Appl Microbiol Biotechnol (2008) 80:555–561 DOI 10.1007/s00253-008-1579-y

MINI-REVIEW

Lysostaphin: an antistaphylococcal agent Jaspal K. Kumar

Received: 12 June 2008 / Accepted: 12 June 2008 / Published online: 8 July 2008 # Springer-Verlag 2008

Abstract Lysostaphin is a zinc metalloenzyme which has a specific lytic action against Staphylococcus aureus. Lysostaphin has activities of three enzymes namely, glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acteyl muramyl-L-alanine amidase. Glycylglycine endopeptidase specifically cleaves the glycine–glycine bonds, unique to the interpeptide cross-bridge of the S. aureus cell wall. Due to its unique specificity, lysostaphin could have high potential in the treatment of antibiotic-resistant staphylococcal infections. This review article presents a current understanding of the lysostaphin and its applications in therapeutic agent as a treatment against antibiotic-resistant S. aureus and methicillin-resistant S. aureus (MRSA) infections, either alone or in combination with other antibiotics. Keywords Staphylococcus aureus . MRSA . Lysostaphin . Therapeutics

Introduction Staphylococcus aureus is a major nosocomial pathogen that causes a range of diseases including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food poisoning, carbuncles, and boils (Archer and Climo 2001; Lowy 1998). The emergence of antibiotic-resistant variants in the treatment of staphylococcal infections has been a serious problem. Above all, the increasing evidence of S. aureus resistance to various antibiotics has been well documented for MRSA (Long 2003). Apart from this, there has been J. K. Kumar (*) Department of Biochemistry, National University of Singapore, Kent Ridge, Singapore e-mail: [email protected]

emergence of clinical isolates of MRSA with reduced susceptibility to vancomycin (Shrinivasan et al. 2002; Smith et al. 1999). Moreover, S. aureus has been shown to be completely resistant to lysozyme. This resistance is due to the modification of S. aureus peptidoglycan by Oacetylation at the C-6 position of the N-acetylmuramic acid (Bera et al. 2005). These problems have prompted a search for new and novel therapeutic agents active against S. aureus. Previous studies have shown lysostaphin to be an effective therapeutic agent for the treatment of various staphylococcal infections. Due to non-availability of homogenous preparations of lysostaphin and its concerns regarding immunogenicity, studies on lysostaphin as therapeutic agents were abandoned. However, the availability of recombinant lysostaphin (r-lysostaphin) that can be produced from Bacillus sphaericus has provided an opportunity to reinvestigate use of lysostaphin as a therapeutic agent for treatment of S. aureus infections. Lysostaphin was first identified in S. simulans (Schindler and Schuhardt 1964). It is a zinc-containing metalloenzyme of 27 kDa (Trayer and Buckley 1970). It has activities of three enzymes such as glycylglycine endopeptidase, endoβ-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase (Browder et al. 1965; Trayer and Buckley 1970; Wadstrom and Vesterberg 1971). Glycylglycine endopeptidase lyses staphylococcal cells by hydrolyzing glycylglycine bonds in the polyglycine bridges which form cross links between glycopeptide chains in the cell wall peptidoglycan of S. aureus cells. The lytic principle of lysostaphin is a peptidase which liberates N-terminal glycine and alanine from S. aureus cell wall (Fig. 1). The hexosaminidase which is present in lysostaphin preparation does not lyse S. aureus and is specific for the gluocainyl–muramic acid bond of the bacterial carbohydrate backbone. The gene of lysostaphin was cloned from a large plasmid in SSL (Heath et al. 1987)

