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 screened a library of Ͼ170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, 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 most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of Ͼ9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 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 both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by Ͼ3.5 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 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 wall and are refractory to resistance development, therefore holding promise as novel antibacterial agents. This study employed a screening approach to identify PGH constructs with high staphylolytic activity in cow's milk within a large collection of enzymes. Our results suggest that the most promising enzymes identified by this strategy hold potential as novel mastitis therapeutics and support their further characterization in animal models.KEYWORDS antibiotic resistance, antimicrobial agents, bovine mastitis, peptidoglycan hydrolases

The Antistaphylococcal Lysin, CF-301, Activates Key Host Factors in Human Blood To Potentiate Methicillin-ResistantStaphylococcus aureusBacteriolysis

“…Extension to other lysins. CF-301 was originally identified as a Streptococcus suis prophage lysin exhibiting a domain arrangement commonly observed in many antistaphylococcal lysins (5,56) and defined by a catalytic N-terminal cysteine-histidinedependent amidohydrolase/peptidase (CHAP) domain linked to a cell wall-binding C-terminal domain belonging to the SH3b family of proteins (57,58). We predicted that lysins of this group, which include enzymes active against both staphylococci and streptococci (like CF-301), as well as enzymes active against only staphylococci (59), would all exhibit the CF-301-like potentiation effect in human blood.…”

Recombinant Endolysins as Potential Therapeutics against Antibiotic-Resistant Staphylococcus aureus: Current Status of Research and Novel Delivery Strategies

“…For this reason, it is not recommended that lysostaphin be used as a single antimicrobial agent. However, combined applications with endolysin-derived proteins, which exploit synergistic effects due to the different peptidoglycan cleavage sites of these enzymes, have been reported and may represent a feasible strategy to decrease the chance of resistance (102,109). Furthermore, components of lysostaphin have been used for the construction of chimeric peptidoglycan hydrolases (74,109).…”

“…However, combined applications with endolysin-derived proteins, which exploit synergistic effects due to the different peptidoglycan cleavage sites of these enzymes, have been reported and may represent a feasible strategy to decrease the chance of resistance (102,109). Furthermore, components of lysostaphin have been used for the construction of chimeric peptidoglycan hydrolases (74,109). The spectrum of specificity of an enzyme can also be extended by a combination of two heterologous CBDs; this has been shown by fusions of CBDs from Listeria phage endolysins with different binding specificities that were fluorescently tagged (110).…”

Csl2, a novel chimeric bacteriophage lysin to fight infections caused by Streptococcus suis, an emerging zoonotic pathogen

“…The modular architecture of endolysins has allowed the successful swapping of functional domains to construct new chimeric proteins, some of them being even improved versions of the parental enzymes 27 – 30 . In particular, Cpl-711 chimera turned out to be the most lethal lysin with muramidase activity against pneumococci described to date.…”