Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

Abstract

Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by low cure rates and increasing antibiotic resistance in bacteria. Therefore, alternative treatment options are highly desirable. PGHs, including bacteriophage endolysins, rapidly and specifically kill selected pathogens by degrading their cell walls and are refractory to resistance development; thus, they have promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk among a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and thus support their further characterization in animal models.

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

“…The observation that cocktail_2_TAT was also significantly more effective than were its individual components suggests that, besides CPP-mediated uptake, synergism also contributed to the potency of this enzyme mixture. Synergistic antibacterial effects when combining PGHs with different PG cleavage specificities have been reported previously (20) and may be explained by the increased accessibility of one PG cleavage site after cleavage of another (46). Here, we demonstrated that synergy can also be observed within intracellular compartments, following CPP-driven PGH uptake.…”

“…Screening identifies PGHs with activity under extra-and intracellular conditions. To develop effective antimicrobials for the treatment of S. aureus infections involving intracellular and drug-resistant bacteria, we selected 322 PGH 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 (phosphate-buffered saline [PBS] and Dulbecco's modified Eagle's medium [DMEM]), cytosolic (intracellular buffer), and lysosomal (lysosomal buffer) conditions.…”

“…CPP-fused PGHs act synergistically to kill intracellular S. aureus. Previous studies have demonstrated that combining PGHs targeting different chemical bonds in the PG, such as those harboring a CHAP domain and an M23 endopeptidase domain, makes it possible to capitalize on synergistic antibacterial effects (17,20). To explore this phenomenon in an intracellular setting, we first defined concentrations for 4 individual enzymes (LST-TAT, GH15-TAT, SEP-TAT, and LysK-TAT) that caused similar reductions in intracellular S. aureus in our coculture model.…”

“…To accommodate these challenges, we screened PGHs for high activity in multiple buffer systems that simulate conditions found in different intracellular niches of S. aureus, such as the cytoplasm or phagolysosomes. While we have previously described a screening approach to identify PGHs with high activity under the desired conditions (20), selection for PGHs with activity in intracellular compartments has thus far not been pursued. This could explain why previous studies by us and other groups that describe intracellular transduction of PGHs via fusion to CPPs only observed moderate intracellular killing efficacy in cell culture infection models (Ͻ1-log reduction) (18,21).…”

“…Therefore, a first step to address this limitation was to identify PGHs with high activity under these conditions. Previous studies have shown that molecular engineering of PGHs, including the generation of chimeras harboring functional domains from different origins, can yield enzymes with such novel and optimized properties (19)(20)(21).…”