MEndoB, a chimeric lysin featuring a novel domain architecture and superior activity for the treatment of staphylococcal infections.

Bacterial infections are a growing global healthcare concern, as an estimated annual 4.95 million deaths are associated with antimicrobial resistance (AMR). Methicillin-resistant Staphylococcus aureus is one of the deadliest pathogens and a high-priority pathogen according to the World Health Organization. Peptidoglycan hydrolases (PGHs) of phage origin have been postulated as a new class of antimicrobials for the treatment of bacterial infections, with a novel mechanism of action and no known resistances. The modular architecture of PGHs permits the creation of chimeric PGH libraries. In this study, the chimeric enzyme MEndoB was selected from a library of staphylococcal PGHs based on its rapid and sustained activity against staphylococci in human serum. The benefit of the presented screening approach was illustrated by the superiority of MEndoB in a head-to-head comparison with other PGHs intended for use against staphylococcal bacteremia. MEndoB displayed synergy with antibiotics and rapid killing in human whole blood with complete inhibition of re-growth over 24 h at low doses. Successful treatment of S. aureus-infected zebrafish larvae with MEndoB provided evidence for its in vivo effectiveness. This was further confirmed in a lethal systemic mouse infection model in which MEndoB significantly reduced S. aureus loads and tumor necrosis factor alpha levels in blood in a dose-dependent manner, which led to increased survival of the animals. Thus, the thorough lead candidate selection of MEndoB resulted in an outstanding second-generation PGH with in vitro, ex vivo, and in vivo results supporting further development.IMPORTANCEOne of the most pressing challenges of our era is the rising occurrence of bacteria that are resistant to antibiotics. Staphylococci are prominent pathogens in humans, which have developed multiple strategies to evade the effects of antibiotics. Infections caused by these bacteria have resulted in a high burden on the health care system and a significant loss of lives. In this study, we have successfully engineered lytic enzymes that exhibit an extraordinary ability to eradicate staphylococci. Our findings substantiate the importance of meticulous lead candidate selection to identify therapeutically promising peptidoglycan hydrolases with unprecedented activity. Hence, they offer a promising new avenue for treating staphylococcal infections.

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

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.

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

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.IMPORTANCE Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells.

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

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.

Are Phage Lytic Proteins the Secret Weapon To Kill Staphylococcus aureus?

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most threatening microorganisms for global human health. The current strategies to reduce the impact of S. aureus include a restrictive control of worldwide antibiotic use, prophylactic measures to hinder contamination, and the search for novel antimicrobials to treat human and animal infections caused by this bacterium. The last strategy is currently the focus of considerable research. In this regard, phage lytic proteins (endolysins and virion-associated peptidoglycan hydrolases [VAPGHs]) have been proposed as suitable candidates. Indeed, these proteins display narrow-spectrum antimicrobial activity and a virtual lack of bacterial-resistance development. Additionally, the therapeutic use of phage lytic proteins in S. aureus animal infection models is yielding promising results, showing good efficacy without apparent side effects. Nonetheless, human clinical trials are still in progress, and data are not available yet. This minireview also analyzes the main obstacles for introducing phage lytic proteins as human therapeutics against S. aureus infections. Besides the common technological problems derived from large-scale production of therapeutic proteins, a major setback is the lack of a proper legal framework regulating their use. In that sense, the relevant health authorities should urgently have a timely discussion about these new antimicrobials. On the other hand, the research community should provide data to dispel any doubts regarding their efficacy and safety. Overall, the appropriate scientific data and regulatory framework will encourage pharmaceutical companies to invest in these promising antimicrobials.

Pneumococcal LytA autolysin, a potent therapeutic agent in experimental peritonitis-sepsis caused by highly beta-lactam-resistant Streptococcus pneumoniae.

The in vitro and in vivo antipneumococcal activities of the main pneumococcal autolysin (LytA) and Cpl-1, a lysozyme encoded by phage Cp-1, were studied. Intraperitoneal therapy with LytA or high-dose Cpl-1 remarkably reduced peritoneal bacterial counts (>5 log(10) CFU/ml) compared with those for the controls. After intravenous injection, LytA was the most effective treatment.