Mini ReviewBacteriophage endolysins: A novel anti-infective to control Gram-positive pathogens
Introduction
After replication inside its bacterial host, a bacteriophage (or phage) is faced with a problem, needs to exit the bacterium to disseminate its progeny phage. To solve this, double-stranded DNA phages have evolved a lytic system to weaken the bacterial cell wall resulting in bacterial lysis. Phage endolysins or lysins are highly efficient molecules that have been refined over millions of years for this very purpose. These enzymes target the integrity of the cell wall and are designed to attack one of the 4 major bonds in the peptidoglycan. With few exceptions (Loessner et al., 1997) lysins do not have signal sequences, so they are not translocated through the cytoplasmic membrane to attack their substrate in the peptidoglycan. This movement is controlled by a second phage gene product in the lytic system, the holin (Wang et al., 2000). During phage development in the infected bacterium, lysin accumulates in the cytoplasm in anticipation of phage maturation. At a genetically specified time, holin molecules are inserted in the cytoplasmic membrane-forming patches, ultimately resulting in generalized membrane disruption (Wang et al., 2003), allowing the cytoplasmic lysin to access the peptidoglycan, thereby causing cell lysis and the release of progeny phage (Wang et al., 2000). Compared to large DNA phage, small RNA and DNA phages use a different release strategy. They call upon a phage-encoded proteins to interfere with bacterial host enzymes responsible for peptidoglycan biosynthesis (Young et al., 2000, Bernhardt et al., 2001) resulting in misassembled cell walls and ultimate lysis. Scientists have been aware of the lytic activity of phage for nearly a century, and while whole phage have been used to control infection (Matsuzaki et al., 2005), not until recently have lytic enzymes been exploited for bacterial control in vivo (Loeffler et al., 2003, Nelson et al., 2001, Schuch et al., 2002). One of the main reasons that such an approach is now even being considered is the sharp increase in antibiotic resistance among pathogenic bacteria. Current data indicate that lysins work only with Gram-positive bacteria, since they are able to make direct contact with the cell wall carbohydrates and peptidoglycan when added externally, whereas the outer membrane of Gram-negative bacteria prevents this interaction. This review will outline the remarkable potency these enzymes have in killing bacteria both in vitro and in vivo.
Most human infections (either viral or bacterial) begin at a mucous membrane site (upper and lower respiratory, intestinal, urogenital, and ocular). In addition, the human mucous membranes are the reservoir (and sometimes the only reservoir) for many pathogenic bacteria found in the environment (i.e., pneumococci, staphylococci, streptococci) some of which are resistant to antibiotics. In most instances, it is this mucosal reservoir that is the focus of infection in the population (Coello et al., 1994, de Lencastre et al., 1999, Eiff et al., 2001). Currently, except for polysporin and mupirocin ointments, which are the most widely used topically, there are no anti-infectives that are approved to control colonizing pathogenic bacteria on mucous membranes (Hudson, 1994); we usually first wait for infection to occur before treating. Because of the fear of increasing the resistance problem, antibiotics are not indicated to control the carrier state of disease bacteria. It is agreed, however, that by reducing or eliminating this human reservoir of pathogens in the community and controlled environments (i.e., hospitals and nursing homes), the incidence of disease will be markedly reduced (Eiff et al., 2001, Hudson, 1994). Towards this goal, lysins may be used to prevent infection by safely and specifically destroying disease bacteria on mucous membranes. For example, based on extensive animal results, enzymes specific for Streptococcus pneumoniae and S. pyogenes, may be used nasally and orally to control these organisms in the community as well as in nursing homes and hospitals to prevent or markedly reduce serious infections caused by these bacteria. This has been accomplished by capitalizing on the efficiency by which phage lysins kill bacteria (Young, 1992). Like antibiotics, which are used by bacteria to control the organisms around them in the environment, phage lysins are the culmination of millions of years of development by the bacteriophage in their association with bacteria. Specific lysins have now been identified and purified that are able to kill specific Gram-positive bacteria seconds after contact (Loeffler et al., 2001, Nelson et al., 2001). For example, nanogram quantities of lysin could reduce 107 S. pyogenes by >6 logs seconds after enzyme addition. No known biological compounds, except chemical agents, kill bacteria that quickly.
