- Email: [email protected]
Enzymes for anti-infective therapy: phage lysins
Vol. 1, No. 4 2004
Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Infectious diseases
Enzymes for anti-infective therapy: phage lysins Rubens Lo´pez*, Ernesto Garcı´a, Pedro Garcı´a Departamento de Microbiologı´a Molecular, Centro de Investigaciones Biolo´gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
Bacterial viruses (bacteriophages or phages) were first isolated 90 years ago and were envisaged early on as curative tools. Bacterial resistance to most available drugs has driven the attention of researchers to test the possibilities of using phages, and/or some of their gene products, as therapeutic agents. The peculiarities of cell-wall lytic enzymes, and recent achievements in using phage-coded lysins for the treatment of severe human infections caused by antibiotic-resistant bacteria are most intriguing. Introduction Virus-infecting bacteria were first reported at the beginning of the 20th century by Fe´lix D’Herelle and Frederick W. Twort although D’Herelle was the first to link phages and bacterial lysis. Phages have been fundamental tools for the spectacular development of molecular biology [1]. In addition, phages were envisaged very early as therapeutic tools to fight pathogenic bacteria. The successful and generalized use of antibiotics to control bacterial infections after World War II, and the difficulties in obtaining purified phage preparations, delayed the use of phages for therapy. However, the emergence of antibiotic-resistant bacteria has now highlighted the need to explore the potential phages (or their gene products) for use as therapeutic agents. Recent literature reveals that we are now in a new dawn for phage therapy [2].
Section Editor: Gary Woodnutt – Diversa Corp., San Diego, CA, USA Anti-bacterial phages have been used successfully for many years in the treatment of infectious disease in some parts of the world. However, the effectiveness and convenience of broad-spectrum anti-bacterials have prevented the more widespread use of phage therapy. The spread of anti-microbial resistance has necessitated re-analysis of alternate therapy and interest in phage is now on the increase. In particular, there is increased interest in understanding the potential for phage lytic enzymes as this can reduce production difficulties by heterologous expression in suitable fermentation hosts. As highlighted by this review, there are clear advantages for this type of therapy, including rapid killing and lysis of bacterial strains. Lopez et al. also discuss the potential advantages of combination therapy with mixtures of enzymes. However, it is probable that the specificity of these lysins for particular bacteria could be a significant issue (phage are perfectly adapted to infect particular species or even sub-sets of species) but there is potential for construction of chimeric enzymes that might increase both activity and anti-bacterial spectrum. If this proves possible, phage lysins have a very exciting future.
additional effects on virulence [3]. The scarlatinal and diphteria toxins were early and historical examples illustrating that bacterial virulence is modified by genes located in temperate bacteriophages. Toxins are among the best-recognized examples of detrimental phage genes in several bacterial pathogens such as those causing cholera, botulism and diphtheria. Interestingly, phages are also involved in alteration of the susceptibility of bacteria to drugs as well as on the enhancement of the capacity of bacterial transmission among humans [4].
Phages as carriers of virulence genes It is currently recognized that phage carriage by lysogenic bacteria (see Glossary) is frequently associated with *Corresponding author: (R. Lo´pez) [email protected] 1740-6773/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2004.09.002
Phage therapy Recently, phages have again come to prominence due to the discovery of the remarkable dynamics underlying their www.drugdiscoverytoday.com
469
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Vol. 1, No. 4 2004
Glossary Capsule: polysaccharide that localizes outside the bacterial cell wall. In the case of the pneumococcus, it is the major contributor to virulence. Chimeric protein: a protein that contains domains from different proteins. These domains may have different evolutionary origins. The genes coding for such proteins are named chimeric genes and are generated by genetic recombination. The underlying recombinational events may be natural or be done through genetic manipulations in the laboratory. Holin: a phage-encoded polypeptide that disrupts the bacterial membrane. The gene coding for this polypeptide is normally located immediately upstream of the lytic gene. Lysogenic bacteria (or lysogen): bacteria that harbors a temperate phage. Microbiota: microscopic life of a region. In microbiology, this term is more appropriate than ‘microflora’. Temperate phages: phages that are in a dormant state in which only few genes are expressed; the genome of which is either integrated into that of the host, or is replicated autonomously.
evolution, role in virulence, and use as therapeutic agents. The ability of virulent phages to rapidly kill and lyse infected bacteria, and the capacity of phages to increase in number during the infection process, have made phages excellent potential agents for fighting bacterial diseases. However, temperate phages (see Glossary) are of little use in this therapy. Although phage therapy is actually an old idea and was widely employed to treat various bacterial diseases both in humans and animals, it is only recently that more rigorous assays have been performed [2–4]. Bacteria frequently change to resist infecting phages, but the rate of this mutation appears lower than that reported against antibiotics. Most interestingly, bacteriophages are the most abundant entities in the biosphere (1031) and, consequently, the use of phage cocktails after a stringent selection of a variety of phages appropriate to combat a given pathogen is a manageable aim [4]. Moreover, Mushtaq et al. [5] have determined that, in a rat model, bacteremia and death from a systemic infection provoked by an Escherichia coli K1 strain could be prevented by an intraperitoneal (IP) administration of a bacteriophagederived endosialidase that selectively degrades the polysialic acid of the capsular polysaccharide of such strain. In summary, there are good reasons to believe that the use of phages for treatment and preventing bacterial infections could be successful in limiting settings, such as acute and chronic infections [6]. By contrast, phage genomes have been used as tools for target discovery and validation, assay development and compound design. Exploiting the concept of phagemediated inhibition of bacterial growth, and taking advance of the detailed knowledge of the sequence of 25 phage genomes infecting Staphylococcus aureus, Liu et al. [7] have recently put forward an ingenious procedure for searching antibiotics with new anti-bacterial mechanisms and identi470
www.drugdiscoverytoday.com
Figure 1. Electron micrograph of intracellular phage development in Streptococcus pneumoniae cells infected with the pneumococcal bacteriophage Dp-1. The central cell shows partial disruption of the cell wall by the phage Pal amidase (arrow) and liberation to the supernatant of some phage particles (arrowhead). The inset shows CsCl-purified Dp-1 particles negatively stained with 1% phosphotungstic acid.
fied 31 proteins (or polypeptides) that inhibit the growth of S. aureus. Furthermore, different phage-based variations to therapy have recently been reported. Hagens and Bla¨ si [8] have shown the feasibility of genetically modified filamentous phages that encode either a restriction endonuclease or an altered holin (see Glossary) to kill susceptible bacteria without cell lysis – a method that minimizes the release of large amounts of endotoxins as a consequence of bacterial lysis.
