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

ABSTRACT The current standard of treatment for chronic staphylococcal osteomyelitis entails high doses of antibiotics over the course of several weeks. Biofilm-associated and intracellular persisters are key factors contributing to therapeutic failure. Additionally, systemic application results in low concentrations of antibiotics at local infection sites due to its general distribution throughout the host. In this study, we explored a targeted approach for the treatment of staphylococcal osteomyelitis, employing a combination of highly active peptidoglycan hydrolases (PGHs) and cell-penetrating homing peptides (CPHPs) with specificity for osteoblasts. In vitro phage display on murine osteoblasts followed by next-generation sequencing led to the identification of 10 putative cell-penetrating homing peptides, which subsequently showed cell-line specific internalization of covalently linked fluorescent molecules into murine osteoblasts. Upon intravenous application, the lead candidate peptide mediated tissue-specific accumulation of an associated PGH in murine bones, confirming its function as an osteotropic peptide with cell-penetrating abilities. Furthermore, we selected three enzymes with high staphylolytic activity in murine serum screened from a set of 28 PGHs highly active against Staphylococcus aureus in human serum and under intracellular conditions: lysostaphin (LST), M23LST(L)_SH3b2638, and CHAPGH15_SH3bAle1. Finally, we demonstrated increased efficacy of the three PGHs modified with two osteotropic CPHPs as compared to their unmodified parentals at reducing bacterial numbers in a murine model of S. aureus deep wound subcutaneous infection leading to dissemination to the bone. Collectively, our findings show that modification of PGHs with tissue-specific CPHPs presents a viable approach for the systemic treatment of localized infections associated with intracellular bacteria. IMPORTANCE 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. 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.