Bacteriophage

The structure of a typical myovirus bacteriophage

A bacteriophage /ˈbækˈtɪər.i.oʊˌfeɪdʒ/ (informally, phage /ˈfeɪdʒ/) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek: φαγεῖν (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.^[1]

Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×10⁸ virions per milliliter have been found in microbial mats at the surface,^[2] and up to 70% of marine bacteria may be infected by phages.^[3] They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France.^[4] They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy).^[5] Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection.^[6]

Classification

Bacteriophages occur abundantly in the biosphere, with different virions, genomes and lifestyles. Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid.

Nineteen families are currently recognized by the ICTV that infect bacteria and archaea. Of these, only two families have RNA genomes and only five families are enveloped. Of the viral families with DNA genomes, only two have single-stranded genomes. Eight of the viral families with DNA genomes have circular genomes, while nine have linear genomes. Nine families infect bacteria only, nine infect archaea only, and one (Tectiviridae) infects both bacteria and archaea.

History

In 1896, Ernest Hanbury Hankin reported that something in the waters of the Ganges and Yamuna rivers in India had marked antibacterial action against cholera and could pass through a very fine porcelain filter. In 1915, British bacteriologist Frederick Twort, superintendent of the Brown Institution of London, discovered a small agent that infected and killed bacteria. He believed the agent must be one of the following:

1. a stage in the life cycle of the bacteria;
2. an enzyme produced by the bacteria themselves; or
3. a virus that grew on and destroyed the bacteria.

Twort's work was interrupted by the onset of World War I and shortage of funding. Independently, French-Canadian microbiologist Félix d'Hérelle, working at the Pasteur Institute in Paris, announced on 3 September 1917, that he had discovered "an invisible, antagonistic microbe of the dysentery bacillus". For d’Hérelle, there was no question as to the nature of his discovery: "In a flash I had understood: what caused my clear spots was in fact an invisible microbe … a virus parasitic on bacteria."^[7] D'Hérelle called the virus a bacteriophage or bacteria-eater (from the Greek phagein meaning to eat). He also recorded a dramatic account of a man suffering from dysentery who was restored to good health by the bacteriophages.^[8] It was D'Herelle who conducted much research into bacteriophages and introduced the concept of phage therapy.^[9]

In 1969, Max Delbrück, Alfred Hershey and Salvador Luria were awarded the Nobel Prize in Physiology and Medicine for their discoveries of the replication of viruses and their genetic structure.^[10]

Phage therapy

Phages were discovered to be antibacterial agents and were used in the former Soviet Republic of Georgia (pioneered there by Giorgi Eliava with help from the co-discoverer of bacteriophages, Felix d'Herelle) and the United States during the 1920s and 1930s for treating bacterial infections. They had widespread use, including treatment of soldiers in the Red Army. However, they were abandoned for general use in the West for several reasons:

• Medical trials were carried out, but a basic lack of understanding of phages made these invalid.^[11]
• Antibiotics were discovered and marketed widely. They were easier to make, store and to prescribe.
• Former Soviet research continued, but publications were mainly in Russian or Georgian languages, and were unavailable internationally for many years.
• Clinical trials evaluating the antibacterial efficacy of bacteriophage preparations were conducted without proper controls and were methodologically incomplete preventing the formulation of important conclusions.

Their use has continued since the end of the Cold War in Georgia and elsewhere in Central and Eastern Europe. Globalyz Biotech is an international joint venture that commercializes bacteriophage treatment and its various applications across the globe. The company has successfully used bacteriophages in administering Phage therapy to patients suffering from bacterial infections, including: Staphylococcus (including MRSA), Streptococcus, Pseudomonas, Salmonella, skin and soft tissue, gastrointestinal, respiratory, and orthopedic infections. In 1923, the Eliava Institute was opened in Tbilisi, Georgia, to research this new science and put it into practice.

The first regulated randomized, double blind clinical trial was reported in the Journal of Wound Care in June 2009, which evaluated the safety and efficacy of a bacteriophage cocktail to treat infected venous leg ulcers in human patients. The study was approved by the FDA as a Phase I clinical trial. Study results satisfactorily demonstrated safety of therapeutic application of bacteriophages, however it did not show efficacy. The authors explain that the use of certain chemicals that are part of standard wound care (e.g. lactoferrin, silver) may have interfered with bacteriophage viability. Another regulated clinical trial in Western Europe (treatment of ear infections caused by Pseudomonas aeruginosa) was reported shortly after in the journal Clinical Otolaryngology in August 2009.[14] The study concludes that bacteriophage preparations were safe and effective for treatment of chronic ear infections in humans. Additionally, there have been numerous animal and other experimental clinical trials evaluating the efficacy of bacteriophages for various diseases, such as infected burns and wounds, and cystic fibrosis associated lung infections, among others. Meanwhile, Western scientists are developing engineered viruses to overcome antibiotic resistance, and engineering the phage genes responsible for coding enzymes which degrade the biofilm matrix, phage structural proteins and also enzymes responsible for lysis of bacterial cell wall.^[2][3][4]

D'Herelle "quickly learned that bacteriophages are found wherever bacteria thrive: in sewers, in rivers that catch waste runoff from pipes, and in the stools of convalescent patients."^[12] This includes rivers traditionally thought to have healing powers, including India's Ganges River.^[13]

Replication

Diagram of the DNA injection process

Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying out both. With lytic phages such as the T4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. Lytic phages are more suitable for phage therapy. Some lytic phages undergo a phenomenon known as lysis inhibition, where completed phage progeny will not immediately lyse out of the cell if extracellular phage concentrations are high. This mechanism is not identical to that of temperate phage going dormant and is usually temporary.