Phage lytic enzymes An alternative to the use of whole phages to destroy bacteria relies on one of the key enzyme phage products, the lysin. Extensive work has been done on the molecular biology and biochemistry of these enzymes in the pneumococcal system. Pneumococcal phages were first isolated in 1975 from throat swabs of healthy children [9]. Later, several pneumococcal bacteriophages from different origins have been isolated and characterized [10,11]. These phages have been useful in expanding our knowledge on the mechanisms of genetic interchange in pneumococcus, mainly on lytic enzymes. The last step of phage infection is the release of mature phage particles by hydrolysis of the bacterial cell wall (Fig. 1). Most often, phages encode murein-degrading enzymes that hydrolyze either the glycosidic linkages between the amino sugars of the peptidoglycan backbone (i.e. lysozymes such as Cpl-1),
Vol. 1, No. 4 2004
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Figure 2. The structure of the bacterial LytA amidase, and phage Pal and Cpl-1 cell-wall hydrolases, and crystal structure of the Cpl-1 lysozyme. (a) Representation of the pneumococcal cell wall, indicating the chemical bonds cleaved by the lytic enzymes Pal and Cpl-1. Abbreviations: G, N-acetylglucosamine; M, N-acetylmuramic acid; ChTA, choline-containing teichoic acid. (b) The domain containing the active center of the enzymes are represented in red or dark blue (amidases), and green (lysozyme). The choline-binding domain (light blue) and the choline-binding repeats are also shown. Note that, whereas seven repeats are predicted in LytA and Pal amidases [13], the Cpl-1 lysozyme only have six repeats plus a short, 16-amino acid residues tail (colored in gray) [15]. Homology between lytic enzymes is indicated by identical colors. The linker regions between the N- and C-terminal domains are also shown. (c) Ribbon diagram of Cpl-1. The catalytic N-terminal domain (N), the CI and CII domains of the ChBD, and the linker region (L) are indicated.
the N-acetylmuramoyl-L-alanine amide bond between the glycan strand and the cross-linking peptide (i.e. amidases such as Pal; Fig. 2a), or the inter-peptide bridge linkages (i.e. endopeptidases); for a review on pneumococcal lytic enzymes see Ref. [12]. Curiously, the lytic enzymes from most phages infecting other Gram-positive bacteria are annotated in databases simply as ‘(endo)lysin’, or ‘cell wall/peptidoglycan hydrolase’. Genes encoding the lytic enzymes of pneumococcal phages were cloned and sequence comparisons revealed that the host and phage-coded enzymes have a highly similar Cterminal domain that is responsible for the binding to the choline residues (Fig. 2b) present in the teichoic acids of the cell-wall substrate [13]. We proposed that this amino-alcohol served as an element of selective pressure to preserve the substrate-recognition domain of all but one of the pneumococcal lytic enzymes characterized so far [12]. Noticeably, Cpl-7 is a lysozyme encoded by phage Cp-7 that has completely changed the C-terminal domain and, consequently, has lost the dependence of choline for activity. As expected, this enzyme shows a completely different C-terminal domain [14] (Fig. 3). The lytic system encoded by Streptococcus pneumoniae and its phages represents one of the most comprehensive examples supporting the modular theory of protein evolution. We have demonstrated that the pneumococcal phage lysins are the result of the fusion of two independent functional modules, one is responsible for the specific recognition of the cell wall, whereas the other localizes the active center of these enzymes [12]. In an effort to better understand how murein hydrolases bind and degrade the peptidoglycan, the 3D structure of the free and choline-bound states of an entire pneumococcal choline-binding protein, the Cpl-1
lysozyme has been elucidated [15]. The polypeptide chain consists of the catalytic and the choline-binding domain (ChBD) joined by an acidic linker comprising residues 189– 199 (Fig. 2c).
Figure 3. Representation of the constructions of chimeric pneumococcal-phage murein hydrolases. The bars represent the lytic genes, and the direction of transcription is from left to right. A new restriction site (PstI) was introduced by site-directed mutagenesis into the linker region of the lytic genes [22]. The relevant characteristics of the corresponding enzymes are also indicated. The active chimeric enzymes have interchanged the biochemical properties of the parental natural enzymes of phage (lysozyme Cpl-7) and bacteria (amidase LytA), as indicated. Observe that the C-terminal domain of Cpl-7 [14] only has three motifs (yellow) that are different of the choline binding motifs (blue), making this enzyme functionally independent of choline in the teichoic acid for activity.
www.drugdiscoverytoday.com
471
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Vol. 1, No. 4 2004
Phage lysins as anti-microbial agents Using an elegant variation of the classical phage therapy approach, Fischetti and co-workers pioneered the use of specific phage-encoded murein hydrolases to kill Streptococcus pyogenes, and defined a new class of antibiotics (‘enzybiotics’) totally distinct from any other previously described [16]. This method of ‘lysis from without’ – using purified phage lytic enzymes (as opposed to the ‘lysis from within’ occurring at the end of the phage lytic cycle, as illustrated in Fig. 1) – appears to be a new and challenging area in antibiotic treatment.
