Targeting Hidden Pathogens: Cell-Penetrating Enzybiotics Eradicate Intracellular Drug-Resistant Staphylococcus aureus

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. aureus. IMPORTANCE 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.

Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

“…To identify PGH constructs featuring high antibacterial activity against S. aureus in human serum, we first modified a previously established protocol ( 50 ) to screen a comprehensive collection of more than 300 parental and engineered staphylococcal PGHs. This procedure has previously been successfully applied to identify lytic enzymes highly active in milk ( 50 ) and under intracellular conditions ( 51 ) and was adapted here for human serum. Applying this protocol in an iterative fashion, using S. aureus Newman as a target strain, enabled us to narrow down the selection of possible candidates to the 25 most promising constructs (PGH-1 to PGH-25; see Table S1 in the supplemental material).…”

Advanced engineering of third-generation lysins and formulation strategies for clinical applications

“…Then, N-terminal fusions of the seven most active lysins to six different CPPs were prepared. Whereas all JDlys-CPP fusions were inactive in the study of Wang, lysin-CPP combinations retained antibacterial activity in the work of R€ ohrig and colleagues (Wang et al 2018;R€ ohrig et al 2020). They also observed that the trans-activating transcription factor of HIV type 1 (Tat) has the least detrimental effect on the in vitro antibacterial activity.…”

“…Another example is the fusion with cell-penetrating peptides (CPPs). Here, the reduction in antibacterial activity was compensated by targeting intracellular S. aureus thereby eradicating a major cause of recurrent, chronic infections (Wang et al 2018;R€ ohrig et al 2020).…”

“…Overview of the third-generation engineering and advanced formulation strategies discussed in this review. CPP to target intracellular S. aureus(Becker et al 2016;Wang et al 2018;R€ ohrig et al 2020) …”

A Significant Question in Cancer Risk and Therapy: Are Antibiotics Positive or Negative Effectors? Current Answers and Possible Alternatives

“…Although agents with dual antimicrobial and anti-tumor activities would be ideal, combined applications of mono-therapeutic agents may easily provide the same effect. Two types of agents to be considered in this class are bacteriophages [184][185][186][187] and enzybiotics [188][189][190][191], which have attracted substantial attention in recent times, and can be used in prophylactic and therapeutic applications in antimicrobial and anticancer protocols. Bacteriophages have been engineered for medical applications [192] in ways allowing them to retain their antibacterial activity [185] and have been used as anticancer drug delivery systems [186,192], their possible effects on anti-tumor immunity and the response to anticancer therapy must be further evaluated.…”