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Fig. 1 Enzymatic activities of lysostaphin: The peptidoglycan of S. aureus showing points of cleavage. Lysostaphin has three enzyme activities 1 endo-βN-acetyl glucosaminidase, 2 N-acetyl-muramyl-L-alanine amidase and 3 endopeptidase. Endopeptidase causes the solubilization of pentaglycine bridges. NAG N-acetyl glucosamine, NAM N-acetyl muramic acid, ala alanine, lys lysine, glu glutamine and (Gly)5 is pentaglycine

and its DNA sequence has been determined (Heinrich et al.1987; Recsei et al. 1987). In addition, lysostaphin is secreted as a proenzyme. Proteolytic cleavage of 13 Nterminal tandem repeats generates mature lysostaphin with two functionally separable domains (Recsei et al. 1987; Heinrich et al. 1987). The N-terminal domain with glycylglycine endopeptidase activity cleaves pentaglycine cross bridges (Schindler and Schuhardt 1964), whereas C-terminal cell-wall-targeting domain promotes bacteriocin binding to staphylococcal peptidoglycan (Baba and Schneewind 1996; Grundling and Schneewind 2006). The peptidoglycan of S. aureus consists of a backbone made up of alternating β-1,4 linked N-acetylglucosamine and N-acetylmuramic acid residues. Tetrapeptide chains consisting of D-alanine, D-glutamine, L-lysine, and Dalanine are bound to the carboxyl groups of the muramic acid residues. These tetrapeptide chains are cross-linked by polyglycine bridges between the ε-amino group of the lysine residues of one chain and the D-alanyl carboxyl group of another chain (Fig. 1).The peptidoglycan is insoluble due to this cross linking of the polymers and hydrolysis of any single chemical linkage in sufficient number within the cross-linked network can bring about solubilization of the cell wall (Strominger and Ghuysen 1967). The extreme mechanical strength of S. aureus cell walls is probably dependent on the high degree of cross linking of the pentaglycine bridge between the ε-amino group of lysine and the terminal D-alanine of an adjacent tetrapeptide. However, this is not the case with other

staphylococcal species. The peptidoglycan of other staphylococcal species contains higher amount of serine than glycine and hence makes them less susceptible to lysostaphin. This unique property of S. aureus cell wall separates it from other staphylococcal species and makes lysostaphin as a novel therapeutic agent against antibiotic-resistant S. aureus infections. Moreover, lysostaphin rapidly lyses actively growing and non-dividing cells including staphylococci in biofilms (Zygmunt and Tavormina 1972; Wu et al. 2003), whereas most antibiotics require actively dividing cells to mediate their action. Hence, due to low toxicity and unique specificity of lysostaphin, it has once again aroused the interest of researchers to investigate the therapeutic values of lysostaphin.

Activity determination of lysostaphin Different methods have been described for the determination of lysostaphin activity viz. dye release assay which uses Remazol Brilliant blue r (RBB)-dyed staphylococcal cells or RBB-dyed staphylococcal peptidoglycan as substrate (Zhou et al. 1988) and turbidometric assay for detection of S. aureus contamination (Jaspal et al. 2001) in foods. Apart from this, lysostaphin lysis procedure has also been used for detection of S. aureus by the firefly bioluminescent ATP method (Tuncan and Martin 1987). Recently, Surovtsev et al. (2004) demonstrated Michaelis-Menten kinetics for determining enzymatic activity of lysostaphin.

