A modular phage vector platform for targeted photodynamic therapy of Gram-negative bacterial pathogens

Abstract

Growing antibiotic resistance has encouraged the revival of phage-inspired antimicrobial approaches. On the other hand, photodynamic therapy (PDT) is considered a very promising research domain for the protection against infectious diseases. Yet, very few efforts have been made to combine the advantages of both approaches in a modular, retargetable platform. Here, we foster the M13 bacteriophage as a multifunctional scaffold, enabling the selective photodynamic killing of bacteria. We took advantage of the well-defined molecular biology of M13 to functionalize its capsid with hundreds of photo-activable Rose Bengal sensitizers and contemporarily target this light-triggerable nanobot to specific bacterial species by phage display of peptide targeting moieties fused to the minor coat protein pIII of the phage. Upon light irradiation of the specimen, the targeted killing of diverse Gram(-) pathogens occurred at subnanomolar concentrations of the phage vector. Our findings contribute to the development of antimicrobials based on targeted and triggerable phage-based nanobiotherapeutics.

Keywords: Bacteriology; Biotechnology; Microbiology; Virology.

Graphical abstract

Figure 1

Orthogonal engineering of M13 phage for selective PDT Phages with targeted tropism against Acinetobacter baumannii (light blue) and against the gram-negative Acinetobacter baumannii and Pseudomonas aeruginosa cells (dark blue) were generated. M13_Aba is retargeted to the biofilm activating protein (BAP) of Acinetobacter baumannii through the specific display on the minor coat protein pIII of the C20 nanobody (C20Nb), whereas M13_Gram− targets the lipopolysaccharides of the Gram-negative outer membrane through the display on the pIII protein of KNYSSSISSIRAC-binding peptide (LPSbp). The amino group of M13_Aba and M13_Gram− major capsid protein pVIII were chemically conjugated with hundreds of RB for the production of ROS, which are highly antimicrobial, upon light irradiation, in the cell proximity.

Figure 2

Conjugation of RB to M13 phage (A–C) Chemical conjugation was performed on (A) M13, (B) M13_Aba, and (C) M13_Gram- phages. Absorption spectra of phages (dashed black line), RB (dashed pink line), and phage conjugated with RB (purple line).

Figure 3

Antimicrobial PDT of M13-RB bioconjugates (A–C) The survival rate of bacterial cells incubated with increasing concentration of either RB (white bars) or M13-RB (black bars) and irradiated with a white led amp for 45 min. PDT assays were performed on (A) E. coli, (B) P. aeruginosa, and (C) A. baumannii. Data are represented as mean ± SD. ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.

Figure 4

Characterization of the anti-A. baumannii phage M13_Aba (A) Schematic representation of the M13 genetic engineering through co-transformation in E. coli of ppK_C20 phagemid and hyperphage genome. (B–D) (B) Immunoblotting of the antiBAP_Nb-pIII fusion (M13_Aba), demonstrating incorporation in the purified virion. The integrity of purified M13_Aba phages was visualized through (C) TEM (scale bar = 200 nm) and (D) AFM (scale bar = 600 nm). (E) M13_Aba length distribution analyzed by AFM. (F) Specificity and selectivity of engineered M13_Aba phages targeting A. baumannii: the binding was determined by SyberGreen real-time PCR using an oligonucleotide that anneals on the pIII minor-coat-protein-coding gene. M13_Aba phages showed significant binding to A. baumannii (cyan), whereas poor binding was observed to S. aureus (orange) and P. aeruginosa (magenta). Data are represented as mean ± SD. ∗∗∗∗ = p < 0.0001.

Figure 5

Photodynamic properties of M13Aba-RB (A) Amplex Red assay for the generation of peroxides using different concentrations of RB (pink) and M13_Aba-RB (purple). (B) Determination of ¹O₂ generation following the decrease of ABMDMA absorbance over the irradiation time under white light irradiation for RB (pink line), M13_Aba-RB (purple line), and PBS (black line). (C) Flow cytometry analysis of refactored and RB-conjugated M13_Aba-RB targeting to S. aureus (orange), P. aeruginosa (magenta), and A. baumannii (cyan). The fluorescence, detected in the PE channel, is related to RB. (D–G) (D) Percentage of fluorescent cells at the flow cytometer. (E) Selective photodynamic killing of S. aureus (orange), P. aeruginosa (magenta), and A. baumannii (cyan) after 45 min of light irradiation following incubation with 0.1 and 0.25 μM (RB equivalents) of photoactive M13_Aba-RB. Live/dead assays were performed on A. baumannii cells preincubated with M13_Aba-RB and (F) kept in dark conditions or (G) irradiated. (H) percentage of live, injured, and dead cells measured in the live/dead assay, 20 min post-irradiation. (I) Metabolic activity of A. baumannii cells after phage-mediated aPDT. Data are represented as mean ± SD. ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.

Figure 6

Characterization of the anti-Gram phage M13_Gram− (A) Schematic representation of the M13 genetic engineering through co-transformation in E. coli of pPK_LPS phagemid and hyperphage genome. (B–F) (B) Immunoblotting of the KNYSSSISSIRAC-pIII fusion (M13_Gram−) demonstrating incorporation in the purified virion. Visualization of purified M13_Gram− phage through (C) TEM (scale bar = 200 nm) and (D) AFM (scale bar = 600 nm). (E) M13_Gram− length distribution analyzed by AFM. (F) Selective tropism of engineered M13_Gram− to Gram-negative bacteria was proved through targeting assays performed on S. aureus (orange), P. aeruginosa (magenta), and A. baumannii (cyan). Data are represented as mean ± SD. ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.

Figure 7

Photodynamic properties of the M13_Gram− phage vector (A) Amplex Red assay for the generation of peroxides using different concentrations of RB (pink) and M13_Gram−-RB (purple). (B) Determination of ¹O₂ generation following the decrease of ABMDMA absorbance over the irradiation time under white light irradiation for RB (pink line), M13_Gram−-RB (purple line), and PBS (black line). (C) Flow cytometry analysis of retargeted and RB-conjugated M13_Gram− phages directed to the lipopolysaccharides of Gram-negative bacteria: dark lines correspond to the control cells treated with PBS, whereas colored ones highlight binding of the engineered M13_Gram−-RB to S. aureus (orange), P. aeruginosa (magenta), and A. baumannii (cyan). (D) Percentage of bacterial fluorescent cells measured by flow cytometry analysis. (E–I) (E) Percentage of bacteria survived to light irradiation after preincubation with M13_Gram−-RB phages. The activity of phages was tested at two concentrations, 0.1 and 0.25 μM, of equivalent RB. Live/dead assay was performed on P. aeruginosa and A. baumannii preincubated with M13_Gram−-RB and (F–H) kept in dark or (G–I) irradiated. (J–M) Percentage of live, injured, and dead cells measured in live/dead assay 20 min after irradiation. Metabolic activity of (K) P. aeruginosa and (M) A. baumannii after phage-mediated aPDT. Data are represented as mean ± SD. ∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001, ∗∗∗∗ = p < 0.0001.