Bacterial specificity of lysins The general background supporting the use of phage lysins for prophylactic purposes is based on the observation that because lytic enzymes specifically kill the species in which they were synthesized (or a small number of closely related bacteria) these enzymes might represent an effective way to control specific pathogens without disturbing the normal microbiota (see Glossary). Lysins have been successfully used to prevent and eliminate the upper respiratory colonization of mice by groupA streptococci [16], to kill pneumococci from nasopharyngeal carrier mice [17], and to rapid detect and kill both vegetative cells and germinating spores of Bacillus anthracis [18]. PlyG lysin, which is encoded by g phage of B. anthracis, specifically kills this bacterium and other members of this cluster of bacilli. Germinating spores turned out to be also susceptible. The specificity of the lytic enzyme also provided a rapid method for the identification of this potential biological weapon. In S. pneumoniae, Fischetti [17] used Pal, the amidase coded by Dp-1 (Figs. 1 and 2), which was very effective in eliminating several serotypes of pneumococci demonstrating that the capsule (see Glossary) did not interfere with the access of the enzyme to the cell wall. It is important to emphasize that, in this case, the rise of resistant strains appears a rare event because Pal is strictly dependent on the presence of cholinecontaining teichoic acids of S. pneumoniae for activity, and this aminoalcohol is necessary for pneumococcal viability in vivo. Hence, it is probable that, over evolution, the ChBD of these lytic enzymes evolved to target a unique and essential molecule in the wall. The specificity of this approach was validated by the observation that only Streptococcus oralis and Streptococcus mitis cultures (which also contain choline in their cell walls) showed lower viability in these assays [17]. It has been proposed that, in the case of streptococci, the eradication of these bacteria from the upper respiratory mucosal epithelium might reduce the associated disease.
Curing bacteremia More recently, we have expanded such experimental approach using a murine sepsis model to study the ability of pneumococcal phage amidases and/or lysozymes to cure 472
www.drugdiscoverytoday.com
Figure 4. Synergistic therapeutic effect of the combined use of Pal and Cpl-1. (a) One hour after IP injection of mice with 5 107 colonyforming units (cfu) (at zero time), 2.5 mg each of Pal and Cpl-1 (red circles), or 5 mg of either Pal (green circle) or Cpl-1 (blue squares) were injected into the mouse peritoneal cavity and survival of mice was followed. (b) The effect of the combined therapy (2.5 mg each of Pal and Cpl-1 were administered intraperitoneally 1 h after challenge) in bacteremia of four mice was estimated as cfu per ml at different times after injection of the lytic enzymes. Each curve indicates the bacteremic counts of a single mouse. Reproduced, with permission, from Ref. [19].
bacteremia produced by an antibiotic-multiresistant 6B strain, the most common pneumococcal serotype isolated from children with bacteremia [19]. The ability of Cpl-1 lysozyme and/or Pal amidase to cure bacteremic animals is a protein-specific function, as indicated by the observation that rescue did not occur with heat-inactivated enzymes. Irrespective of the enzymatic activity of the enzyme used, our results revealed that a single IP injection of the corresponding lysin rescued mice from death due to S. pneumoniae even when bacteremia was already established. We observed that effective protection was achieved in mice when a single dose of at least 10 mg of Cpl-1 (or Pal) was administered 1 h after the S. pneumoniae strain was inoculated. The protective
Vol. 1, No. 4 2004
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Table 1. Current concerns on phage lysin therapy Pros
Cons
Latest developments (including drug therapies in progress and failures)
Who is working on this strategy (include web address) (group/institute/company)
Refs.
Target alteration
Create new active lysins
Resistance
Natural or engineered chimeric enzymes
Fischetti/Rockefeller Univ. ([email protected]): Lo´ pez/Centro Inv. Biol./CSIC ([email protected])
[17,19,24–27]
Shock reactions
Nonea
Possible death
Not needed
Fischetti and Lo´ pez groups
[20,28]
Antibody neutralization
a
New enzymes required
Not needed
Fischetti and Lo´ pez groups
[19,20,28]
None
Disease prevention Avoid infection Increased resistance Enzyme encapsulation in micelles Fischetti/Rockefeller Univ. ([email protected]) [28] a
Means that this response has not been found.
effect of both enzybiotics was notable when the enzymes were injected 2 h after pneumococcal challenge, although the extent of this effect was related to the pre-treatment outcome. Moreover, a complete pneumococcal clearance was observed in most surviving mice after a six to eightday follow-up period. An IP injection of either lytic enzyme, delayed up to 4 h after challenge, had a partial protective effect by prolonging the life of the severely ill mice for several days [19]. Provided that mice are most susceptible to pneumococcal infections, the results obtained in these experiments are encouraging because our findings, and those of others [20], support the development of modified procedures in a near future to make use of lysins more efficient. Most noticeably, the simultaneous use of both enzymes, rather than being competitive, enhanced the destruction of the cell wall and, hence, shows a synergistic lytic action on S. pneumoniae in the murine sepsis model used. The positive interaction of the two muralytic enzymes, with only 2.5 mg each of Cpl-1 and Pal, could be because of the increased access of these enzymes, with different catalytic domains, to their cleavage sites (Fig. 2a) as recently suggested [20,21]. Survival rates of animals that received both enzymes were significantly higher than those of animals receiving only Cpl-1 or Pal (Fig. 4). These experiments extend previous observations on the bacterial carrier state to the sepsis model and also reveal the lack of toxicity of the pneumococcal enzybiotics in mice. The combined use of two lytic enzymes would make more unlikely the intrinsic resistance of the bacteria to these proteins that target essential cell-wall molecules [19].
Future prospects: strategy comparison and recombinant lysins In contrast to our observations, Loeffler et al. [21] found that intravenous injections (IV) of Cpl-1 in a mouse model of pneumococcal bacteremia resulted in longer survival of infected mice, but failed to block final killing. The use of IV route rather than the IP one for mice infection and rescue by Cpl-1 and the presence of an endotoxin in the lysozyme preparation employed [20] might account for the uneven
results reported by these two groups for rescue of pneumococcal bacteremia in mice [19,21]. We have demonstrated that the modular design exhibited by the lytic enzymes of pneumococcus and its phages facilitates the construction of chimeric enzymes. As illustrated in Fig. 3, bacterial specificities and activities can be combined by enzymatic swapping [22,23]. Successful construction of chimeric proteins (see Glossary) exhibiting two lytic activities [24] or intergeneric active chimera between bacterial species [25] have also been documented by our laboratory. This experimental approach anticipates the use of genetically engineered enzymes that combine an optimal enzymatic activity with the capacity to recognize different and specific receptors in bacteria. A novel strategy that might extend lysin therapy to a broader range of bacteria by taking advantage of the versatility provided by the modular organization of lytic enzymes is illustrated in the model depicted in Fig. 3. A preliminary illustration in this direction has been reported recently [26] (Table 1).