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Therapeutic applications of lysostaphin S. aureus has been a serious cause of nosocomial and wound infections. Several studies have demonstrated using lysostaphin as a therapeutic agent for the treatment of S. aureus infections (Patron et al. 1999; Kokai-Kun et al. 2003; Climo et al. 1998, 2001; Dajcs et al. 2000; Zygmunt and Tavormina 1972). In addition, the potential use of lysostaphin–lysozyme combination for topical therapy of staphylococcal infections resistant to other antibiotics has been demonstrated by Cisani et al. (1982). Moreover, the most endangered species MRSA are also susceptible to lyses by lysostaphin and, hence, has been considered for treatment of infections caused by MRSA strains (Huber and Huber 1989). Furthermore, Huan (1992) suggests the use of lysostaphin as a topical antimicrobial to control burn wound infections with S. aureus in mice and against MRSA in burned patients (Huan et al. 1994). Besides this, synergistic combinations of recombinant lysostaphin and antibiotics has been suggested in controlling cutaneous staphylococcal infections and MRSA carriage (Polak et al. 1993) as lysostaphin activity is additive when combined either with vancomycin, gentamicin, tetracycline, or erythromycin. Recently, studies by Kokai-Kun et al. (2007) demonstrate that lysostaphin is an effective treatment for eradicating S. aureus from the blood and from the organs of infected mice. Furthermore, studies by Climo et al. (1998) demonstrated that lysostaphin alone or in combination with vancomycin is more effective in the treatment of experimental MRSA aortic valve endocarditis than vancomycin alone. In addition, studies by Graham and Coote (2007) suggests that combination of lysostaphin and the cationic peptide ranalexin could represent a novel route to target wounds infected with drug-resistant MRSA via dressings impregnated with the two compounds. Apart from this, Patron et al. (1999) shows that lysostaphin is an effective alternative for the treatment of experimental aortic valve endocarditis caused by a clinical vancomycin intermediate S. aureus strain. In addition, lysostaphin has been shown to be a potent therapy for treatment of the keratitis and endophthalmitis` mediated by MRSA in rabbit (Dajcs et al. 2000, 2001). Recently, Oluola et al. (2007) demonstrated the use of lysostaphin in the treatment of neonatal S. aureus infections. Bovine mastitis is one of the major diseases of animal agriculture. The main pathological agent for mastitis is S. aureus. The intramammary infections caused by S. aureus are usually chronic and subclinical (Buddle and Cooper 1978; McDonald 1977). In addition, the emergence of antibiotic-resistant S. aureus strains has attributed to major economic loss to the dairy industry. Apart from this, Bramley and Foster (1990) suggest the considerable potential of lysostaphin for the therapeutic or prophylactic

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control of staphylococcal mastitis. In addition, Oldham and Daley (1991) suggests the use of r-lysostaphin as an effective alternative to antibiotic therapy for bovine mastitis. Moreover, intramammary infusions of r-lysostaphin did not elicit any observable effects on the host animal or on the potential efficacy of the recombinant molecule (Daley and Oldham 1992). Hence, intramammary recombinant proteins may be suitable, effective, and safe infusion products that provide an alternative to classical antibiotic therapy. Hence, the above studies suggest the use of lysostaphin as a novel therapeutic in the treatment of various S. aureus infections either in the form of topical ointments or oral administrations. However, further studies are required to explore the proper formulations of lysostaphin as a therapeutic drug against antibiotic-resistant S. aureus infections.

Other applications of lysostaphin Besides using lysostaphin as a therapeutic agent in the treatment of S. aureus infections, it has been successfully used as an antistaphylococcal agent in clinical laboratories. For instance, in the phagocytosis of S. aureus, lysostaphin proved to be useful for the differentiation between engulfed and extracellular staphylococci, particularly those attached to the surface of polymorphonuclear granulocytes. It enabled a better recognition of the phagocytosized staphylococci and, therefore, a more precise analysis of phagocytosis experiments (Dorner et al. 1977). Similarly, Steuden and Szymanice (1988) demonstrated that use of lysostaphin helps to remove non-phagocytized granulocyte-adherent bacteria and thus allows more precise calculation of the percentage of the ingested as well as killed bacteria in the process of phagocytosis. In addition, Huan et al. (1995) showed the ability of lysostaphin to prevent immunosuppression of phagocyte in burn mice and hence is effective in improving phagocytosis by phagocytes. Moreover, lysostaphin lyses both capsulated and uncapsulated S. aureus strains at similar rates (King et al. 1980). Apart from this, lysostaphin has been commonly used as a rapid screening test to differentiate S. aureus from other species of Staphylococci and Micrococci (Sevarance et al. 1980; Knight and Shales 1983; Poutrel and Caffin 1981; Geary and Stevens 1986) in blood cultures. In addition, Walencka et al. (2005) demonstrated the effectiveness of lysostaphin in the treatment of biofilm, built by S. aureus and S. epidermidis strains on the flat surfaces of the microplates and on the catheter’s surface. Apart from being cause of concern for wound and hospital acquired infections, S. aureus is a common pathogen responsible for food contaminations and, hence,

