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Peptidoglycan hydrolase enzyme fusions for treating multi-drug resistant pathogens

Figure 1. PG structure and sites of hydrolase cleavage. S. aureus peptidoglycan is depicted with the generic cut sites for amidases and lysozyme-like glycosidases. Glucosaminidase and muramidase are examples of glycosidases that cleave between N-acetyl glucosamine (NGlu) and N-acetyl muramic acid (NMur). Amidases cleave between the NMur and the first amino acid of the peptide. The cut sites for LysK and lysostaphin have been determined, and are noted. Inset: Gram-positive cell walls can have very thick layers of this sugar-protein structure. Figure adapted from [2].
Figure 2. LysK-lysostaphin (LysK-Lyso) contains three active lytic domains and eradicates S. aureus ex vivo in heparinsed rat blood. A. Lysostaphin and LysK were fused head to tail using standard PCR cloning techniques. Following DNA sequence verification, the fusion was expressed in E. coli using the pET21a expression and NiNTA nickel chromatography purification sytem (Novagen; Qiagen). Lysostaphin glycyl-glycine endopeptidase domain: horizontal stripes; CHAP Endopeptidase: vertical stripes; amidase: diagonal stripes; black box: SH3b cell wall binding domain. B. S. aureus PG was digested with either LysK (top panel) or the fusion (bottom panel) and examined via electrospray ionisation mass spectrometry. The presence of the m/z 702 peak shows that both LysK endopeptidase and amidase domains are active. The peaks at m/z 645 (A2QKG4), 588 (A2QKG3), and 531 (A2QKG2) are the result of Gly-Gly cleavages by lysostaphin [see Figure 1 for cut sites]. C. Lysins kill S. aureus in heparinised rat blood. Rat blood inoculated with ~1000 cfu/mL S. aureus strain Newbould 305 was added to tubes containing between 10 and 45 µg of each enzyme (less than 10% final reaction volume). Samples were taken at 0, 90 and 180 min. serial diluted, and then plated in triplicate onto TSB agar to determine the number of colony forming units per mL. Buffer control: ●; LysK: ♦; Lysostaphin: ■; LysK-Lyso:▲

by Dr D. M. Donovan, Dr S. C. Becker, Dr S. Dong, Dr J. R. Baker, Dr J. Foster-Frey and Dr D. G.Pritchard
Pathogens with multiple resistance to antibiotics are a world-wide concern for human and animal health. Bacteriophage lytic enzymes are a potent new source of antimicrobials for treating these pathogens. Phages are viruses that infect bacteria. Survival of the phage relies on phage-encoded endolysins to degrade host peptidoglycan (PG) — a major structure of bacterial cell walls — so that nascent phage particles can escape and reinfect new host cells. Bacteria-phage co-evolution has yielded endolysins that target immutable PG bonds such that no hosts with resistance to the phage endolysins have been identified. The lysins are modular enzymes with lytic domains that maintain their parental specificities when fused. These qualities and the ability to kill pathogens in biofilms position the PG hydrolases as a potent new class of antimicrobials uniquely suited to eradicate multi-drug resistant pathogens.


Bacterial resistance to conventional antibiotics is a world-wide concern. The problem is not limited to human health but is showing up with increasing frequency in live stock and companion animals. The existence of animal pathogens harbouring antibiotic resistance genes with a high potential for transfer to human pathogens is a reality, with many current scientific meetings devoted to methicillin resistant Staphylococcus aureus (MRSA) in animals (http://www.asm.org/Meetings/index.asp?bid=59440). The United States Department of Agriculture, NIH, FDA and CDC all support the identification of antimicrobials that are refractory to resistance development (CDC Action Plan: www.cdc.gov/drugresistance/actionplan/html/product.htm), and many governments world-wide are banning the use of broad range antibiotics in animal feed.

Peptidoglycan (PG) hydrolases are a potential source of novel antimicrobials. PG is the major structural component of bacterial cell walls and can be many layers thick. Autolytic PG hydrolases modify the PG to allow the bacterial cell to grow and divide. Other well-studied groups of PG hydrolases are enzyme bacteriocins (like lysostaphin) and bacteriophage lytic enzymes. Phages are bacterial viruses. Phage-encoded PG hydrolases degrade the host bacterial cell wall and allow nascent phage to escape and infect new hosts. When externally exposed to purified phage lysins, Gram-positive bacteria undergo exolysis or “lysis from without.” This was first described in 1940 by M. Delbruck; at high phage titres, PG degrading proteins on the surface of the phage particles caused premature lysis of the bacterial host. The use of these enzymes as antimicrobials has not been reported for treatment of Gram-negative bacteria, presumably due to the presence of an outer membrane that prevents access to the PG [1].
PG is unique to bacteria and has a complex structure with a sugar backbone of alternating units of N-acetylglucosamine (NGlu) and N-acetylmuramic acid (NMur). Each NMur residue is linked to a short pentapeptide chain. Characteristic of S. aureus is the pentaglycine cross-bridge that connects the L-Lys of the stem peptide to the D-Ala at position 4 of a neighbouring subunit [Figure 1].  