In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo lysogeny are known as temperate phages. Their viral genome will integrate with host DNA and replicate along with it fairly harmlessly, or may even become established as a plasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of nutrients; then, the endogenous phages (known as prophages) become active. At this point they initiate the reproductive cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is reproduced in all of the cell’s offspring. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle is the phage lambda of E. coli.^[14]

Sometimes prophages may provide benefits to the host bacterium while they are dormant by adding new functions to the bacterial genome in a phenomenon called lysogenic conversion. Examples are the conversion of harmless strains of Corynebacterium diphtheriae or Vibrio cholerae by bacteriophages to highly virulent ones, which cause Diphtheria or cholera, respectively.^[15][16] Strategies to combat certain bacterial infections by targeting these toxin-encoding prophages have been proposed.^[17]

Attachment and penetration

In this electron micrograph of bacteriophages attached to a bacterial cell, the viruses are the size and shape of coliphage T1.

To enter a host cell, bacteriophages attach to specific receptors on the surface of bacteria, including lipopolysaccharides, teichoic acids, proteins, or even flagella. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn determines the phage's host range. Host growth conditions also influence the ability of the phage to attach and invade them.^[18] As phage virions do not move independently, they must rely on random encounters with the right receptors when in solution (blood, lymphatic circulation, irrigation, soil water, etc.).

Myovirus bacteriophages use a hypodermic syringe-like motion to inject their genetic material into the cell. After making contact with the appropriate receptor, the tail fibers flex to bring the base plate closer to the surface of the cell; this is known as reversible binding. Once attached completely, irreversible binding is initiated and the tail contracts, possibly with the help of ATP present in the tail,^[3] injecting genetic material through the bacterial membrane. Podoviruses lack an elongated tail sheath similar to that of a myovirus, so they instead use their small, tooth-like tail fibers to enzymatically degrade a portion of the cell membrane before inserting their genetic material.

Synthesis of proteins and nucleic acid

Within minutes, bacterial ribosomes start translating viral mRNA into protein. For RNA-based phages, RNA replicase is synthesized early in the process. Proteins modify the bacterial RNA polymerase so it preferentially transcribes viral mRNA. The host’s normal synthesis of proteins and nucleic acids is disrupted, and it is forced to manufacture viral products instead. These products go on to become part of new virions within the cell, helper proteins that help assemble the new virions, or proteins involved in cell lysis. Walter Fiers (University of Ghent, Belgium) was the first to establish the complete nucleotide sequence of a gene (1972) and of the viral genome of bacteriophage MS2 (1976).^[19]

Virion assembly

In the case of the T4 phage, the construction of new virus particles involves the assistance of helper proteins. The base plates are assembled first, with the tails being built upon them afterwards. The head capsids, constructed separately, will spontaneously assemble with the tails. The DNA is packed efficiently within the heads. The whole process takes about 15 minutes.

Diagram of a typical tailed bacteriophage structure

Release of virions

Phages may be released via cell lysis, by extrusion, or, in a few cases, by budding. Lysis, by tailed phages, is achieved by an enzyme called endolysin, which attacks and breaks down the cell wall peptidoglycan. An altogether different phage type, the filamentous phages, make the host cell continually secrete new virus particles. Released virions are described as free, and, unless defective, are capable of infecting a new bacterium. Budding is associated with certain Mycoplasma phages. In contrast to virion release, phages displaying a lysogenic cycle do not kill the host but, rather, become long-term residents as prophage.

Genome structure

Given the millions of different phages in the environment, phages genomes come in a variety of forms and sizes. RNA phage such as MS2 have the smallest genomes of only a few kilo bases. However, some DNA phages such as T4 may have large genomes with hundreds of genes.

Bacteriophage genomes can be highly mosaic, i.e. the genome of many phage species appear to be composed of numerous individual modules. These modules may be found in other phage species in different arrangements. Mycobacteriophages – bacteriophages with mycobacterial hosts – have provided excellent examples of this mosaicism. In these mycobacteriophages, genetic assortment may be the result of repeated instances of site-specific recombination and illegitimate recombination (the result of phage genome acquisition of bacterial host genetic sequences).^[20] It should be noted, however, that evolutionary mechanisms shaping the genomes of bacterial viruses vary between different families and depend on the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the viral life cycle.^[21]

Systems biology

Phage often have dramatic effects on their hosts. As a consequence, the transcription pattern of the infected bacterium may change considerably. For instance, infection of Pseudomonas aeruginosa by the temperate phage PaP3 changed the expression of 38% (2160/5633) of its host's genes. Many of these effects are probably indirect, hence the challenge becomes to identify the direct interactions among bacteria and phage.^[22]