Section snippets
Mechanism of action
Lysin-treated bacteria examined by thin section electron microscopy revealed that lysins exert their lethal effects by forming holes in the cell wall through peptidoglycan digestion. The high internal pressure of bacterial cells (roughly 15–25 atmospheres) is controlled by the highly cross-linked cell wall. Any disruption in the wall's integrity will result in the extrusion of the cytoplasmic membrane and ultimate hypotonic lysis. Catalytically, a single enzyme molecule should be sufficient to
Endolysin structure
Lysins from DNA phage that infect Gram-positive bacteria are generally between 25 and 40 kDa in size except the PlyC LYSIN for streptococci which is 114 kDa. This enzyme is unique because it is composed of 2 separate gene products, PlyCA and PlyCB. Based on biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of 8 PlyCB subunits for each PlyCA (Nelson et al., 2006). A feature of all other Gram-positive phage lysins is their two-domain structure (Diaz et al.,
Endolysin efficacy
In most instances, lysins only kill the species (or subspecies) of bacteria from which they were produced. For instance, enzymes produced from streptococcal phage kill certain streptococci, and enzymes produced by pneumococcal phage kill pneumococci (Loeffler et al., 2001, Nelson et al., 2001). Specifically, a lysin from a group C streptococcal phage (PlyC) will kill group C streptococci as well as groups A and E streptococci, the bovine pathogen S. uberis and the horse pathogen, S. equi, but
Synergy
When the pneumococcal lysin Cpl-1 was used in combination with certain antibiotics, a synergistic effect was seen. Cpl-1 and gentamicin were found to be increasingly synergistic in killing pneumococci with a decreasing penicillin MIC, while Cpl-1 and penicillin showed synergy against an extremely penicillin-resistant strain (Djurkovic et al., 2005). Synergy was also observed with a staphylococcal-specific enzyme and glycopeptide antibiotics (Rashel et al., 2007). Thus, the right combination of
Effects of antibodies
The pharmacokinetics of lysins like other foreign proteins delivered systemically to animals is about 20 min (Loeffler et al., 2003). Thus, if lysins are to be used systemically, they will need to be modified to extend their half-life, or they need to be delivered frequently or by IV infusion. An additional concern in the use of lysins is the development of neutralizing antibodies which could reduce the in vivo levels of enzyme during treatment. Unlike antibiotics, which are small molecules that
Animal models
Animal models of mucosal colonization were used to test the capacity of lysins to kill organisms on these surfaces; perhaps the most important use for these enzymes. An oral colonization model was developed for S. pyogenes (Nelson et al., 2001), a nasal model for pneumococci (Loeffler et al., 2001), and a vaginal model for group B streptococci (Cheng et al., 2005). In all 3 cases, when the animals were colonized with their respective bacteria and treated with a single dose of lysin, specific
Sepsis, pneumonia, endocarditis, and meningitis
Similar to other proteins delivered intravenously to animals and humans, lysins have a short half-life (approximately 15–20 min) (Loeffler et al., 2003). However, the action of lysins for bacteria is so rapid, that this may be sufficient time to observe a therapeutic effect (Jado et al., 2003, Loeffler et al., 2003). Mice intravenously infected with type 14 S. pneumoniae and treated 1 h later with a single bolus of 2.0 mg of Cpl-1 survived through the 48 h endpoint, whereas the median survival time
Secondary infections
Secondary bacterial infections following upper respiratory viral infections such as influenza, are a major cause of morbidity and mortality (Brundage and Shanks, 2007, Brundage and Shanks, 2008). The organisms responsible for most of these complications are S. aureus and S. pneumoniae. Furthermore, otitis media due to S. pneumoniae is a leading cause of morbidity and health care expenditures worldwide and also increases after an upper respiratory viral infection (McCullers, 2006). By
Bacterial resistance to lysins
Exposure of bacteria grown on agar plates to low concentrations of lysin did not lead to the recovery of resistant strains even after over 40 cycles. Organisms in colonies isolated at the periphery of a clear lytic zone created by a 10-μl drop of dilute lysin on a lawn of bacteria always resulted in enzyme-sensitive bacteria. Enzyme-resistant bacteria could also not be identified after >10 cycles of bacterial exposure to low concentrations of lysin (from 5 to 20 units) in liquid culture (
Conclusion
Lysins are a new reagent to control bacterial pathogens, particularly those found on the human mucosal surface. For the first time, we may be able to specifically kill pathogens on mucous membranes without affecting the surrounding normal flora thus reducing a significant pathogen reservoir in the population. Since this capability has not been previously available, its acceptance may not be immediate. Nevertheless, like vaccines, we should be striving to developing methods to prevent rather
Acknowledgments
I wish to acknowledge the members of my laboratory who are responsible for much of the phage lysin work, Qi Chang, Mattias Collin, Anu Daniel, Sherry Kan, Jutta Loeffler, Daniel Nelson, Jonathan Schmitz, Raymond Schuch, and Pauline Yoong, and the excellent technical assistance of Peter Chahales, Adam Pelzek, Rachel Shively, Mary Windels, and Shiwei Zhu. I am indebted to my collaborators Philippe Moreillon, Stephen Leib, Jon McCullars, and Martin Witzenrath, and members of their laboratory for
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