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The New Phage Biology: From Genomics to Applications

from McAuliffe et al (2007) The New Phage Biology: From Genomics to Applications. In: Mc Grath, S. and and van Sinderen, D. (eds) Bacteriophage: Genetics and Molecular Biology Caister Academic Press ISBN: 978-1-904455-14-1
Phage Therapy
Edited by: Jan Borysowski, Ryszard Międzybrodzki and Andrzej Górski
Full and comprehensive coverage of phage therapy with a focus on current research and emerging applications.
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Bacterial viruses (or bacteriophages) are found in all habitats in the world where bacteria proliferate. They are estimated to be the most widely distributed biological entity in the biosphere, with an estimated viral population of greater than 1031 or approximately 10 million per cubic centimeter of any environmental niche where bacteria or archaea reside. Most of these viruses are bacteriophages. The dsDNA tailed phages, or Caudovirales, account for 95% of all the phages reported in the scientific literature, and possibly make up the majority of phages on the planet. However, there are other phages that occur abundantly in the biosphere, phages with different virions, genomes and lifestyles. Over the past three decades, phage research has revealed the abundance of phages in nature, the diversity of their genomes, their impact on evolution of microbial diversity, their control of infectious diseases and their influence in regulating the microbial balance in every ecosystem where this has been explored. These findings have led to a resurgence of interest in phage research. Since their discovery in 1915 and 1917 by Fredrick Twort and Felix d'Herelle respectively, bacteriophages have been studied in many laboratories and used in a variety of practical applications. Indeed, phage research has played a central role in some of the most significant discoveries in biological sciences, from the identification of DNA as the genetic material, to the deciphering of the genetic code, to the development of the science of molecular biology over 50 years ago. Research on phages has continually broken new ground in our understanding of the basic molecular mechanisms of gene action and biological structure. In more recent times, phage genomics is revealing novel biochemical mechanisms for replication, maintenance and expression of the genetic material and is providing new insights into origins of infectious disease and the potential use of phage gene products and even whole phage as therapeutic agents.

As mentioned above, the ubiquity and prevalence of bacteriophages in nature and the diversity of their genomes are just two of the reasons for the renewed interest and excitement in phage research. Studies to date have revealed that phages are incredibly varied in their properties, from host range, genetic content, regulatory mechanisms, and physiological effects. Indeed, most of the genes identified on genome sequencing of cultured phages and metagenomic analysis of uncultured phage communities are unidentifiable, i.e. they show no similarity to anything in the currently available databases. At one level, there is diversity in the types of phages that infect individual or interrelated bacterial species. At another level, there is diversity among genomically related phages that do not share the same bacterial hosts. A classic example is the well-studied group of dsDNA T4 phages. This group of phages contains relatives that infect species such as Aeromonas, Vibrio, Acinetobacter, marine and other bacteria. The genomes of a few T4-like phages have been sequenced and found to share homologies with T4 itself, but to also differ from one another in size, organization of the T4-like genes and content of other putative genes. The evidence suggests that phage families like the T4-related phages have learned to cross bacterial species barriers through possession of dynamic genomes that acquire and lose genetic cassettes. Since phages share a long evolutionary history with their hosts, it is tempting to speculate that the genomes of the dsDNA phages are repositories of the genetic diversity of all microorganisms in nature. In addition to their role as carriers of genetic information, bacteriophages have a strong influence on microbial populations: these populations fluctuate with nutritional inputs and there is a dynamic relationship between phage population sizes and host numbers and physiology.

In addition, phages have an impact on the performance of microbial food webs and biogeochemical cycles, affecting nutrient cycling, system respiration, biodiversity and species distribution, and genetic transfer. In the oceans, phages exert significant control on marine bacterial and phytoplankton communities, with respect to both biological production and species composition, influencing the pathways of matter and energy transfer in these systems. Studies on the evolution of phages and their role in natural ecosystems are on the increase. With the data emerging from these studies, researchers can ask more practical questions, such as how to use phages to combat infectious diseases that are caused by bacteria, how to eradicate phage pests in the food and agricultural sectors and what role they have in the causation of human diseases. Interest in phage and phage gene products as potential therapeutic agents is increasing rapidly and is likely to have profound impact on the pharmaceutical industry and biotechnology in general over the coming years. There is a general sense that the best is yet to come out of phage research.

A historical perspective

Since the discovery of phages by Frederick Twort in 1915 and independently in 1917 by Felix d'Herelle, phages have been key players in scientific research. D'Herelle pioneered two important areas of phage research. He observed at an early stage that phages had the potential to kill bacteria that cause diseases in humans, as well as in agriculturally important plants and animals, and advocated phages as therapeutic agents in the pre-antibiotic era. In 1933, he co-founded an institute for phage research in the Soviet Republic of Georgia, together with George Eliava. This establishment continued to supply phage for therapeutic uses to the entire Soviet Union until its recent breakup. In the West, research on such 'phage therapy' was abandoned when penicillin and other chemical antibiotics were discovered in the 1940s, though there has been some renewed interest in phage therapy in recent years as antibiotic resistance of pathogenic bacteria has become a more prominent threat to public health. D'Herelle's second research program concentrated on the biological nature of the bacteriophage itself, proving the concept that phages are obligate intracellular parasites.

