Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria

Abstract

The prevalence of multidrug-resistant Pseudomonas aeruginosa has stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria. However, lysins against Gram-negatives are generally deterred by the outer membrane and their inability to work in serum. One solution involves exploiting evolved delivery systems used by colicin-like bacteriocins (e.g., S-type pyocins of P. aeruginosa) to translocate through the outer membrane. Following surface receptor binding, colicin-like bacteriocins form Tol- or TonB-dependent translocons to actively import bactericidal domains through outer membrane protein channels. With this understanding, we developed lysocins, which are bioengineered lysin-bacteriocin fusion molecules capable of periplasmic import. In our proof-of-concept studies, components from the P. aeruginosa bacteriocin pyocin S2 (PyS2) responsible for surface receptor binding and outer membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin. PyS2-GN4 delivered the GN4 lysin to the periplasm to induce peptidoglycan cleavage and log-fold killing of P. aeruginosa with minimal endotoxin release. While displaying narrow-spectrum antipseudomonal activity in human serum, PyS2-GN4 also efficiently disrupted biofilms, outperformed standard-of-care antibiotics, exhibited no cytotoxicity toward eukaryotic cells, and protected mice from P. aeruginosa challenge in a bacteremia model. In addition to targeting P. aeruginosa, lysocins can be constructed to target other prominent Gram-negative bacterial pathogens.

Keywords: ESKAPE; Pseudomonas aeruginosa; antibiotic resistance; antimicrobial; endolysin; lysin; lysocin; peptidoglycan hydrolase; protein delivery.

FIG 1

GN4 and PyS2-GN4 purification and antipseudomonal activity. (A) To construct the PyS2-GN4 lysocin, PyS2 domain IV (aa 559 to 690) was deleted and replaced with the GN4 lysin (aa 1 to 144). (B) The GN4 lysin (16 kDa) and the wild-type PyS2-GN4 (76 kDa), PyS2-GN4_ΔTBB (75 kDa), and PyS2-GN4_KO (76 kDa) lysocins were purified to homogeneity according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. (C) The muralytic activity of purified GN4 and PyS2-GN4 was determined by spotting 25 pmol of each on autoclaved Pseudomonas. CEWL and buffer were, respectively, used as positive and negative controls. Clearing zones signify PG degradation. (D to G) Using the plate lysis assay, the antipseudomonal activities of GN4, PyS2-GN4, PyS2-GN4_ΔTBB, and PyS2-GN4_KO, as indicated, were determined in growth medium by spotting 0.01 to 400 pmol of purified protein on P. aeruginosa strain 453. (H) The plate lysis assay was further used to analyze the antipseudomonal activity of 0.01 to 400 pmol of PyS2-GN4 against P. aeruginosa strain 453 in 50% HuS. Growth inhibition zones observed using the plate lysis assay indicate antipseudomonal activity.

FIG 2

PyS2-GN4 killing kinetics and antibiofilm efficacy. The dose-response lysocin killing kinetics were determined by incubating P. aeruginosa strain 453 at 10⁶ CFU/ml statically with 0.01 to 100 μg/ml PyS2-GN4 for 24 h at 37°C. Bacterial viability was assessed (A) in 2-h increments over the first 12 h in growth medium only and (B) at 24 h with or without HuS. (C) P. aeruginosa strain PAO1 biofilms were grown for 72 h at 37°C in CAAg medium and subsequently treated for 24 h with buffer or 0.03 to 500 μg/ml GN4, PyS2-GN4, or tobramycin. The residual biomass of the biofilms was qualitatively measured by means of crystal violet staining. SC, sterility control; GC, growth control. All error bars correspond to ±SEM, while dashed lines mark the limits of detection.

FIG 3

Visualizing lysocin-treated P. aeruginosa by TEM. P. aeruginosa strain 453 was incubated with 50 μg/ml PyS2-GN4 in CAA medium with EDDHA for a total of 1 h at 37°C. The bacteria were then fixed and visualized by TEM at 0, 30, and 60 min posttreatment. Total magnifications of ×2,600 (scale bar, 1 μm) and ×13,000 (insets; scale bar, 200 nm) are shown.

FIG 4

Lysocin cytotoxicity toward eukaryotic cells and bacterial endotoxin release. (A) hRBCs were incubated in triplicate with buffer or 0.5 to 256 μg/ml PyS2-GN4 for 8 h at 37°C, and hemolysis, as a function of hemoglobin release, was assayed spectrophotometrically at 405 nm. Triton X-100 was used as a positive control for hemolysis. (B) Human promyeloblast HL-60 cells were incubated in triplicate with buffer or 0.5 to 256 μg/ml PyS2-GN4 for 8 h at 37°C, and viability, as a function of formazan product formation, was measured spectrophotometrically at 570 nm. Triton X-100 was used as a control for cytotoxicity. (C) Endotoxin release was measured in duplicate experiments after treating P. aeruginosa strain 453 at 10⁶ CFU/ml for 1 or 4 h at 37°C in growth medium with 0.2× and 5× MIC of PyS2-GN4, colistin, meropenem, or tobramycin. An untreated control was included. All error bars correspond to ±SEM. P values were calculated using a one-way analysis of variance. EU, endotoxin units.

FIG 5

Lysocin antipseudomonal in vivo efficacy using a murine model of bacteremia. Mice (n =100) were i.p. infected with 10⁸ CFU of P. aeruginosa strain 453. (A) The bacterial counts in organs were determined 3 h postinfection in order to confirm the animals were bacteremic. (B) Infected mice were i.p. treated at 3 h postinfection with either buffer (n = 35) or 2.5 (n = 15), 5 (n = 15), 12.5 (n = 15), or 25 mg/kg (n = 20) lysocin. Survival was monitored over 10 days. All error bars correspond to ±SEM. P values were calculated using a log rank (Mantel-Cox) test.

FIG 6

Schematic overview of the proposed mechanism of PyS2-GN4 antipseudomonal activity. (A) When added extrinsically as a purified recombinant protein, domain I of PyS2-GN4 binds to the FpvAI receptor located on the surface of target P. aeruginosa bacteria. (B) This protein-protein interaction induces a conformational change in the receptor structure, resulting in the FpvAI TBB in the periplasm to recruit and bind TonB1. (C) The formation of this complex allows for the PMF-dependent unfolding of the labile half of the FpvAI plug domain. (D) Next, the unstructured region of lysocin domain I passes through the channel created in order to enable its own TBB to bind another nearby TonB1 protein in the periplasm. (E) The newly formed lysocin-TonB1 translocon stimulates the PMF-driven unfolding and import of the remainder of the lysocin into the periplasm. (F) Upon refolding, GN4 is proteolytically released and cleaves the PG to provoke partial membrane destabilization, cytoplasmic leakage, PMF disruption, and cell death.