Engineered Bacteriophages Containing Anti-CRISPR Suppress Infection of Antibiotic-Resistant P. aeruginosa

Abstract

The therapeutic use of bacteriophages (phages) provides great promise for treating multidrug-resistant (MDR) bacterial infections. However, an incomplete understanding of the interactions between phages and bacteria has negatively impacted the application of phage therapy. Here, we explored engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) by introducing Type I anti-CRISPR (AcrIF1, AcrIF2, and AcrIF3) genes into the P. aeruginosa bacteriophage DMS3/DMS3m to render the potential for blocking P. aeruginosa replication and infection. In order to achieve effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo, the inhibitory concentration for EATPs was 1 × 10⁸ PFU/mL with a multiplicity of infection value of 0.2. In addition, the EATPs significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection. Collectively, these findings provide evidence that engineered phages may be an alternative, viable approach by which to treat patients with an intractable bacterial infection, especially an infection by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy. IMPORTANCE Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic Gram-negative bacterium that causes severe infection in immune-weakened individuals, especially patients with cystic fibrosis, burn wounds, cancer, or chronic obstructive pulmonary disease (COPD). Treating P. aeruginosa infection with conventional antibiotics is difficult due to its intrinsic multidrug resistance. Engineered bacteriophage therapeutics, acting as highly viable alternative treatments of multidrug-resistant (MDR) bacterial infections, have great potential to break through the evolutionary constraints of bacteriophages to create next-generation antimicrobials. Here, we found that engineered anti-CRISPR (Acr) gene-containing phages (EATPs, eat Pseudomonas) display effective antibacterial activities along with high safety against clinically isolated MDR P. aeruginosa through an anti-CRISPR immunity mechanism in vitro and in vivo. EATPs also significantly suppressed the antibiotic resistance caused by a highly antibiotic-resistant PA14 infection, which may provide novel insight toward developing bacteriophages to treat patients with intractable bacterial infections, especially infections by clinically MDR bacteria that are unresponsive to conventional antibiotic therapy.

Keywords: Acr; CRISPR-Cas; P. aeruginosa; anti-CRISPR; bacteriophages; multidrug resistance bacterial infection.

FIG 1

The evolutionary race between EATPs and CRISPR-Cas immunity. (A) Schematic illustration of the strategy to acquire EATPs. (B) Phage plaque assays were performed on PA14 (Phage: 1 × 10⁷ PFU/mL, PA14: 5 × 10⁸ CFU/mL, MOI = 0.02). The growth kinetics of PA14 (C) and the inhibitory rates (%) of EATPs (D) were evaluated within 48 h after treatment with 1 × 10⁷ DMS3, DMS3m phages, and EATPs (MOI = 0.02), respectively. (E) Phage titers were measured by PFU. Data were presented as mean ± standard error of the mean (SEM), determined from biological triplicates and analyzed via a one-way analysis of variance (ANOVA) compared against a control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 2

Acrs-mediated CRISPR-Cas inactivation requires a critical phage concentration. (A) PA14 was treated with DMS3_acrIF1/DMS3m_acrIF1 for 12 h (1 × 10⁷ PFU, MOI = 0.02) and infected with DMS3_acrIF1/DMS3 or DMS3m_acrIF1/DMS3m for 36 h (1 × 10⁷ PFU, MOI = 0.02). Growth kinetics upon phage infection were continuously monitored from the initial DMS3_acrIF1 challenge to 48 h. (B) Inhibitory rates (%) of DMS3_acrIF1 on PA14 were evaluated within 48 h after treatment with DMS3_acrIF1 for 12 h and were infected with DMS3 or DMS3_acrIF1 for 36 h (1 × 10⁷ PFU, MOI = 0.02). (C) DMS3_acrIF1 of different titers was used to infect PA14 (5 × 10⁸ CFU/mL), and the growth kinetics upon phage infection were calculated by measuring the OD₆₀₀ nm at 20 min intervals up to 48 h at 37°C with constant shaking. Data were presented as mean ± standard error of the mean (SEM), determined from biological triplicates and analyzed via a one-way ANOVA compared against a control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 3

DMS3_acrIF1 mitigates clinically isolated P. aeruginosa infection. The clinically isolated P. aeruginosa (5 × 10⁸ CFU/mL) were treated with DMS3_acrIF1 or control phages for 48 h (1 × 10⁸ PFU/mL, MOI = 0.2). The growth kinetics of P. aeruginosa upon phage infection were obtained by measuring the OD₆₀₀ nm at 20 min intervals up to 48 h at 37°C with constant shaking. Data were presented as mean ± standard error of the mean (SEM), determined from biological triplicates and analyzed via a one-way ANOVA compared against a control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 4