Conclusions It has been recently emphasized that, during the long history of using phages administered by different routes in several countries, there have been almost no reports of serious complications related to their use [2]. As phages are common entities in the environment and regularly consumed in foods, the development of neutralizing antibodies should not be, in theory, a significant obstacle during the initial treatment of acute infections because the kinetics of phage action or lytic enzymes is much faster than production of neutralizing antibodies by the host. Successful antibiotic therapy in infected mice requires an appropriate dosing interval of administration to maintain pharmacological concentrations in serum. Most interestingly, a single IP injection of a lytic enzyme(s) is sufficient for a complete clearance of invading pneumococci from surviving mice. This property, referred to as therapeutic efficacy, provides a rapid and specific activity and makes www.drugdiscoverytoday.com
473
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Related articles Anonymous. (2003) We haven’t heard much about anthrax since the mail attacks in 2001. Are there any new treatments for it? Mayo Clin. Health Lett. 21, 8 Dixon, B. (2004) New dawn for phage therapy. Lancet Infect. Dis. 4, 186 Joerger, R.D. (2003) Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poult. Sci. 82, 640–647 Rosovitz, M.J. et al. (2002) Virus deals anthrax a killer blow. Nature 418, 825–826 Sone, R. (2002) Stalin’s forgotten cure. Science 298, 728–731 Thiel, K. (2004) Old dogma, new tricks – 21st Century phage therapy. Nat. Biotechnol. 22, 31–36
these proteins very promising candidates for current antimicrobial therapies. The lack of toxicity of phage lysins and the observation that hyperimmune sera do not neutralize these enzymes in the mice model are encouraging and warrant future research, although testing in humans will be required. A better understanding of phage biology, combined with the availability of highly purified phage lytic enzymes and the experience attained in several Eastern European countries, where rigorously controlled trials are currently being carried out [27], should improve our armamentarium to combat infectious diseases that are currently the first cause of death in the world.
Acknowledgements We thank J.A. Hermoso for preparation of Fig. 2c. This work was supported by a grant from the Direccio´ n General de Investigacio´ n Cientı´fica y Te´ cnica (BCM2003-00074).
References 1 Summers, W.C. (1999) Fe´lix D’Herelle and the Origins of Molecular Biology. Yale University Press 2 Sulakvelidze, A. et al. (2001) Bacteriophage therapy. Antimicrob. Agents Chemother. 45, 149–659 3 Wagner, P.L. and Waldor, M.K. (2002) Bacteriophage control of bacterial virulence. Infect. Immun. 70, 3985–3993 4 Summers, W.C. (2001) Bacteriophage therapy. Annu. Rev. Microbiol. 55, 437–451 5 Mushtaq, N. et al. (2004) Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob. Agents Chemother. 48, 1503–1508 6 Levin, B.R. and Bull, J.J. (2004) Population and evolutionary dynamics of phage therapy. Nat. Microbiol. Rev. 2, 166–173
474
www.drugdiscoverytoday.com
Vol. 1, No. 4 2004
7 Liu, J. et al. (2004) Antimicrobial drug discovery through bacteriophage genomics. Nat. Biotechnol. 22, 185–191 8 Hagens, S. and Bla¨si, U. (2003) Genetically modified filamentous phage as bactericidal agents: a pilot study. Lett. Appl. Microbiol. 37, 318–323 9 McDonnell, M. et al. (1975) ‘‘Diplophage’’: a bacteriophage of Diplococcus pneumoniae. Virology 63, 577–582 10 Ronda, C. et al. (1981) Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae. J. Virol. 40, 551–559 11 Garcı´a, P. et al. (2000) Bacteriophages of Streptococcus pneumoniae: a molecular approach. In Streptococcus pneumoniae – Molecular Biology & Mechanisms of Disease (Tomasz, A. ed), pp. 211–222, Mary Ann Liebert, Inc 12 Lo´pez, R. et al. (2004) Cell wall murein hydrolases. In The Pneumococcus (Tuomanen, E.I., Mitchell, T.J., Morrison, D., Spratt, B.G. eds), pp. 75– 88, ASM Press 13 Ferna´ndez-Tornero, C. et al. (2001) A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat. Struct. Biol. 8, 1020–1024 14 Garcı´a, P. et al. (1990) Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Gene 86, 81–88 15 Hermoso, J.A. et al. (2003) Structural basis for selective recognition of the pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 11, 1239–1249 16 Nelson, D. et al. (2001) Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc. Natl. Acad. Sci. USA 98, 4107–4112 17 Loeffler, J.M. et al. (2001) Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294, 2170–2172 18 Schuch, R. et al. (2002) A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418, 884–889 19 Jado, I. et al. (2003) Phage lytic enzymes as therapy of antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J. Antimicrob. Chemother. 52, 967–973 20 Loeffler, J.M. and Fischetti, V.A. (2003) Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillinsensitive and -resistant Streptococcus pneumoniae strains. Antimicrob. Agents Chemother. 47, 375–377 21 Loeffler, J.M. et al. (2003) Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. Infect. Immun. 71, 6199–6204 22 Dı´az, E. et al. (1990) Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc. Natl. Acad. Sci. USA 87, 8125–8129 23 Dı´az, E. et al. (1991) Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits. J. Biol. Chem. 266, 5464–5471 24 Sanz, J.M. et al. (1996) Construction of a multifunctional pneumococcal murein hydrolase by module assembly. Eur. J. Biochem. 235, 601–605 25 Croux, C. et al. (1993) Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal–clostridial cell wall lytic enzyme. Mol. Microbiol. 9, 1019–1025 26 Yoong, P. et al. (2004) Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J. Bacteriol. 186, 4808–4812 27 Carlton, R.M. (1999) Phage therapy: past history and future prospects. Arch. Immunol. Ther. Exp. 47, 267–274 28 Larkin, M. (2004) Vincent Fischetti – following phages for life. The Lancet Infect. Dis. 4, 246–249
Drug Discovery Today: Therapeutic Strategies Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY
TODAY THERAPEUTIC
STRATEGIES
Infectious diseases
Enzymes for anti-infective therapy: phage lysins Rubens Lo´pez*, Ernesto Garcı´a, Pedro Garcı´a Departamento de Microbiologı´a Molecular, Centro de Investigaciones Biolo´gicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
Bacterial viruses (bacteriophages or phages) were first isolated 90 years ago and were envisaged early on as curative tools. Bacterial resistance to most available drugs has driven the attention of researchers to test the possibilities of using phages, and/or some of their gene products, as therapeutic agents. The peculiarities of cell-wall lytic enzymes, and recent achievements in using phage-coded lysins for the treatment of severe human infections caused by antibiotic-resistant bacteria are most intriguing. Introduction Virus-infecting bacteria were first reported at the beginning of the 20th century by Fe´lix D’Herelle and Frederick W. Twort although D’Herelle was the first to link phages and bacterial lysis. Phages have been fundamental tools for the spectacular development of molecular biology [1]. In addition, phages were envisaged very early as therapeutic tools to fight pathogenic bacteria. The successful and generalized use of antibiotics to control bacterial infections after World War II, and the difficulties in obtaining purified phage preparations, delayed the use of phages for therapy. However, the emergence of antibiotic-resistant bacteria has now highlighted the need to explore the potential phages (or their gene products) for use as therapeutic agents. Recent literature reveals that we are now in a new dawn for phage therapy [2].
