Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection

Abstract

The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.

Keywords: MRSA; Staphylococcus aureus; antibiotic resistance; bacteriophages; cell-penetrating homing peptide; endolysin; osteomyelitis; peptidoglycan hydrolase; phage display; protein therapeutics; tissue-targeting.

Fig 1

Screening of pre-selected PGHs for high staphylolytic activity in murine serum. Twenty-eight highly active enzymes were expressed in a microwell plate format and challenged with 10⁷ CFU/mL S. aureus Newman in murine serum for 30 minutes. Each enzyme was scored based on the number of surviving bacteria (>20 colonies = 0; 10–20 colonies = 1; <10 colonies = 2; and 0 colonies = 3). Experiments were performed in technical and biological triplicates, resulting in a maximum possible score of 27 (dashed line). Stacked bars indicate the scores reached in individual biological replicates. As controls, pET21a_LST (+) and pET21a (horizontal gray bar) were used. PGHs are sorted according to their enzymatic activity profile, the domain structure is detailed in Table S2.

Fig 2

Comparison of in vitro staphylolytic activity of PGHs in murine and human sera. S. aureus Cowan I was treated with 20 nM of eight selected PGHs with high activity in murine and human sera. Bacterial counts were determined after 10, 60, and 180 minutes by plating. (A) Representative graphs for three PGHs show the mean (±SEM) CFU/mL determined at each timepoint in biological triplicates. Y-axis was cut at the detection limit (44 CFU/mL). (B) Table of average log(CFU/mL) reductions (±SEM) reached after 180 minutes compared to the untreated control for all tested enzymes. Averages were calculated from biological triplicates. Cases where the bacterial load was reduced to the detection limit are specified as ^a.

Fig 3

Selection and characterization of CPHP candidates. (A) CPHP candidates were identified using a cell culture-based phage display approach followed by next-generation sequencing and bioinformatics analysis. Ten CPHP candidates were C-terminally fused to eGFP (B) or LST (C) to assess their cell-line specificity and ability to translocate an active PGH cargo. (B) The cell line-specific uptake of eGFP and CPHP-modified eGFP was assessed by CLSM. MC3T3-E1 (top row) and a non-target cell line (Caco-2, bottom row) were treated for 60 minutes with 5 µM eGFP_CPHP or the controls eGFP and eGFP_TAT (a non-tissue-specific cell-penetrating peptide). Extracellular proteins were removed with three DPBS washes. Cells were stained with FM4-64 (membrane, magenta) and Hoechst 33342 (DNA, blue). Images for the three CPHP candidates that were subsequently used for in vivo experiments are shown. For ease of visualization, an overlay including the stained membrane is shown separately (inset) to the overlay of the eGFP and nucleic acid signals. (C) MC3T3-E1 cells were infected with S. aureus Cowan I (MOI = 5, 1 hour) and treated with LST, LST_TAT, or different LST_CPHP constructs (2 µM, 4 hours). Intracellular bacteria were enumerated by plating, and the average log(CFU/mL) reduction compared to an untreated control was determined (±SEM). The dashed line indicates a 95% reduction in intracellular CFUs. Experiments were conducted in biological triplicates. *P ≤ 0.05; **P ≤ 0.01; and ****P < 0.0001.

Fig 4

Biodistribution of a parental PGH and CPHP-modified variants thereof in murine tissues. Europium-labeled LST and LST_CPHPs were injected in C57BL/6 mice through the tail vein (10 mg/kg of body weight). Tissues were harvested 12 hours post-injection. Protein levels were determined by measuring europium signals in homogenized tissues by time-resolved fluorescence (TRF) spectrophotometry. Measurements were fitted to a standard curve determined for each tissue individually. (A) The amount of protein per mass of tissue (±SD) for bone, liver, and kidney is shown. Each individual data point represents one animal. Asterisks represent significant differences to the LST control. *P ≤ 0.05; **P ≤ 0.01; and ****P < 0.0001. (B) Summary of the numerical data obtained for all tested tissues (± SEM).

Fig 5

Treatment efficacy of parental and CPHP-modified PGHs in a murine deep wound subcutaneous infection model. (A) Experimental design of the in vivo efficacy study. Hind legs of mice were infected subcutaneously with S. aureus Cowan I, and infection of the bone was established over the course of 48 hours. Mice were treated with a single dose of parental PGH or PGH_CPHP (150 µL, 75 µM) or PBS as a control. At 14–16 hours post-treatment, the femur, fibula, and tibia were harvested, homogenized, and total CFUs normalized to the weight of the bone were determined by plating. (B) S. aureus CFUs (±SEM) in murine bones after treatment with parental PGHs and their CPHP-modified variants as compared to a PBS control. *P ≤ 0.05 and **P ≤ 0.01.