antibiotics Article Enzybiotics LYSSTAPH-S and LYSDERM-S as Potential Therapeutic Agents for Chronic MRSA Wound Infections Lukáš Vacek 1,2 ,Šárka Kobzová 1, Richard Cmelˇ ík 3, Roman Pant ˚uˇcek 4 and Lubomír Janda 1,* 1 Clinical Immunology and Immunology of Infectious Diseases, Veterinary Research Institute, Brno, Hudcova 70, 62100 Brno, Czech Republic; [email protected] (L.V.); [email protected] (Š.K.) 2 Department of Microbiology, St. Anne’s University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Pekaˇrská 53, 65691 Brno, Czech Republic 3 Institute of Analytical Chemistry of the Czech Academy of Sciences, Veveˇrí 97, 60200 Brno, Czech Republic; [email protected] 4 Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-533-331-344 Received: 30 June 2020; Accepted: 12 August 2020; Published: 15 August 2020 Abstract: Antibacterial antibiotic therapy has played an important role in the treatment of bacterial infections for almost a century. The increasing resistance of pathogenic bacteria to antibiotics leads to an attempt to use previously neglected antibacterial therapies. Here we provide information on the two recombinantly modified antistaphylococcal enzymes derived from lysostaphin (LYSSTAPH-S) and endolysin (LYSDERM-S) derived from kayvirus 812F1 whose target sites reside in the bacterial cell wall. LYSSTAPH-S showed a stable antimicrobial effect over 24-h testing, even in concentrations lower than 1 µg/mL across a wide variety of epidemiologically important sequence types (STs) of methicillin-resistant Staphylococcus aureus (MRSA), especially in the stationary phase of growth (status comparable to chronic infections). LYSDERM-S showed a less potent antimicrobial effect that lasted only a few hours at concentrations of 15 µg/mL and higher. Our data indicate that these antimicrobial enzymes could be of substantial help in the treatment of chronic MRSA wound infections. Keywords: enzyme therapy; enzybiotics; lysostaphin; LYSSTAPH-S; endolysin; LYSDERM-S; MRSA 1. Introduction Antibiotic resistance poses an increased threat for patients’ health, prolongs stays in hospitals, and consumes more healthcare resources. In response to this threat, the WHO in 2015 announced the Global action plan on antimicrobial resistance, which sets out strategic objectives to reduce antimicrobial resistance impacts. According to this effort, research and development are needed to produce a novel approach to treatments that could be deployed against antibiotic-resistant infections [1–6]. Following this endeavor, this article focuses on promising antimicrobial enzymes LYSSTAPH-S (lysostaphin derivative) and LYSDERM-S (kayvirus 812F1 endolysin derivative) and their antibacterial potential against epidemiologically important sequence types (ST) of methicillin-resistant Staphylococcus aureus commonly found in chronic infections. Not surprisingly, the bacterial cell wall is the target of antibacterial therapies. The cell wall of S. aureus is primarily composed of peptidoglycan, teichoic acids, and various surface proteins. Peptidoglycan itself consists of glycan chains of alternating β-(1,4)-linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM; a variant of NAG with a D-lactate attached to the C-3 by an ether Antibiotics 2020, 9, 519; doi:10.3390/antibiotics9080519 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, x FOR PEER REVIEW 2 of 16 Not surprisingly, the bacterial cell wall is the target of antibacterial therapies. The cell wall of S. aureus is primarily composed of peptidoglycan, teichoic acids, and various surface proteins. Antibiotics 2020, 9, 519 2 of 16 Peptidoglycan itself consists of glycan chains of alternating β-(1,4)-linked N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM; a variant of NAG with a D-lactate attached to the C-3 by an etherbond). bond). Stem Stem pentapeptides pentapeptides (L-Ala-D-iso-Gln-L-Lys-D-Ala-D-Ala) (L-Ala-D-iso-Gln-L-Lys-D-Ala-D-Ala) are covalently are covalently linked to linked the carboxyl to the carboxylcarbon of carbon the D-lactyl of the groupD-lactyl of NAM,group andof NAM, interpeptide and interpeptide bridges (pentaglycines, bridges (pentaglycines, i.e., chain ofi.e., five chain glycine of fiveresidues) glycine connect residues) the L-Lys connect component the L-Lys of one comp stemonent peptide of one to the stem D-Ala peptide of a neighboring to the D-Ala stem of peptide, a neighboringFigure1. Instem this peptide, manner, Figure glycan 1. strands In this aremanner, cross-linked glycan bystrands stem are peptides cross-linked and pentaglycines by stem peptides and form and apentaglycines complex net ofand peptidoglycan form a complex (murein) net of sacculuspeptidoglycan [7]. (murein) sacculus [7]. FigureFigure 1. The 1. cellThe wall cell of Staphylococcus wall of Staphylococcus aureus with aureus the detailedwiththe structure detailed of peptidoglycan. structure of peptidoglycan. Cleavage sites Cleavageof endolysin, sites lysostaphin, of endolysin, and lysostaphin, lysozyme and and target lysozyme site of andholins target are marked site of holins1 to 4 respectively. are marked 1 to 4 respectively. Further, the antimicrobial enzymes lysostaphin and endolysin LysK, from which LYSSTAPH-S and LYDERM-SFurther, were the antimicrobial derived, will enzymesbe described. lysostaphin and endolysin LysK, from which LYSSTAPH-S and LYDERM-S were derived, will be described. 