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Bacteriophage Types Phage Therapy and Applications

Types Biological Traits and Discovery of Bacteriophage (phage)

Meaning of Bacteriophage (phage): It is a virus that infects bacteria and is also genetic material that imparts biological traits to the host bacteria. The phage must be parasitic within the living bacteria and have strict host specificity, which depends on the molecular structure and complementarity of the phage adsorption organ and the surface receptor of the recipient bacteria. Bacteriophages are the most common and widely distributed group of viruses. Bacteriophages are usually found in places full of bacterial communities, such as soil and animal intestines.

Bacteriophage Definition: A virus kind that infects bacteria and is also genetic material that imparts biological traits to the host bacteria.

Name:    Bacteriophage

English Name:    Phage

Alias:    Bacterial virus

Subject:    Molecular biology / Virology

Discoverer:    F. d'Herelle and F. W. Twort

    What is the Introduction of Bacteriophage?

    Bacteriophage (phage) is a general term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, or spirochaetes. 
    Because they can cause the host bacteria to lyse, they are called phages.
     It was first discovered in Staphylococcus and Shigella early this century. 

    As a type of virus, phages have some of the characteristics of a virus: they are small individuals. They do not have a complete cellular structure. They contain only a single nucleic acid. Can be regarded as a kind of "predator" bacteria. 

    The phage genome contains many genes, but all known phages use bacterial ribosomes, various factors required for protein synthesis, various amino acids, and energy production systems in bacterial cells to achieve their own growth and proliferation. 

    Once out of the host cell, the phage can neither grow nor replicate. Bacteriophage is a kind of virus.
    Its special feature is that it uses bacteria as its host.

    The more known phage is T2 phage with E. coli as its host. Like other viruses, phages are just a mass of genetic material wrapped in a protein shell.

    Most phages also have a "tail" that is used to inject genetic material into the host. Bacteriophages are a ubiquitous organism and are often accompanied by bacteria.
     Phages can usually be found in places full of bacterial communities, such as dirt and the internal organs of animals.
    The most abundant phage in the world is seawater.

    Bacteriophage (Phage) Chart Diagram

    What are the Biological Traits of Bacteriophage?

    Bacteriophages are small in size, and their morphology includes tadpoles, microspheres, and thin rods, which are more common in tadpoles.
     Phages are made up of nucleic acids and proteins. 
    Proteins protect nucleic acids and determine the shape and surface characteristics of phages. 
    There is only one type of nucleic acid, namely DNA or RNA, double- or single-stranded, circular or linear.

    What Kind of Virus is Bacteriophage?

    Protein structure

    Icosahedron without tail structure: This bacteriophage is an icosahedron with an outer surface composed of regularly arranged protein subunits, the capsid, and the nucleic acid is enclosed inside.

    Icosahedron with tail structure: In addition to the head of an icosahedron, this bacteriophage has a tail consisting of a hollow needle-like structure and an outer sheath, and a base consisting of tail filaments and tail needles.

    Linear body: This bacteriophage is linear and has no obvious head structure, but a spiral structure composed of shell particles.

    Most of the phages known so far are icosahedrons with tail structures. This is because regular polyhedra are the simplest structure in polyhedra and the easiest to set up, so viruses prefer to use regular polyhedra. 
    There are only five kinds of regular polyhedrons, which are regular 4, 6, 8, 12, and 20 polyhedrons. Among them, the regular 20 polyhedron is the closest to a spherical shape, that is, under the same volume, it requires less material and saves more.

    Nucleic acid characteristics

    ss RNA: The nucleic acid contained in the phage is single-stranded RNA.

    ds RNA: The nucleic acid contained in the phage is double-stranded RNA.

    ss DNA: The nucleic acid contained in the phage is single-stranded DNA.

    ds DNA: The nucleic acid contained in the phage is double-stranded DNA.
    Phage. Type of Virus Bacteriophage

    What are the Reproduction Characteristics of Bacteriophage?

    1. Toxic phage

    Refers to replication and proliferation in the host bacteria, producing many progeny phages, and eventually lysing the bacteria. 

    The proliferation mode of toxic phage is replication, and its proliferation process undergoes three stages: adsorption and penetration, biosynthesis and mature release.

    The phage nucleic acid entering the bacterial cell is firstly transcribed to produce an early protein, and the progeny nucleic acid is copied, and then the late transcript is generated to produce the phage structural protein. 

    When the progeny phage reaches a certain number, due to the lysis of phage synthase enzymes, the bacterial cells suddenly lyse, and the released phage infects other sensitive bacteria.

    2. Mild phage

    It does not proliferate after infection with host bacteria. Its gene is integrated on the bacterial chromosome, that is, the prophage, which is replicated as the bacterial chromosome is replicated, and is distributed to the progeny bacteria's chromosome as the bacterium divides. 

    Mild phages have a lysogenic cycle and a lysolytic cycle. Occasionally, spontaneously or under the influence of certain physical, chemical or biological factors, the integrated prophage detaches from the host chromosome. Then it enters the lysolytic cycle and causes the bacteria to lyse and produce new maturity Phage.

    Discovery of Bacteriophage

    What is the Discovery History of Bacteriophage?

    Early period: 1915-1940

    In 1915, Frederick W. Twort became director of the Brown Institute in London. In his research, Twart sought to find a variant of the vacciina virus used for smallpox vaccine, which may replicate in living extracellular media. 
    He vaccinated part of the smallpox vaccine in a culture dish containing nutrient agar in an experiment.

    Although the virus failed to replicate, bacterial contaminants grew quickly in the agar plate, Twote continued his cultivation and noticed that some bacterial colonies showed a "water-like appearance" (ie, became more transparent).
    Such colonies can no longer replicate when further cultured (ie, the bacteria are killed). 

