for wildtype T4 (control) and T4::sfgfp phage conditions (n=6 mice per condition). (C) Mean fluorescence intensity of the murine mucosa was significantly higher for mice colonized with T4::sfgfp phage compared to wildtype T4 (n=6 mice per condition). (D) Fluorescence was also visually evident within fluorescence microscopy images of unfixed colonic sections, with minimal autofluorescence in mice receiving wildtype T4 phage, while (E) mice colonized with T4::sfgfp had substantially greater mucosal fluorescence. Scale bars = 50 μm. Dashed white lines represent the intestinal mucosa. (F-O) Colonic sections were fixed and fluorescently stained with nuclear (blue) and mucin (red) stains (n=6 mice per condition). Images shown are representative results from n=3 mice per condition. Scale bars = 10 μm. (F,K) Macroscopic and (G,L) higher magnification views show overlays of DAPI, FITC, and TRITC channels whereas individual high magnification channels for (H,M) FITC, (I,N) TRITC, and (J,O) DAPI are shown separately. All bars represent mean values. Statistical analysis was performed by a two-tailed unpaired t-test.

Figure 4.

Reduction of DSS-induced colitis via Serpin production. (A) Schematic of 1.5% DSS-induced colitis mouse model, assessing Serpin reduction of inflammation and NE activity. (B) Fecal bacteria and (C) phage concentrations for the wildtype T4 (control) and T4::serpin phage conditions was consistent throughout the experiment, including during DSS administration (n=5 mice per condition). (D) Weight change and (E) fecal neutrophil elastase concentration based on protease activity was determined after 7 d of DSS, on Day 9 (n=5 mice per condition). (F) Analyses of colonic cells by flow cytometry measured neutrophil fractions, and the density of (G) CD24 markers on these neutrophils (n=4 mice for the wildtype T4 phage condition and n=5 mice for the T4::serpin condition). Statistical analyses were accomplished via two-tailed unpaired t-test.

Figure 5.

The efficacy of T4::clpB to reduce food consumption and weight gain. (A) Schematic representation of the in vivo experiment using a murine model of diet-induced obesity with C57BL/6 mice. Mice were colonized with E. coli alone “no phage” (control, n=8 mice), wildtype T4 phage (control, n=9 mice), or T4::clpB phage (n=9 mice). (B) Phage measured by plaque assay and (C) bacteria measured by selective culture show that wildtype T4 and T4::clpB are able to coexist with their E. coli host in vivo and that the presence of phage does not markedly alter E. coli colonization (n=8 mice for the E. coli alone condition and n=9 mice for wildtype T4 and T4::clpB conditions). (D) Fluorescence microscopy of E. coli mixed with wildtype T4 (control) or T4::clpB phage after fixation and labeling of liquid cultures for DNA (DAPI), and ClpB (anti-α-MSH antibodies) and (E) quantification of ClpB fluorescence from individual bacteria (n=27 cells for wildtype T4 and n=40 cells for T4::clpB). (F) Fluorescence microscopy of murine intestinal samples labeled for DNA, mucin (WGA/UEI) and ClpB, and (G) its quantification of ClpB fluorescence in mice colonized with wildtype T4 (control) or T4::clpB phage. Scale bars = 10 μm. Of wildtype T4 mice (n=3), 20, 15 and 20 cells were counted from each mouse. Of T4::clpB mice (n=3), 20, 22 and 20 cells were counted from each mouse. Different shading of symbols represent different mice. (H) Longitudinal weight measurements were collected for E. coli (n=8), E. coli + wildtype T4 phage (n=9) and E. coli + T4::clpB phage mice (n=9), and for a subset of mice, (I) food consumed on a per day basis (n= 5 mice per condition) and (J) overall activity was measured over a 24 h period before sacrifice for E. coli (n=4), E. coli + wildtype T4 phage (n=5) and E. coli + T4::clpB phage mice (n=5). (K) Cytokine panel of serum at sacrifice. Samples with undetectable concentrations were set to the limit of detection. (B,C,E,K) Lines represent median values and (H,I,J) bars represent mean values. Statistical analyses were performed by (E) two-tailed unpaired t-test , (G) mixed model analysis , (H,J,K) two-way ANOVA or (I) one-way ANOVA with (H,I,J,K) Tukey’s multiple comparison test.