Section Editor: Gary Woodnutt – Diversa Corp., San Diego, CA, USA Anti-bacterial phages have been used successfully for many years in the treatment of infectious disease in some parts of the world. However, the effectiveness and convenience of broad-spectrum anti-bacterials have prevented the more widespread use of phage therapy. The spread of anti-microbial resistance has necessitated re-analysis of alternate therapy and interest in phage is now on the increase. In particular, there is increased interest in understanding the potential for phage lytic enzymes as this can reduce production difficulties by heterologous expression in suitable fermentation hosts. As highlighted by this review, there are clear advantages for this type of therapy, including rapid killing and lysis of bacterial strains. Lopez et al. also discuss the potential advantages of combination therapy with mixtures of enzymes. However, it is probable that the specificity of these lysins for particular bacteria could be a significant issue (phage are perfectly adapted to infect particular species or even sub-sets of species) but there is potential for construction of chimeric enzymes that might increase both activity and anti-bacterial spectrum. If this proves possible, phage lysins have a very exciting future.
additional effects on virulence [3]. The scarlatinal and diphteria toxins were early and historical examples illustrating that bacterial virulence is modified by genes located in temperate bacteriophages. Toxins are among the best-recognized examples of detrimental phage genes in several bacterial pathogens such as those causing cholera, botulism and diphtheria. Interestingly, phages are also involved in alteration of the susceptibility of bacteria to drugs as well as on the enhancement of the capacity of bacterial transmission among humans [4].
Phages as carriers of virulence genes It is currently recognized that phage carriage by lysogenic bacteria (see Glossary) is frequently associated with *Corresponding author: (R. Lo´pez) [email protected] 1740-6773/$ ß 2004 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddstr.2004.09.002
Phage therapy Recently, phages have again come to prominence due to the discovery of the remarkable dynamics underlying their www.drugdiscoverytoday.com
469
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Vol. 1, No. 4 2004
Glossary Capsule: polysaccharide that localizes outside the bacterial cell wall. In the case of the pneumococcus, it is the major contributor to virulence. Chimeric protein: a protein that contains domains from different proteins. These domains may have different evolutionary origins. The genes coding for such proteins are named chimeric genes and are generated by genetic recombination. The underlying recombinational events may be natural or be done through genetic manipulations in the laboratory. Holin: a phage-encoded polypeptide that disrupts the bacterial membrane. The gene coding for this polypeptide is normally located immediately upstream of the lytic gene. Lysogenic bacteria (or lysogen): bacteria that harbors a temperate phage. Microbiota: microscopic life of a region. In microbiology, this term is more appropriate than ‘microflora’. Temperate phages: phages that are in a dormant state in which only few genes are expressed; the genome of which is either integrated into that of the host, or is replicated autonomously.
evolution, role in virulence, and use as therapeutic agents. The ability of virulent phages to rapidly kill and lyse infected bacteria, and the capacity of phages to increase in number during the infection process, have made phages excellent potential agents for fighting bacterial diseases. However, temperate phages (see Glossary) are of little use in this therapy. Although phage therapy is actually an old idea and was widely employed to treat various bacterial diseases both in humans and animals, it is only recently that more rigorous assays have been performed [2–4]. Bacteria frequently change to resist infecting phages, but the rate of this mutation appears lower than that reported against antibiotics. Most interestingly, bacteriophages are the most abundant entities in the biosphere (1031) and, consequently, the use of phage cocktails after a stringent selection of a variety of phages appropriate to combat a given pathogen is a manageable aim [4]. Moreover, Mushtaq et al. [5] have determined that, in a rat model, bacteremia and death from a systemic infection provoked by an Escherichia coli K1 strain could be prevented by an intraperitoneal (IP) administration of a bacteriophagederived endosialidase that selectively degrades the polysialic acid of the capsular polysaccharide of such strain. In summary, there are good reasons to believe that the use of phages for treatment and preventing bacterial infections could be successful in limiting settings, such as acute and chronic infections [6]. By contrast, phage genomes have been used as tools for target discovery and validation, assay development and compound design. Exploiting the concept of phagemediated inhibition of bacterial growth, and taking advance of the detailed knowledge of the sequence of 25 phage genomes infecting Staphylococcus aureus, Liu et al. [7] have recently put forward an ingenious procedure for searching antibiotics with new anti-bacterial mechanisms and identi470
www.drugdiscoverytoday.com
Figure 1. Electron micrograph of intracellular phage development in Streptococcus pneumoniae cells infected with the pneumococcal bacteriophage Dp-1. The central cell shows partial disruption of the cell wall by the phage Pal amidase (arrow) and liberation to the supernatant of some phage particles (arrowhead). The inset shows CsCl-purified Dp-1 particles negatively stained with 1% phosphotungstic acid.
fied 31 proteins (or polypeptides) that inhibit the growth of S. aureus. Furthermore, different phage-based variations to therapy have recently been reported. Hagens and Bla¨ si [8] have shown the feasibility of genetically modified filamentous phages that encode either a restriction endonuclease or an altered holin (see Glossary) to kill susceptible bacteria without cell lysis – a method that minimizes the release of large amounts of endotoxins as a consequence of bacterial lysis.