Three classes of PG hydrolase domains have been identified: endopeptidases, amidases and glycosidases [3] [Figure 1]. Alignment of conserved domain sequences from multiple PG hydrolase proteins has revealed a high degree of inter-genus conservation, including non-variant amino acid positions that, when mutated, destroy the hydrolytic activity of the domain.  Chimeric PG hydrolases have also been created by the exchange of cell wall binding (CWB) domains [4], or the fusion of streptococcal phage lysins to lysostaphin [5]. The results of these works indicate that PG hydrolases have evolved a modular design, with both lytic and CWB domains. When fused the domains can maintain their parental specificities for both the PG bond cleaved, and the species of cell wall recognised.

These modular enzymes are strong candidate antimicrobials for several reasons. The Gram-positive PG is located on the cell surface. Many mechanisms of resistance development take advantage of the ability to inactivate the antimicrobial inside the cell, such as the acquisition of enzymes that: inactivate the
antibiotic; reduce membrane permeability; facilitate active efflux of the antimicrobial from the cell; modify the target protein to a resistant form; or produce higher quantities of the target protein. Alternatively, the original target can be altered via a mutational or recombination event at the endogenous gene to an antibiotic-resistant form [6]. Thus, targets outside the cytoplasmic membrane reduce the possible mechanisms by which resistance can develop.

Despite repeated attempts to identify them, no strains of host bacteria have been reported that can resist the lytic activities of their bacteriophage endolysins [7]. It is likely that the bacteriophages have coevolved with their host bacteria such that in order to guarantee escape from the bacterium, the phages have targeted PG bonds that the host can not alter. Resistance to the phage lysins is thus expected to be a very rare event.

The near-species specificity of phage lysins also avoids many pitfalls associated with broad range antimicrobial treatments.  These lead to selection for resistant strains, not just in the target pathogen, but also in co-resident commensal bacteria exposed to the drug. The acquisition of antibiotic resistance is often accomplished by the transfer of DNA sequences from a resistant strain to a susceptible strain. This transfer is not necessarily species or genus limited, and can lead to commensal bacteria that are both antibiotic resistant and can serve as carriers of these DNA elements for propagation to neighbouring bacteria. Those neighbouring strains (potential pathogens) with newly acquired resistance elements can emerge as antibiotic resistant strains during future treatment episodes. Thus, in order to reduce the spread of antibiotic resistance, it is recommended to avoid subjecting commensal bacterial communities to broad range antibiotics.  Phage lysins are usually genus- or even species-specific, thus avoiding many of the caveats associated with broad range antimicrobial use.

A high level of antimicrobial resistance is also achieved by many pathogens through the multi-faceted changes that accompany growth in a biofilm. Sessile forms of bacterial colonies, biofilms attach to a mechanical or prosthetic device or a layer of mammalian cells. NIH estimates that 80% of human bacterial infections involve biofilms (http://grants.nih.gov/grants/guide/pa-files/PA-06-537.html). Bacteria in biofilms can be orders of magnitude more resistant to antibiotic treatment than their planktonic (liquid culture) counterparts [8].
Several mechanisms are thought to contribute to the antimicrobial resistance associated with biofilms: delayed or restricted penetration of antimicrobial agents through the biofilm exopolysaccharide matrix; decreased metabolism and growth rate of biofilm organisms which resist killing by compounds that only attack actively growing cells; increased accumulation of antimicrobial-degrading enzymes; enhanced exchange rates of drug resistance genes; and increased antibiotic tolerance (as opposed to resistance) through expression of stress response genes, phase variation and biofilm specific phenotype development.
 
Biofilms also show heightened resistance to host defence mechanisms. S. aureus grown in biofilms express a polymer of beta-1,6-linked N-acetylglucosamine (PNAG) in large amounts. Biofilm cultures are believed to exhibit reduced activation of complement (compared to planktonic cultures), and the aggregation of bacteria makes them less susceptible to phagocytosis. Altered gene expression of binding factors, cell surface PG, glycoprotein synthesising enzymes, and stress related proteins involved in the detoxification of formate, urea and reactive oxygen species, are likely factors involved in persistence and resistance of cells in a biofilms. Treatment with antibiotics, especially at subinhibitory concentrations, can actually foster the formation of biofilms [9]. There is clearly a current need for enzymes to break down biofilms for more efficient treatment of biofilm-associated staphylococcal infections [10].

Two PG hydrolases, namely staphylococcal phage endolysin phi11 and lysostaphin, are both reported to disrupt S. aureus biofilms [11]. We have identified other phage lysins that also disrupt biofilms (unpublished data).