DMS3_acrIF1 potently inhibits clinically isolated P. aeruginosa infection. The clinically isolated P. aeruginosa strains were treated with DMS3_acrIF1 or control phages for 48 h (1 × 10⁸ PFU/mL, MOI = 0.2). The inhibitory rate (%) of DMS3_acrIF1 on P. aeruginosa was evaluated upon phage infection by measuring CFU at 4 h intervals up to 48 h at 37°C with constant shaking. Data were presented as mean ± standard error of the mean (SEM), determined from biological triplicates and analyzed via a one-way ANOVA compared against a control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 5

DMS3_acrIF1 mitigates tissue injury and improve survival. HEK293T cells grown to subconfluence (1 × 10⁵ cells in a 96-well plate) were infected with PA154197 for 1 h (1 × 10⁶ CFU/mL, MOI = 10) and treated with 2 × 10⁵ PFU/mL DMS3_acrIF1 (MOI = 0.2). Cell viability (A) and toxicity (B) were measured by MTT and LDH assays in HEK293T. C57BL/6N mice (n = 10, both sexes) were anesthetized with ketamine (45 mg/kg), intranasally infected with 6 × 10⁶ CFU/25g clinically isolated MDR P. aeruginosa PA154197 for 2 h, and intraperitoneally injected with 1.2 × 10⁶ PFU/mL DMS3_acrIF1 (MOI = 0.2). Survival was monitored within 14 days postinfection and is represented by Kaplan-Meier survival curves (C). Bacterial burden (D) and histological analysis (E) were also measured. Data were presented as mean ± standard error of the mean (SEM), determined from biological triplicates and analyzed via a one-way ANOVA compared against a control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 6

DMS3_acrIF1 ameliorates P. aeruginosa antibiotic resistance. (A) PA14 (OD₆₀₀ = 0.5) was evenly spread on a plate containing different antibiotics (Amp⁺: 100 μg/mL, Kan⁺: 100 μg/mL, Gen⁺: 100 μg/mL, Str⁺: 100 μg/mL), and antibiotic resistance was tested by observing the growth of bacteria on the plate. (B) PA14 (5 × 10⁸ CFU/mL) was cultured in lysogeny broth (LB) containing different antibiotics (Amp⁺: 100 μg/mL, Kan⁺: 100 μg/mL, Gen⁺: 100 μg/mL, Str⁺: 100 μg/mL) or treated with 1 × 10⁸ PFU/mL EATPs/DMS3 (MOI = 0.2). The growth kinetics of PA14 were continuously monitored at 4 h intervals up to 48 h at 37°C with constant shaking. (C) The left panel is a schematic diagram of the antibiotic resistance experiment. PA14 (PA14: 5 × 10⁸ CFU/mL) was treated with gradient concentrations of antibiotic (Gen⁺ or Str⁺), gradient concentrations of antibiotic and DMS3 (MOI = 0.02) (Gen⁺/Str⁺ + DMS3) or Gen⁺/Str⁺ + DMS3_acrIF1. The growth kinetics of PA14 were measured. (D) PA14 that acquired antibiotic resistance were picked out and grown to the mid-logarithmic phase (OD₆₀₀ = 0.4 to 0.6) in LB at 37°C with 220 rpm shaking. The growth kinetics of the acquired antibiotic resistance of PA14 were measured after treatment with DMS3_acrIF1 (MOI = 0.2) or with 100 μg/mL Gen^+/Str⁺ antibiotics. Data were presented as mean ± standard error of the mean (SEM), determined from biological triplicates and analyzed via a one-way ANOVA compared against a control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 7

Schematic illustration of EATPs in suppressing P. aeruginosa infection. Normally, phage infection activates CRISPR-Cas adaptive immunity, which blocks phage replication through the cleavage of phage genomes, resulting in only a small amount of proliferation of phages, eventually preserving the bacterial homeostasis. EATPs suppress the activity of CRISPR-Cas systems to protect their associated phage genomes by producing Acrs, resulting in a large quantity proliferation of phages, eventually, the bacteria are lysed. EATPs have great potential for treating antibiotic-resistant P. aeruginosa infection by lysing bacteria.