Abstract
Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. Intracellular localization of S. aureus plays a key role in recurrent infections by protecting the pathogens from antibiotics and immune responses. Peptidoglycan hydrolases (PGHs) are highly specific bactericidal enzymes active against both drug-sensitive and -resistant bacteria. However, PGHs able to effectively target intracellular S. aureus are not yet available. To overcome this limitation, we first screened 322 recombineered PGHs for staphylolytic activity under conditions found inside eukaryotic intracellular compartments. The most active constructs were modified by fusion to different cell-penetrating peptides (CPPs), resulting in increased uptake and enhanced intracellular killing (reduction by up to 4.5 log units) of various S. aureus strains (including methicillin-resistant S. aureus [MRSA]) in different tissue culture infection models. The combined application of synergistic PGH-CPP constructs further enhanced their intracellular efficacy. Finally, synergistically active PGH-CPP cocktails reduced the total S. aureus by more than 2.2 log units in a murine abscess model after peripheral injection. Significantly more intracellular bacteria were killed by the PGH-CPPs than by the PGHs alone. Collectively, our findings show that CPP-fused PGHs are effective novel protein therapeutics against both intracellular and drug-resistant S. aureusIMPORTANCE The increasing prevalence of antibiotic-resistant bacteria is one of the most urgent problems of our time. Staphylococcus aureus is an important human pathogen that has acquired several mechanisms to evade antibiotic treatment. In addition, S. aureus is able to invade and persist within human cells, hiding from the immune response and antibiotic therapies. For these reasons, novel antibacterial strategies against these pathogens are needed. Here, we developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. Our results suggest that cell-penetrating enzybiotics are a promising new class of therapeutics against staphylococcal infections.
Highlights
Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance
To develop effective antimicrobials for the treatment of S. aureus infections involving intracellular and drug-resistant bacteria, we selected 322 Peptidoglycan hydrolases (PGHs) constructs with presumptive staphylolytic activity from an extensive library and assessed them in a microtiter plate-based screening assay [20] under conditions simulating relevant physiological settings
Each construct was rated based on its ability to eradicate or reduce S. aureus numbers under extracellular, cytosolic, and lysosomal conditions
Summary
Staphylococcus aureus is a major concern in human health care, mostly due to the increasing prevalence of antibiotic resistance. We developed lytic enzymes which are able to effectively target drug-resistant and intracellular S. aureus Fusion of these so-called enzybiotics to cell-penetrating peptides enhanced their uptake and intracellular bactericidal activity in cell culture and in an abscess mouse model. These enzymes cleave specific bonds within the peptidoglycan (PG) of the bacterial cell wall, thereby inducing bacterial lysis This active killing mechanism enables killing of metabolically inactive persisters and drug-resistant bacteria [9]. When applied from the outside, they rapidly and effectively kill Gram-positive bacteria by degrading the externally exposed PG; for this reason, they are deemed a promising new class of antimicrobials [12] Their major advantages include high specificity for their target bacteria, nontoxicity to eukaryotic cells, and very low risk of resistance development due to their highly conserved PG target bonds [13]
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