Phage lytic enzymes An alternative to the use of whole phages to destroy bacteria relies on one of the key enzyme phage products, the lysin. Extensive work has been done on the molecular biology and biochemistry of these enzymes in the pneumococcal system. Pneumococcal phages were first isolated in 1975 from throat swabs of healthy children [9]. Later, several pneumococcal bacteriophages from different origins have been isolated and characterized [10,11]. These phages have been useful in expanding our knowledge on the mechanisms of genetic interchange in pneumococcus, mainly on lytic enzymes. The last step of phage infection is the release of mature phage particles by hydrolysis of the bacterial cell wall (Fig. 1). Most often, phages encode murein-degrading enzymes that hydrolyze either the glycosidic linkages between the amino sugars of the peptidoglycan backbone (i.e. lysozymes such as Cpl-1),
Vol. 1, No. 4 2004
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Figure 2. The structure of the bacterial LytA amidase, and phage Pal and Cpl-1 cell-wall hydrolases, and crystal structure of the Cpl-1 lysozyme. (a) Representation of the pneumococcal cell wall, indicating the chemical bonds cleaved by the lytic enzymes Pal and Cpl-1. Abbreviations: G, N-acetylglucosamine; M, N-acetylmuramic acid; ChTA, choline-containing teichoic acid. (b) The domain containing the active center of the enzymes are represented in red or dark blue (amidases), and green (lysozyme). The choline-binding domain (light blue) and the choline-binding repeats are also shown. Note that, whereas seven repeats are predicted in LytA and Pal amidases [13], the Cpl-1 lysozyme only have six repeats plus a short, 16-amino acid residues tail (colored in gray) [15]. Homology between lytic enzymes is indicated by identical colors. The linker regions between the N- and C-terminal domains are also shown. (c) Ribbon diagram of Cpl-1. The catalytic N-terminal domain (N), the CI and CII domains of the ChBD, and the linker region (L) are indicated.
the N-acetylmuramoyl-L-alanine amide bond between the glycan strand and the cross-linking peptide (i.e. amidases such as Pal; Fig. 2a), or the inter-peptide bridge linkages (i.e. endopeptidases); for a review on pneumococcal lytic enzymes see Ref. [12]. Curiously, the lytic enzymes from most phages infecting other Gram-positive bacteria are annotated in databases simply as ‘(endo)lysin’, or ‘cell wall/peptidoglycan hydrolase’. Genes encoding the lytic enzymes of pneumococcal phages were cloned and sequence comparisons revealed that the host and phage-coded enzymes have a highly similar Cterminal domain that is responsible for the binding to the choline residues (Fig. 2b) present in the teichoic acids of the cell-wall substrate [13]. We proposed that this amino-alcohol served as an element of selective pressure to preserve the substrate-recognition domain of all but one of the pneumococcal lytic enzymes characterized so far [12]. Noticeably, Cpl-7 is a lysozyme encoded by phage Cp-7 that has completely changed the C-terminal domain and, consequently, has lost the dependence of choline for activity. As expected, this enzyme shows a completely different C-terminal domain [14] (Fig. 3). The lytic system encoded by Streptococcus pneumoniae and its phages represents one of the most comprehensive examples supporting the modular theory of protein evolution. We have demonstrated that the pneumococcal phage lysins are the result of the fusion of two independent functional modules, one is responsible for the specific recognition of the cell wall, whereas the other localizes the active center of these enzymes [12]. In an effort to better understand how murein hydrolases bind and degrade the peptidoglycan, the 3D structure of the free and choline-bound states of an entire pneumococcal choline-binding protein, the Cpl-1
lysozyme has been elucidated [15]. The polypeptide chain consists of the catalytic and the choline-binding domain (ChBD) joined by an acidic linker comprising residues 189– 199 (Fig. 2c).
Figure 3. Representation of the constructions of chimeric pneumococcal-phage murein hydrolases. The bars represent the lytic genes, and the direction of transcription is from left to right. A new restriction site (PstI) was introduced by site-directed mutagenesis into the linker region of the lytic genes [22]. The relevant characteristics of the corresponding enzymes are also indicated. The active chimeric enzymes have interchanged the biochemical properties of the parental natural enzymes of phage (lysozyme Cpl-7) and bacteria (amidase LytA), as indicated. Observe that the C-terminal domain of Cpl-7 [14] only has three motifs (yellow) that are different of the choline binding motifs (blue), making this enzyme functionally independent of choline in the teichoic acid for activity.
www.drugdiscoverytoday.com
471
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Vol. 1, No. 4 2004
Phage lysins as anti-microbial agents Using an elegant variation of the classical phage therapy approach, Fischetti and co-workers pioneered the use of specific phage-encoded murein hydrolases to kill Streptococcus pyogenes, and defined a new class of antibiotics (‘enzybiotics’) totally distinct from any other previously described [16]. This method of ‘lysis from without’ – using purified phage lytic enzymes (as opposed to the ‘lysis from within’ occurring at the end of the phage lytic cycle, as illustrated in Fig. 1) – appears to be a new and challenging area in antibiotic treatment.
