Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy

Abstract

Antimicrobial drug development is increasingly lagging behind the evolution of antibiotic resistance, and as a result, there is a pressing need for new antibacterial therapies that can be readily designed and implemented. In this work, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. We show that suppressing the SOS network in Escherichia coli with engineered bacteriophage enhances killing by quinolones by several orders of magnitude in vitro and significantly increases survival of infected mice in vivo. In addition, we demonstrate that engineered bacteriophage can enhance the killing of antibiotic-resistant bacteria, persister cells, and biofilm cells, reduce the number of antibiotic-resistant bacteria that arise from an antibiotic-treated population, and act as a strong adjuvant for other bactericidal antibiotics (e.g., aminoglycosides and beta-lactams). Furthermore, we show that engineering bacteriophage to target non-SOS gene networks and to overexpress multiple factors also can produce effective antibiotic adjuvants. This work establishes a synthetic biology platform for the rapid translation and integration of identified targets into effective antibiotic adjuvants.

Fig. 1.

Engineered ϕ_lexA3 bacteriophage enhances killing of wild-type E. coli EMG2 bacteria by bactericidal antibiotics. (A) Schematic of combination therapy with engineered phage and antibiotics. Bactericidal antibiotics induce DNA damage via hydroxyl radicals, leading to induction of the SOS response. SOS induction results in DNA repair and can lead to survival (8). Engineered phage carrying the lexA3 gene (ϕ_lexA3) under the control of the synthetic promoter P_LtetO and an RBS (27) acts as an antibiotic adjuvant by suppressing the SOS response and increasing cell death. (B) Killing curves for no phage (black diamonds), unmodified phage ϕ_unmod (red squares), and engineered phage ϕ_lexA3 (blue circles) with 60 ng/ml ofloxacin (oflox) (solid lines, closed symbols). 10⁸ pfu/ml phage was used. A growth curve for E. coli EMG2 with no treatment (dotted line, open symbols) is shown for comparison. ϕ_lexA3 greatly enhanced killing by ofloxacin by 4 h of treatment. (C) Killing curves for no phage (black diamonds), ϕ_unmod (red squares), and ϕ_lexA3 (blue circles) with 5 μg/ml gentamicin (gent). 10⁹ pfu/ml phage was used. ϕ_lexA3 phage greatly increases killing by gentamicin. (D) Killing curves for no phage (black diamonds), ϕ_unmod (red squares), and ϕ_lexA3 (blue circles) with 5 μg/ml ampicillin (amp). 10⁹ pfu/ml phage was used. ϕ_lexA3 phage greatly increases killing by ampicillin.

Fig. 2.

Engineered ϕ_lexA3 bacteriophage enhances killing of quinolone-resistant E. coli RFS289 bacteria by ofloxacin. Killing curves for no phage (black diamonds), unmodified phage ϕ_unmod (red squares), and engineered phage ϕ_lexA3 (blue circles) with 1 μg/ml ofloxacin (oflox). 10⁸ pfu/ml phage was used. ϕ_lexA3 greatly enhanced killing by ofloxacin by 1 h of treatment.

Fig. 3.

Engineered ϕ_lexA3 bacteriophage increases survival of mice infected with bacteria. (A) Female Charles River CD-1 mice were inoculated with i.p. injection of 8.8 * 10⁷ cfu/mouse E. coli EMG2 bacteria. After 1 h, the mice received no treatment or i.v. treatment with 0.2 mg/kg ofloxacin plus no phage, plus unmodified phage ϕ_unmod, or plus engineered phage ϕ_lexA3 (10⁹ pfu/mouse phage was used). The mice were observed for 5 days, and deaths were recorded at the end of each day to generate survival curves. [Mouse drawing reproduced under a Creative Commons Attribution 2.5 license (53).] (B) Survival curves for infected mice treated with phage and/or ofloxacin demonstrate that engineered phage ϕ_lexA3 plus ofloxacin (closed blue circles with solid line) significantly increases survival of mice compared with unmodified phage ϕ_unmod plus ofloxacin (closed red squares with solid line), no phage plus ofloxacin (closed black diamonds with solid line), or no treatment (open black diamonds with dashed line).

Fig. 4.

Engineered bacteriophage targeting single and multiple gene networks (other than the SOS network) as adjuvants for ofloxacin treatment (oflox). (A) Ofloxacin stimulates superoxide generation, which normally is countered by the oxidative stress response, coordinated by SoxR (8). Engineered phage producing SoxR (ϕ_soxR) enhances ofloxacin-based killing by disrupting regulation of the oxidative stress response. (B) Killing curves for no phage (black diamonds), unmodified phage ϕ_unmod (red squares), and engineered phage ϕ_soxR (blue downward-pointing triangles) with 60 ng/ml ofloxacin (solid lines, closed symbols). 10⁸ pfu/ml phage was used. The killing curve for ϕ_unmod and a growth curve for E. coli EMG2 with no treatment (dotted line, open symbols) are reproduced from Fig. 1B for comparison and show that ϕ_soxR enhances killing by ofloxacin. (C) CsrA suppresses the biofilm state in which bacterial cells tend to be more resistant to antibiotics (35). OmpF is a porin used by quinolones to enter bacterial cells (37). Engineered phage producing both CsrA and OmpF simultaneously (ϕ_csrA-ompF) enhances antibiotic penetration via OmpF and represses biofilm formation and antibiotic tolerance via CsrA to produce an improved dual-targeting adjuvant for ofloxacin. (D) Killing curves for ϕ_csrA (black diamonds), ϕ_ompF (red squares), and ϕ_csrA-ompF (brown upward-pointing triangles) with 60 ng/ml ofloxacin. 10⁸ pfu/ml phage was used. Phage expressing both csrA and ompF (ϕ_csrA-ompF) is a better adjuvant for ofloxacin than phage expressing csrA (ϕ_csrA) or ompF alone (ϕ_ompF).