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the cause for food poisoning. A few studies have demonstrated use of lysostaphin as an antistaphylococcal agent in the food industry. Sperber (1976), in his review, demonstrates the use of lysostaphin for identification of S. aureus in food. Furthermore, Jaspal et al. (2001) developed a simple rapid turbidometric method for the detection of S. aureus in foods using lysostaphin. Similarly, Cavadini et al. (1998) demonstrates the potential use of lysostaphinproducing strains of Lactobacillus curvatus to prevent food-borne illness by S. aureus. Apart from this, Rizzo and Korkeala (1984) demonstrate the use of lysostaphin for improvement of lipid extraction of staphylococcal cells. All these studies suggest that lysostaphin, besides being used as a therapeutic, can also be used as an antistaphylococcal agent in various industries such as a preservative in food industry, to detect S. aureus contamination in food and clinical laboratories. However, further studies are required to standardize the use of lysostaphin as an antistaphylococcal in other industries particularly in foods.

Immunogenicity The increasing evidence of microbial resistance to various antibiotics has led to intense research to develop novel proteins as new therapeutic agents. Besides this, S. aureus infections are becoming increasingly more difficult to treat because of changes in the frequency of isolation, distribution in the population, cell wall properties of antibioticresistant strains and the increasing incidence of nosocomial and community-acquired infections due to MRSA. Several studies have shown that systemic infusion of lysostaphin in a number of different animal models leads to eradication of disease and is an alternative to currently available therapy. Although a few studies have shown lysostaphin to be an effective agent for the treatment of experimental MRSA keratitis and endophthalmitis (Dajcs et al. 2000, 2001), there have been concerns regarding enzyme degradation, and immunogenicity of lysostaphin in terms of its safety and efficacy. However, several studies (Dajcs et al. 2002; Harrison et al. 1975; Zygmunt and Tavormina 1972) demonstrate that lysostaphin is able to retain its bactericidal activity in vivo, without any undesirable immune reaction, despite the presence of highneutralizing antibody titer. This suggests that lysostaphin can be used as a therapeutic agent in cases of MRSA keratitis or endophthalmitis and other infections caused by S. aureus. Apart from this, recent studies show that conjugation of polyethylene glycol (PEG) to lysostaphin (PEG gyration) increased serum drug half-life and reduced its binding to antilysostaphin antibodies while also maintaining the enzyme’s staphylolytic activity (Walsh et al. 2002). These

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studies demonstrate that binding of lysostaphin to PEG protects the enzyme from immune neutralization clearance and repeated dosing. These two properties significantly enhance the therapeutic values of lysostaphin as an alternative treatment against S. aureus infections. Furthermore, immunogenicity to purified lysostaphin could be demonstrated in a variety of species when administered parentally, while oral administration failed to elicit a significant immunological response. In addition, intramammary infusion of r-lysostaphin did not elicit any observable effects on the host animal or on the potential therapy of the r-molecule. All these studies suggest lysostaphin as a safe and effective therapeutic agent for staphylococcal infections particularly for MRSA and other antibiotic-resistant S. aureus infections. However, further clinical studies are required to increase the efficiency of lysostaphin as a therapeutic agent for S. aureus both orally as well as when administered parentally. Moreover, r-lysostaphin can be significantly important for its use as an antistaphylococcal agent. However, the stability and ingestion of r-lysostaphin in vivo requires further investigations.