Several attempts have been made to map protein-protein interactions among phage and their host. For instance, bacteriophage lambda was found to interact with its host E. coli by 31 interactions. However, a large-scale study revealed 62 interactions, most of which were new. Again, the significance of many of these interactions remains unclear but these studies suggest that there are most likely several key interactions and many indirect interactions whose role remains uncharacterized.^[23]

In the environment

Metagenomics has allowed the in-water detection of bacteriophages that was not possible previously.^[24]

Bacteriophages have also been used in hydrological tracing and modelling in river systems, especially where surface water and groundwater interactions occur. The use of phages is preferred to the more conventional dye marker because they are significantly less absorbed when passing through ground waters and they are readily detected at very low concentrations.^[25] Non-polluted water may contain ca. 2×10⁸ bacteriophages per mL.^[26]

Other areas of use

Since 2006, the United States Food and Drug Administration (FDA) and United States Department of Agriculture (USDA) have approved several bacteriophage products. LMP-102 (Intralytix) was approved for treating ready-to-eat (RTE) poultry and meat products. In that same year, the FDA approved LISTEX (developed and produced by Micreos) using bacteriophages on cheese to kill Listeria monocytogenes bacteria, giving them generally recognized as safe (GRAS) status.^[27] In July 2007, the same bacteriophage were approved for use on all food products.^[28] In 2011 USDA confirmed that LISTEX is a clean label processing-aid and is included in USDA.^[29] Research in the field of food safety is continuing to see if lytic phages are a viable option to control other food-borne pathogens in various food products.

In 2011 the FDA cleared the first bacteriophage-based product for in vitro diagnostic use.^[30] The KeyPath MRSA/MSSA Blood Culture Test uses a cocktail of bacteriophage to detect Staphylococcus aureus in positive blood cultures and determine methicillin resistance or susceptibility. The test returns results in about 5 hours, compared to 2–3 days for standard microbial identification and susceptibility test methods. It was the first accelerated antibiotic susceptibility test approved by the FDA.^[31]

Government agencies in the West have for several years been looking to Georgia and the former Soviet Union for help with exploiting phages for counteracting bioweapons and toxins, such as anthrax and botulism.^[32] Developments are continuing among research groups in the US. Other uses include spray application in horticulture for protecting plants and vegetable produce from decay and the spread of bacterial disease. Other applications for bacteriophages are as biocides for environmental surfaces, e.g., in hospitals, and as preventative treatments for catheters and medical devices prior to use in clinical settings. The technology for phages to be applied to dry surfaces, e.g., uniforms, curtains, or even sutures for surgery now exists. Clinical trials reported in the Lancet^[33] show success in veterinary treatment of pet dogs with otitis.

Phage display is a different use of phages involving a library of phages with a variable peptide linked to a surface protein. Each phage's genome encodes the variant of the protein displayed on its surface (hence the name), providing a link between the peptide variant and its encoding gene. Variant phages from the library can be selected through their binding affinity to an immobilized molecule (e.g., botulism toxin) to neutralize it. The bound, selected phages can be multiplied by reinfecting a susceptible bacterial strain, thus allowing them to retrieve the peptides encoded in them for further study.

The SEPTIC bacterium sensing and identification method uses the ion emission and its dynamics during phage infection and offers high specificity and speed for detection.

Phage-ligand technology makes use of proteins, which are identified from bacteriophages, characterized and recombinantly expressed for various applications such as binding of bacteria and bacterial components (e.g. endotoxin) and lysis of bacteria.^[34]

Bacteriophages are also important model organisms for studying principles of evolution and ecology.^[35]

Model bacteriophages

The following bacteriophages are extensively studied:

Cultural references

• In 1925 in the Pulitzer Prize-winning novel Arrowsmith, Sinclair Lewis fictionalized the discovery and application of bacteriophages as a therapeutic agent.
• The 1999 Greg Bear novel Darwin's Radio deals with an epidemic in the form of long-dormant sections of human DNA, introduced in prehistoric times by lysogenic bacteriophages, which begin to express themselves. The sequel, Darwin's Children, takes place in the post-epidemic world.

See also

• Bacterivore
• CrAssphage
• DNA viruses
• Phage ecology
• Phage monographs (a comprehensive listing of phage and phage-associated monographs, 1921 – present)
• Polyphage
• RNA viruses
• Transduction
• Viriome
• CRISPR

References

External links

• Animation of bacteriophage targeting E. coli bacteria
• Häusler, T. (2006) "Viruses vs. Superbugs", Macmillan
• Phage.org general information on bacteriophages
• bacteriophages illustrations and genomics
• Bacteriophages get a foothold on their prey
• NPR Science Friday podcast, "Using 'Phage' Viruses to Help Fight Infection", April 2008
• Animation by Hybrid Animation Medical for a T4 Bacteriophage targeting E. coli bacteria.
• Bacteriophages: What are they. Presentation by Professor Graham Hatfull, University of Pittsburgh on YouTube