Endolysins with two active domains are expected to be even more refractory to resistance development since the cell will need to mutate or modify multiple target bonds to resist the lytic action of two activities [7]. The use of two PG hydrolases, the staphylococcal phage K endolysin (LysK) and lysostaphin, has been reported to have a synergistic effect in vitro in the killing of USA300, a multi-drug resistant form of a methicillin resistant S. aureus (MRSA) [12]. Similar results have been obtained with streptococcal pathogens in vivo in a mouse sepsis model [13]. This is consistent with better cure rates observed in models of S. aureus infections in which experimental animals are treated with either two antibiotics or lysostaphin plus an antibiotic.  Synergistic bactericidal activity has also been demonstrated with an endolysin and an antibiotic against Streptococcus pneumoniae [14]. We predict that creating a fusion lysin with three unique, synergistic lytic activities would virtually ensure no resistance development in the target pathogen.
 
We have created several triple-lytic-domain anti-staphylococcal fusion constructs using the synergistic enzymes LysK and lysostaphin. Lysostaphin and LysK cleavage sites are listed in Figure 1. LysK, lysostaphin are known to be active against multiple MRSA strains.  The LysK-Lyso triple lytic domain construct depicted in Figure 2A is highly active against both S. aureus, MRSA and numerous coagulase negative staphylococci (unpublished data). Most importantly, all three lytic domains are active in the fusion construct, as demonstrated by electron spray ionisation mass spectrometry of PG digestion products [Figure 2B]. This fusion construct can eradicate S. aureus from ex vivo rat blood [Figure 2C]. Lysostaphin appears to have an even faster blood-clearing rate than the fusion, but there are known resistance mechanisms to lysostaphin. Studies are underway to determine the efficacy of this and other triple-lytic-domain fusion lysins in animal models of staphylococcal infection, and to test for resistance development in the bacteria retrieved from those in vivo models.

Multi-drug resistant super-bugs have ‘raised the bar’ by establishing a higher set of requirements for new antibiotics.New antimicrobial agents should ideally eradicate multi-drug resistant pathogens, including those in biofilms, and successfully prevent further resistance development. PG hydrolases and their fusions have unique properties that make them ideal candidates for this much needed new class of therapeutics.

REFERENCES
1. Loessner MJ. Bacteriophage endolysins--current state of research and applications. Curr Opin Microbiol 2005; 8: 480-487.
2. Navarre WW, Ton-That H, Faull KF, Schneewind O. Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity. J Biol Chem 1999; 274: 15847-15856.
3. Lopez R, Garcia E. Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes, and bacteriophage. FEMS Microbiol Rev 2004; 28: 553-580.
4. Diaz E, Lopez R, Garcia JL. Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc Natl Acad Sci U SA 1990; 87: 8125-8129.
5. Donovan DM, Dong S, Garrett W, Rousseau GM, Moineau S, Pritchard DG. Peptidoglycan hydrolase fusions maintain their parental specificities. Appl Environ Microbiol 2006; 72:  2988-2996.
6. Spratt BG. Resistance to antibiotics mediated by target alterations. Science 1994; 264:  388-393.
7. Fischetti VA. Bacteriophage lytic enzymes: novel anti-infectives. Trends Microbiol 2005; 13: 491-496.
8. Amorena B, Gracia E, Monzon M, Leiva J, Oteiza C, Perez M, Alabart JL, Hernandez-Yago J. Antibiotic susceptibility assay for Staphylococcus aureus in biofilms developed in vitro. J Antimicrob Chemother 1999; 44: 43-55.
9. Rachid S, Ohlsen K, Witte W, Hacker J, Ziebuhr W. Effect of subinhibitory antibiotic concentrations on polysaccharide intercellular adhesin expression in biofilm-forming Staphylococcus epidermidis. Antimicrob Agents Chemother 2000; 44: 3357-3363.
10. Otto M. Bacterial evasion of antimicrobial peptides by biofilm formation. Curr Top Microbiol Immunol 2006; 306: 251-258.
11. Sass P, Bierbaum G. Lytic Activity of Recombinant Bacteriophage {phi}11 and {phi}12 Endolysins on Whole Cells and Biofilms of Staphylococcus aureus. Appl Environ Microbiol 2007; 73, 347-352.
12. Becker SC, Foster-Frey J, Donovan DM. The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA. FEMS Microbiol Lett 2008.
13. Jado I, Lopez R, Garcia E, Fenoll A, Casal J, Garcia P. Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J Antimicrob Chemother 2003; 52: 967-973.
14. Becker SC, Dong S, Baker JR, Foster-Frey J, Pritchard DG, Donovan DM. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiology letters, In Press.

THE AUTHORS
David M. Donovan1*, Stephen C. Becker1, Shengli Dong2, John R. Baker2, Juli Foster-Frey1, David G. Pritchard2

1Animal Biosciences and Biotechnology Laboratory, Animal and Natural Resources Institute, BARC, ARS, USDA, 10300 Baltimore Ave, Beltsville, MD 20705-2350, USA

2Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

Corresponding author:
David D Donovan
Tel +1 301-504-9150
Fax +1 301-504-8571
Mobile:+1 443-690-4251
e-mail: david.donovan@ars.usda.gov


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