Bacterial specificity of lysins The general background supporting the use of phage lysins for prophylactic purposes is based on the observation that because lytic enzymes specifically kill the species in which they were synthesized (or a small number of closely related bacteria) these enzymes might represent an effective way to control specific pathogens without disturbing the normal microbiota (see Glossary). Lysins have been successfully used to prevent and eliminate the upper respiratory colonization of mice by groupA streptococci [16], to kill pneumococci from nasopharyngeal carrier mice [17], and to rapid detect and kill both vegetative cells and germinating spores of Bacillus anthracis [18]. PlyG lysin, which is encoded by g phage of B. anthracis, specifically kills this bacterium and other members of this cluster of bacilli. Germinating spores turned out to be also susceptible. The specificity of the lytic enzyme also provided a rapid method for the identification of this potential biological weapon. In S. pneumoniae, Fischetti [17] used Pal, the amidase coded by Dp-1 (Figs. 1 and 2), which was very effective in eliminating several serotypes of pneumococci demonstrating that the capsule (see Glossary) did not interfere with the access of the enzyme to the cell wall. It is important to emphasize that, in this case, the rise of resistant strains appears a rare event because Pal is strictly dependent on the presence of cholinecontaining teichoic acids of S. pneumoniae for activity, and this aminoalcohol is necessary for pneumococcal viability in vivo. Hence, it is probable that, over evolution, the ChBD of these lytic enzymes evolved to target a unique and essential molecule in the wall. The specificity of this approach was validated by the observation that only Streptococcus oralis and Streptococcus mitis cultures (which also contain choline in their cell walls) showed lower viability in these assays [17]. It has been proposed that, in the case of streptococci, the eradication of these bacteria from the upper respiratory mucosal epithelium might reduce the associated disease.
Curing bacteremia More recently, we have expanded such experimental approach using a murine sepsis model to study the ability of pneumococcal phage amidases and/or lysozymes to cure 472
www.drugdiscoverytoday.com
Figure 4. Synergistic therapeutic effect of the combined use of Pal and Cpl-1. (a) One hour after IP injection of mice with 5 107 colonyforming units (cfu) (at zero time), 2.5 mg each of Pal and Cpl-1 (red circles), or 5 mg of either Pal (green circle) or Cpl-1 (blue squares) were injected into the mouse peritoneal cavity and survival of mice was followed. (b) The effect of the combined therapy (2.5 mg each of Pal and Cpl-1 were administered intraperitoneally 1 h after challenge) in bacteremia of four mice was estimated as cfu per ml at different times after injection of the lytic enzymes. Each curve indicates the bacteremic counts of a single mouse. Reproduced, with permission, from Ref. [19].
bacteremia produced by an antibiotic-multiresistant 6B strain, the most common pneumococcal serotype isolated from children with bacteremia [19]. The ability of Cpl-1 lysozyme and/or Pal amidase to cure bacteremic animals is a protein-specific function, as indicated by the observation that rescue did not occur with heat-inactivated enzymes. Irrespective of the enzymatic activity of the enzyme used, our results revealed that a single IP injection of the corresponding lysin rescued mice from death due to S. pneumoniae even when bacteremia was already established. We observed that effective protection was achieved in mice when a single dose of at least 10 mg of Cpl-1 (or Pal) was administered 1 h after the S. pneumoniae strain was inoculated. The protective
Vol. 1, No. 4 2004
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Table 1. Current concerns on phage lysin therapy Pros
Cons
Latest developments (including drug therapies in progress and failures)
Who is working on this strategy (include web address) (group/institute/company)
Refs.
Target alteration
Create new active lysins
Resistance
Natural or engineered chimeric enzymes
Fischetti/Rockefeller Univ. ([email protected]): Lo´ pez/Centro Inv. Biol./CSIC ([email protected])
[17,19,24–27]
Shock reactions
Nonea
Possible death
Not needed
Fischetti and Lo´ pez groups
[20,28]
Antibody neutralization
a
New enzymes required
Not needed
Fischetti and Lo´ pez groups
[19,20,28]
None
Disease prevention Avoid infection Increased resistance Enzyme encapsulation in micelles Fischetti/Rockefeller Univ. ([email protected]) [28] a
Means that this response has not been found.
effect of both enzybiotics was notable when the enzymes were injected 2 h after pneumococcal challenge, although the extent of this effect was related to the pre-treatment outcome. Moreover, a complete pneumococcal clearance was observed in most surviving mice after a six to eightday follow-up period. An IP injection of either lytic enzyme, delayed up to 4 h after challenge, had a partial protective effect by prolonging the life of the severely ill mice for several days [19]. Provided that mice are most susceptible to pneumococcal infections, the results obtained in these experiments are encouraging because our findings, and those of others [20], support the development of modified procedures in a near future to make use of lysins more efficient. Most noticeably, the simultaneous use of both enzymes, rather than being competitive, enhanced the destruction of the cell wall and, hence, shows a synergistic lytic action on S. pneumoniae in the murine sepsis model used. The positive interaction of the two muralytic enzymes, with only 2.5 mg each of Cpl-1 and Pal, could be because of the increased access of these enzymes, with different catalytic domains, to their cleavage sites (Fig. 2a) as recently suggested [20,21]. Survival rates of animals that received both enzymes were significantly higher than those of animals receiving only Cpl-1 or Pal (Fig. 4). These experiments extend previous observations on the bacterial carrier state to the sepsis model and also reveal the lack of toxicity of the pneumococcal enzybiotics in mice. The combined use of two lytic enzymes would make more unlikely the intrinsic resistance of the bacteria to these proteins that target essential cell-wall molecules [19].
Future prospects: strategy comparison and recombinant lysins In contrast to our observations, Loeffler et al. [21] found that intravenous injections (IV) of Cpl-1 in a mouse model of pneumococcal bacteremia resulted in longer survival of infected mice, but failed to block final killing. The use of IV route rather than the IP one for mice infection and rescue by Cpl-1 and the presence of an endotoxin in the lysozyme preparation employed [20] might account for the uneven
results reported by these two groups for rescue of pneumococcal bacteremia in mice [19,21]. We have demonstrated that the modular design exhibited by the lytic enzymes of pneumococcus and its phages facilitates the construction of chimeric enzymes. As illustrated in Fig. 3, bacterial specificities and activities can be combined by enzymatic swapping [22,23]. Successful construction of chimeric proteins (see Glossary) exhibiting two lytic activities [24] or intergeneric active chimera between bacterial species [25] have also been documented by our laboratory. This experimental approach anticipates the use of genetically engineered enzymes that combine an optimal enzymatic activity with the capacity to recognize different and specific receptors in bacteria. A novel strategy that might extend lysin therapy to a broader range of bacteria by taking advantage of the versatility provided by the modular organization of lytic enzymes is illustrated in the model depicted in Fig. 3. A preliminary illustration in this direction has been reported recently [26] (Table 1).