1.1. Lysostaphin 1.1. Lysostaphin Lysostaphin (EC 3.4.24.75), a glycyl-glycine endopeptidase, is a bacteriocin secreted by the Gram-positiveLysostaphin bacterium (EC Staphylococcus 3.4.24.75), a glycyl-glycinesimulans. Bacteriocins, endopeptidase, in general, is aare bacteriocin antibacterial secreted proteins by the (andGram-positive peptides) produced bacterium by bactStaphylococcuseria as a competitive simulans. Bacteriocins, advantage over in general, closely are related antibacterial species proteinsand cause(and growth peptides) inhibition produced or kill by bacteriaother bacterial as a competitive species. Lysostaphin advantage over possesses closely relatedbacteriolytic species activity and cause againstgrowth staphylococci inhibition with or kill pentaglycine other bacterial interpeptide species.Lysostaphin bridges because possesses it specifically bacteriolytic cleaves activity the bond against betweenstaphylococci two glycines with in pentaglycine the interpeptide interpeptide bridge. As bridges mentioned because above, it specifically this is a cleavesdistinct thefeature bond of between the S. aureustwo glycines cell wall in and, the interpeptidethus, target selectivity bridge. As is mentioned provided. above, Though this its is disc a distinctovery featureand antimicrobial of the S. aureus activitycell against wall and, S. thus,aureus target has been selectivity reported is as provided. early as Thoughthe 1960s its by discovery Schindler and and antimicrobialSchuhardt, its activity X- ray crystalagainst structureS. aureus hashas beenbeen reportedreported asonly early recently. as the 1960sMature by lysostaphin Schindler and consists Schuhardt, of an itsN-terminal X-ray crystal catalyticstructure domain has with been Zn reported2+-coordinating only recently. amino Matureacid residues lysostaphin and aconsists C-terminal of an cell N-terminal wall targeting catalytic 2+ domaindomain [8,9]. with Zn -coordinating amino acid residues and a C-terminal cell wall targeting domain [8,9]. As shownAs shown earlier, earlier, every every clinical clinical application application of the of theantimicrobial antimicrobial drug drug so far so involved far involved the the developmentdevelopment of antimicrobial of antimicrobial resistance. resistance. Mechanisms Mechanisms associated associated with with lysost lysostaphinaphin resistance resistance usually usually involveinvolve mutations mutations in fem in femgenes.genes. Fem Fem factors factors catalyze catalyze the thenon- non-ribosomalribosomal synthesis synthesis of pentaglycine of pentaglycine cross-bridges,cross-bridges, which which are essential are essential for the for addition the addition of glycine of glycine residues residues 2 and 2 3 and (FemA) 3 (FemA) and 4 andand 45 and (FemB).5 (FemB). Mutations Mutations affecting affecting femA femArendersrenders this gene this genenon-functional, non-functional, resulting resulting in the in formation the formation of of monoglycinemonoglycine cross-bridges cross-bridges instead instead of of pentaglycine ones,ones, thus thus conveying conveying partial partial or complete or complete resistance to lysostaphin. Resistance can also be achieved by the incorporation of serines in place of some glycines in the cross-bridge [10–12]. Antibiotics 2020, 9, 519 3 of 16 Morikawa et al. [13] also identified the sigB gene as a regulator influencing the cell wall thickness. Cells depleted in sigB developed thinner envelopes and demonstrated increased sensitivity to lysostaphin. Conversely, overexpression of sigB led to increased resistance to lysostaphin [13]. In addition, Koehl et al. [14] connected an increased lysostaphin resistance of S. aureus to the thickened cell wall found in vancomycin intermediate-resistant S. aureus (VISA) strains, and also to a decreased autolytic activity. Gründling et al. [15] later identified another gene, lyrA, that caused a high degree of lysostaphin resistance [13,14]. Even though there are several reports of resistance development in S. aureus to lysostaphin, it