Concurrent with d'Herelle's work, research on the nature of phages continued. The viral nature of the bacteriophage was clearly established, the chemical composition of the virions was confirmed as protein and DNA, and progress was made in understanding the phage life cycle. One of the first scientific applications of the electron microscope was the visualization of bacteriophages and their interaction with bacterial cells. This research demonstrated that specific viruses have characteristic morphologies and led to further studies on the morphogenesis and supramolecular assembly of phages. However, the 'modern' era of bacteriophages in biology is said to have begun with the work of Max Delbrück in the late 1930s when he established the lytic mechanism by which some bacteriophages replicate and studied the genetic changes that occur when phages infect bacteria. Until 1952, scientists did not know which part of the virus, the protein or the DNA, carried the information regarding viral replication. It was then that Al Hershey, working with Martha Chase at the Carnegie Laboratory of Genetics, performed a series of experiments using bacteriophages, proving that DNA is the molecule that transmits genetic information. For these discoveries concerning the structure and replication of viruses, Delbrück, Hershey and Salvadore Luria shared the Nobel Prize for Physiology or Medicine in 1969. From this point forward, it was possible to ask and answer complex biological questions using bacteriophage as a model, such as what is the nature of a gene, how do mutations affect genes, how do mutations arise, how do genes replicate, and how are genes expressed.

Around the early 1970s, the world of biological research began to be transformed by the 'recombinant DNA revolution.' The suite of laboratory techniques that made this revolution possible was developed largely through research on phages. The science of molecular biology has produced some profound changes in bacteriophage research, as in all other areas of biological research. For one thing, the number of researchers working primarily on phages decreased dramatically as it became possible to study the genes of more complex, particularly eukaryotic, organisms with nearly the same ease as had been possible previously with simpler organisms, such as phages and bacteria. At the same time, the number of biological researchers using some form of phage in their research has increased substantially, since many of the tools of modern molecular biological research are phages or phage-derived. Thus, for those scientific problems where phages provide advantageous experimental systems, bacteriophage research is still vigorous and in many cases leading the field.

Phage in medicine and therapeutics

Phage therapy

With the recent development of antibiotic resistance within the microbial population, the need for new antibacterials and alternative strategies to control microbial infections is of increasing urgency. One possible option is the use of bacteriophage as antimicrobial agents. Lytic phage kill bacteria via mechanisms that differ from those of antibiotics, and therefore, can be considered as antibacterials with a 'novel mode of action', a concept desired for all new antibacterial agents. The use of phages to treat bacterial infections in animals and humans is an old idea. In Eastern Europe and the former Soviet Union, phage therapy has been used successfully to treat bacterial dysentery, staphylococcal lung infections, surgical wound infections, among others. Phage therapy was exploited for both diarrheal disease and the treatment of traumatic infections during and after World War II. During the 1920s and 1930s, therapeutic phage applications spread rapidly in response to a desperate need for treatment of bacterial infections in Western Europe and the USA. Orally administered phage preparations were reported to effectively treat patients infected with dysentery. Patients suffering from staphylococcal septicemia were also successfully treated by intravenous administration of anti-staphylococcal phages. Phages were reported to reduce the severity of staphylococcal meningitis and eliminate S. aureus from the cerebrospinal fluid. However, with the development of antibiotics for the treatment of infections in the early 1940s and their concomitant widespread use, early clinical trials were abandoned in the West. There is a current renewed interest in bacteriophage therapy in Western Europe and the USA in light of the emergence of drug-resistant pathogenic bacteria and there are strong indications that phages may yet have an important role to play in the treatment of bacterial infection in western countries.

Phage lysins as antimicrobials

A number of recent studies have shown the enormous potential of the use of phage endolysins, rather than the intact phage, as potential therapeutics. Phage endolysins, or lysins, are enzymes that damage the cell walls' integrity by hydrolyzing the four major bonds in its peptidoglycan component. The majority of phage lysins studied to date are modular in structure, composed of at least two distinctly separate functional domains: a C-terminal cell-wall binding domain, which directs the enzyme to its target, and an N-terminal catalytic domain. The catalytic domain can comprise one or more of the following types of peptidoglycan hydrolases: endopeptidases, muramidases (lysozyme), N-acetylmuramoyl- L-alanine amidases and glucosamidases. Most of the lysins studied to date are amidases.

Phage display

Phage display technology is a particularly powerful molecular tool that has had a major impact on drug discovery, pharmacology, immunology and plant science. It is a technique by which foreign peptides, proteins or antibody fragments are expressed at the surface of phage particles. The heterologous peptide or protein is cloned into a phage or phagemid genome as a transcriptional fusion with one of the coat protein genes. These phages then become vehicles for expression that not only carry within them the nucleotide sequence encoding the expressed proteins, allowing the gene sequence to be retrieved, but also have the capacity to replicate.

Vaccines

A novel and exciting use of phages is the use of whole phage particles to deliver vaccines in the form of immunogenic peptides attached to modified phage coat proteins, or as delivery vehicles for DNA vaccines. Phage display is useful for the identification of immunogenic epitopes or mimotopes on displayed peptides which could, in turn, become the basis of peptide vaccines. A study carried out comparing the humoral immune response of animals immunized with a recombinant hepatitis B vaccine or with mimotopes generated by phage display demonstrated that the mimotopes could induce a response similar to that induced by the original antigen; in fact, the mimotopes induced the most reproducible and potent response. Bastien et al. investigated whether a recombinant phage displaying a known protective epitope to the human syncytial virus could protect against infectious challenge in mice. The authors reported that complete protection against the corresponding pathogen could be elicited through mucosal delivery of a filamentous phage displaying the vaccine peptide. This study supports the usefulness of phage display of defined epitopes in prophylactic vaccination. Vaccination with phagedisplaying immunogenic peptides has a number of advantages over the use of recombinant peptides, such as the stimulation of both the cellular and humoral arms of the immune system.

Detection of pathogens

The specific interaction of a bacteriophage and its host lends itself to using phages for the detection of bacteria, in particular, pathogenic bacteria. Unlike other detection systems such as ELISA and PCR, detection with phage is a natural system whereby the phages specifically recognize and bind to their host cells.

Further reading: Bacteriophage: Genetics and Molecular Biology (2007)