Conclusions It has been recently emphasized that, during the long history of using phages administered by different routes in several countries, there have been almost no reports of serious complications related to their use [2]. As phages are common entities in the environment and regularly consumed in foods, the development of neutralizing antibodies should not be, in theory, a significant obstacle during the initial treatment of acute infections because the kinetics of phage action or lytic enzymes is much faster than production of neutralizing antibodies by the host. Successful antibiotic therapy in infected mice requires an appropriate dosing interval of administration to maintain pharmacological concentrations in serum. Most interestingly, a single IP injection of a lytic enzyme(s) is sufficient for a complete clearance of invading pneumococci from surviving mice. This property, referred to as therapeutic efficacy, provides a rapid and specific activity and makes www.drugdiscoverytoday.com
473
Drug Discovery Today: Therapeutic Strategies | Infectious diseases
Related articles Anonymous. (2003) We haven’t heard much about anthrax since the mail attacks in 2001. Are there any new treatments for it? Mayo Clin. Health Lett. 21, 8 Dixon, B. (2004) New dawn for phage therapy. Lancet Infect. Dis. 4, 186 Joerger, R.D. (2003) Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poult. Sci. 82, 640–647 Rosovitz, M.J. et al. (2002) Virus deals anthrax a killer blow. Nature 418, 825–826 Sone, R. (2002) Stalin’s forgotten cure. Science 298, 728–731 Thiel, K. (2004) Old dogma, new tricks – 21st Century phage therapy. Nat. Biotechnol. 22, 31–36
these proteins very promising candidates for current antimicrobial therapies. The lack of toxicity of phage lysins and the observation that hyperimmune sera do not neutralize these enzymes in the mice model are encouraging and warrant future research, although testing in humans will be required. A better understanding of phage biology, combined with the availability of highly purified phage lytic enzymes and the experience attained in several Eastern European countries, where rigorously controlled trials are currently being carried out [27], should improve our armamentarium to combat infectious diseases that are currently the first cause of death in the world.
Acknowledgements We thank J.A. Hermoso for preparation of Fig. 2c. This work was supported by a grant from the Direccio´ n General de Investigacio´ n Cientı´fica y Te´ cnica (BCM2003-00074).
References 1 Summers, W.C. (1999) Fe´lix D’Herelle and the Origins of Molecular Biology. Yale University Press 2 Sulakvelidze, A. et al. (2001) Bacteriophage therapy. Antimicrob. Agents Chemother. 45, 149–659 3 Wagner, P.L. and Waldor, M.K. (2002) Bacteriophage control of bacterial virulence. Infect. Immun. 70, 3985–3993 4 Summers, W.C. (2001) Bacteriophage therapy. Annu. Rev. Microbiol. 55, 437–451 5 Mushtaq, N. et al. (2004) Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob. Agents Chemother. 48, 1503–1508 6 Levin, B.R. and Bull, J.J. (2004) Population and evolutionary dynamics of phage therapy. Nat. Microbiol. Rev. 2, 166–173
474
www.drugdiscoverytoday.com
Vol. 1, No. 4 2004
7 Liu, J. et al. (2004) Antimicrobial drug discovery through bacteriophage genomics. Nat. Biotechnol. 22, 185–191 8 Hagens, S. and Bla¨si, U. (2003) Genetically modified filamentous phage as bactericidal agents: a pilot study. Lett. Appl. Microbiol. 37, 318–323 9 McDonnell, M. et al. (1975) ‘‘Diplophage’’: a bacteriophage of Diplococcus pneumoniae. Virology 63, 577–582 10 Ronda, C. et al. (1981) Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae. J. Virol. 40, 551–559 11 Garcı´a, P. et al. (2000) Bacteriophages of Streptococcus pneumoniae: a molecular approach. In Streptococcus pneumoniae – Molecular Biology & Mechanisms of Disease (Tomasz, A. ed), pp. 211–222, Mary Ann Liebert, Inc 12 Lo´pez, R. et al. (2004) Cell wall murein hydrolases. In The Pneumococcus (Tuomanen, E.I., Mitchell, T.J., Morrison, D., Spratt, B.G. eds), pp. 75– 88, ASM Press 13 Ferna´ndez-Tornero, C. et al. (2001) A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat. Struct. Biol. 8, 1020–1024 14 Garcı´a, P. et al. (1990) Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Gene 86, 81–88 15 Hermoso, J.A. et al. (2003) Structural basis for selective recognition of the pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 11, 1239–1249 16 Nelson, D. et al. (2001) Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc. Natl. Acad. Sci. USA 98, 4107–4112 17 Loeffler, J.M. et al. (2001) Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294, 2170–2172 18 Schuch, R. et al. (2002) A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418, 884–889 19 Jado, I. et al. (2003) Phage lytic enzymes as therapy of antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J. Antimicrob. Chemother. 52, 967–973 20 Loeffler, J.M. and Fischetti, V.A. (2003) Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillinsensitive and -resistant Streptococcus pneumoniae strains. Antimicrob. Agents Chemother. 47, 375–377 21 Loeffler, J.M. et al. (2003) Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. Infect. Immun. 71, 6199–6204 22 Dı´az, E. et al. (1990) Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc. Natl. Acad. Sci. USA 87, 8125–8129 23 Dı´az, E. et al. (1991) Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits. J. Biol. Chem. 266, 5464–5471 24 Sanz, J.M. et al. (1996) Construction of a multifunctional pneumococcal murein hydrolase by module assembly. Eur. J. Biochem. 235, 601–605 25 Croux, C. et al. (1993) Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal–clostridial cell wall lytic enzyme. Mol. Microbiol. 9, 1019–1025 26 Yoong, P. et al. (2004) Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J. Bacteriol. 186, 4808–4812 27 Carlton, R.M. (1999) Phage therapy: past history and future prospects. Arch. Immunol. Ther. Exp. 47, 267–274 28 Larkin, M. (2004) Vincent Fischetti – following phages for life. The Lancet Infect. Dis. 4, 246–249