Bacteriophage lysins as effective antibacterials

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Lysins are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.

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Background

Bacteriophages or phages are viruses that specifically infect bacteria. After replication inside its bacterial host the phage is faced with a problem, it 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 lytic enzymes or lysins are highly efficient molecules that have been refined over millions of years of evolution for this very purpose. These

Structure

Lysins from DNA-phage that infect Gram-positive bacteria are generally between 25 and 40 kDa in size except the PlyC for streptococci that is 114 kDa. This enzyme is unique because it is composed of two separate gene products, PlyCA and PlyCB. On the basis of biochemical and biophysical studies, the catalytically active PlyC holoenzyme is composed of eight PlyCB subunits for each PlyCA [16]. A feature of all other Gram-positive phage lysins is their two-domain structure (Figure 1) [17, 18••].

Mode of action

Thin section electron microscopy of lysin-treated bacteria reveals that lysins exert their lethal effects by forming holes in the cell wall through peptidoglycan digestion. The high internal pressure of bacterial cells (roughly 3–5 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 (Figure 2). Catalytically, a single enzyme molecule should be sufficient to

Lysin efficacy

In general, 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 [15, 7••]. 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 essentially no effect on streptococci

Antibiotic and lysin synergy

Several lysins have been identified from pneumococcal bacteriophage that are classified into two groups: amidases and lysozymes. Exposure of pneumococci to either of these enzymes leads to efficient lysis. Both enzymes have very different N-terminal catalytic domains but share a similar C-terminal choline cell binding domain. These enzymes were tested to determine whether their simultaneous use is competitive or synergistic [40].

To accomplish this, three different methods of analyses were used

Effects of antibodies

A concern in the use of lysins is the development of neutralizing antibodies that could reduce the in vivo activity of enzyme during treatment. Unlike antibiotics, which are small molecules that are not generally immunogenic, enzymes are proteins that stimulate an immune response, when delivered mucosally or systemically, which could interfere with the lysin’s activity. To address this, rabbit hyperimmune serum raised against the pneumococcal-specific enzyme Cpl-1 was assayed for its effect on

Animal models of infection

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 [7••], a nasal model for pneumococci [15], and a vaginal model for group B streptococci [44]. In all three cases, when the animals were colonized with their respective bacteria and treated with a single dose of lysin, specific for the colonizing organism, these organisms were

Sepsis, Pneumonia, Endocarditis, and Meningitis

Similar to other proteins delivered intravenously to animals and humans, lysins have a short half-life (c.a. 15–20 min) [8]. However, the action of lysins for bacteria is so rapid that this may be sufficient time to observe a therapeutic effect [8•, 42]. 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 of buffer-treated mice was only 25 h, and only 20% survival at

Anthrax

Because lysins are able to kill pathogenic bacteria rapidly; they may be a valuable tool in controlling biowarfare bacteria. To determine the feasibility of this approach a lytic enzyme was identified from the gamma phage, a lytic phage that is highly specific for Bacillus anthracis [50]. The gamma lysin, termed PlyG, was purified to homogeneity by a two-step chromatography procedure and tested for its lethal action on gamma phage-sensitive bacilli [9]. Three seconds after contact, as little as

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 that 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 (5–20 units) in liquid culture [9, 15].

Secondary bacterial infections

Secondary bacterial infections following upper respiratory viral infections such as influenza, are a major cause of morbidity and mortality [52, 53]. The organisms responsible for most of these complications are S. aureus and S. pneumonia. Furthermore, otitis media due to S. pneumonia is a leading cause of morbidity and health care expenditures worldwide and also increases after an upper respiratory viral infection [54]. By eliminating or reducing the bacterial burden by these organisms, will

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 develop methods to prevent rather than

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

I wish to acknowledge the members of my laboratory, Qi Chang, Mattias Collin, Anu Daniel, Sherry Kan, Jutta Loeffler, Daniel Nelson, Jonathan Schmitz, Raymond Schuch, and Pauline Yoong, who are responsible for much of the phage lysin work and Peter Chahales, Adam Pelzek, Rachel Shively, Mary Windels, and Shiwei Zhu for the excellent technical assistance. I am indebted to my collaborators Philippe Moreillon, John McCullars, Stephen Leib, and Martin Witzenrath for their excellent work with the

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