Limitations Several studies have demonstrated lysostaphin as a novel antistaphylococcal agent for the treatment of S. aureus infections. However, there are certain limitations to the use of lysostaphin. For instance, certain environmental factors can influence growth conditions such as culture media have been shown to influence capsular polysaccharide production in S. aureus (Dassy et al. 1991; O’Riordan and Lee 2004; Poutrel et al. 1995; Stringfellow et al. 1991). The environmentally induced capsular polysaccharide production of different S. aureus strains may limit the access of lysostaphin to pentaglycine bridges in the peptidoglycan cell wall and, therefore, may render strains less susceptible to lysostaphin depending on the assay used. Another mechanism of resistance to lysostaphin involves mutations that affect femA, the gene responsible for addition of the second and third glycine to the pentaglycine cross bridges. Mutations affecting femA renders this gene non-functional resulting in monoglycine cross bridges instead of pentaglycine bridges (De Jonge et al. 1993; Ehlert et al. 2000; Kopp et al. 1996; Maidhof et al. 1991 and Stranden et al. 1997; Kusuma et al. 2007). This causes the S. aureus cells to be either partially or completely resistant to lysostaphin. In addition to this, another mechanism involves acquisition of the gene for the lysostaphin immunity factor (lif) which is found on the pACK1 plasmid which also encodes lysostaphin in S. simulans biovar staphylolyticus. This factor is also called the endopeptidase resistant gene (epr)

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and results in substitution of serines for glycines in the pentaglycine cross bridges (DeHart et al. 1995, Ehlert et al. 2000 and Sugei et al. 1997). These serine substitutions results in a strain of S. aureus that is of intermediate susceptibility to lysostaphin. This gene, however, has not been found in S. aureus outside of the lab. No other mechanisms have been reported that results in reduced susceptibility of S. aureus to lysostaphin. In spite of these limitations, lysostaphin remains as a novel therapeutic agent for treatment of S. aureus because of its unique specificity.

toxicity, increasing stability and efficiency, r-lysostaphin, particularly, could be a potential therapy for treatment of various S. aureus infections in humans and in bovines. In addition, lysostaphin can also be used as an antistaphylococcal agent in various industries, such as a preservative in food industry and in clinical labs for rapid screening. However, further understanding of the lysostaphin structural and functional properties may help in the standardization of drug formulations either alone or in combination with other antibiotics to be used against antibiotic-resistant S. aureus.

Stability

Acknowledgement I am thankful to Prof. P. R. Kulkarni, Retired Head, Food and Fermentation department, UDCT, Mumbai, for the critical review of the manuscript.

Lysostaphin has been found to have the greatest stability at pH 4 and 5°C. The half-life of the staphylolytic activity of lysostaphin was estimated to be approximately 2 months. As a preservative, 0.02% sodium azide was added to all preparations, thus showing no influence on the enzymatic activities (Iversen and Grov 1973). In addition, r-lysostaphin was found to be stable at 37°C for 72 h (Oldham and Daley 1991).

Manufacture and preparations Lysostaphin is commercially available as a dried powder from Sigma chemicals. Besides this, lysostaphin can be purified from S. simulans strains. In addition, purification of lysostaphin has been reported by various methods (Szweda et al. 2001; Marova and Dadak 1993; Schindler and Shuhradt 1965). Besides this, recently, Mierau et al. (2005) used the nisin-controlled gene expressions system (NICE) of Lactococcus lactis to produce a large amount of lysostaphin.

Conclusions and future prospects Lysostaphin is unique among antistaphylococcal agents in that it destroys bacteria, whether they are active or resting, capsular, or non-capsular, and it is thus capable of killing a large number of organisms. Hence, it may be useful in instances of endocarditis and other conditions where an initial and rapid reduction in bacterial count is necessary. More significantly, since the in vivo effectiveness of this enzyme against MRSA has been demonstrated, lysostaphin might prove useful in the treatment of MRSA staphylococcal infections alone or in combination with antibiotics. Ultimately, it could have potential in the treatment and prevention of many resistant staphylococcal infections in the future. Apart from this, due to its unique specificity, low

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