    Twetter calls this phenomenon glassy transformation. He went on to show that infection with a normal bacterial colony using the principle of transparent transformation would kill the bacterium.
    This transparent body can easily pass through a ceramic filter, which can be diluted one million times, and it will restore its strength, or titer, when placed on fresh bacteria.

    Twott published a short article describing the phenomenon, explaining that what he observed was explained by the presence of a bacterial virus. As a result of serving in World War I, Twort's research was interrupted. 
    After returning to London, he did not continue the research and therefore made no further contributions in this area. 

    At the same time, Canadian medical bacteriologist Felix d’ Herelle was working at the Pasteur Institute in Paris. 
    In August 1915, a French cavalry squadron was stationed in Maisons-Lafitte outside Paris.

    A severe Shigella-induced dysentery devastated the entire army. Deherel filtered the patient's stool and quickly isolated and cultured the dysenteriae from the filtered emulsion. Bacteria continue to grow and cover the surface of the petri dish.

    De Herreel occasionally observed clear dots, with no bacteria growing on them. He called these things taches vierges, or plaques. De Herel followed the entire infection process of a patient to see when the bacteria were the most and when the spots appeared. Interestingly, the patient's condition started to improve on the fourth day after infection.

    Deherel called these viruses bateriophage, and he invented methods in the field of virology. He made a limited dilution of the plaques and determined the virus concentration. 
    His reasoning was that spots appeared to indicate that the virus was a particle or called a corpuscular.

    De Herrell also demonstrated in his research that the first step in viral infection is the attachment (adsorption) of host cells to the pathogen. He demonstrated this by co-precipitating the virus with host cells. (He also proved that the virus does not exist in the supernatant.)

    The attachment of a virus only appeared when the bacteria were sensitive to the virus mixed with it, suggesting that a virus has a specific range of adsorption to host cells.
    He also described the release of lysis in very modern terms. De Herrell is one of the founders of modern virological principles in many ways.

    By 1921, more and more lysogenic bacterial strains were isolated, and in some experiments it was no longer possible to separate the virus from its host.
    This led Jules Bordet of the Pasteur Institute in Brussels to believe that the infectious pathogen described by De Herre was nothing more than a bacterial enzyme that promotes self-reproduction.
    Although this is a false conclusion, it is quite close to the view of prion structure and replication.

    In the 1920s and 1930s, De Herrere focused on the medical application of his research results, but to no avail.
    The basic research carried out at that time was often influenced by the explanations produced by the strong personalities of individual scientists in the field.

    Obviously there are many different phages, some are lytic and others are lysogenic, but the relationship between them is still unclear.
    An important discovery of this period was Max Schlesinger, who demonstrated that purified phages had a maximum linear dimension of 0.1 microns and a mass of approximately 4x10 grams.

    They consisted of protein and DNA in roughly equal proportions. No one knew exactly how to take advantage of this observation in 1936, but it had a significant impact in the next 20 years.
    Modern Analysis on Phage or Bacteriophage

    Modern: 1938-1970

    Max Delbruck is a physicist trained at the University of Gittinge. His first job was at the Wilhelm Institute for Chemistry in Berlin, where he actively discussed the relationship between quantum physics and genetics with some researchers. 
    Delbrück's interest in this field led him to invent a guantum mechanical model of gene.

    In 1937, he applied for and received a scholarship to study at the California Institute of Technology.
    As soon as he arrived at the California Institute of Technology, he began collaborating with another researcher, Emory Ellis.
    Ellis was working on a group of phages-T2, T4, T6 (T-even phages). Delbrück quickly realized that these viruses were suitable for studying viral replication.

    These phages are a way to explore how genetic information determines the structure and function of an organism. From the beginning, these viruses were seen as a typical system for understanding cancer viruses, and even how sperm fertilize eggs and develop into a new type of object.

    Eric and Delbruck designed a one-step growth curve test. In this test, an infected bacteria released a large number of phage after a half-hour latent period or eclipse period.
     This test defines the incubation period, when the virus loses infectivity. This became an experimental example for this phage research team.

    After the outbreak of World War II, Delbrück stayed in the United States (at Vanderbilt University) and met Italian refugee Salvador E. Luria. 
    Luria fled to the United States asylum while studying T1 and T2 phages at Columbia University in New York.
    They met at a conference in Philadelphia on December 28, 1940, and planned trials at Columbia University the next two days.

    The two scientists will recruit and lead an increasing number of researchers to focus on the use of bacterial viruses as a model for understanding life processes.
    The key to their success was that in the summer of 1941 they were invited to experiment in Cold Spring Harbor Laboratory. In this way, a German physicist and an Italian geneticist have been collaborating during World War II, traveling around the United States to recruit a new generation of biologists. 
    These people later became known as the phage research team.
    Shortly thereafter, Tom Anderson, an electron microscopy scientist at the RCA Laboratory in Princeton, New Jersey, met Delbruck.

    By March 1942, they had for the first time a clear picture of the phage. Around the same time, these phage variants were isolated and identified for the first time.

    By 1946, the Cold Spring Harbor Laboratory had opened its first phage course, and in March 1947, eight people had attended the first phage conference.

    Molecular biology has developed from these slow beginnings. The focus of this science is on bacterial hosts and their viruses.

    The next 25 years (1950-1975) were fruitful periods of virological research with phage.

    Hundreds of virologists have published thousands of papers, focusing on three areas:

    (a) E. coli lysogenic infection research with T-even phages.
    (b) lysogenic studies using lambda phages, and (c) Study of replication and characteristics of several unique phages, such as ФX174 (single-stranded circular DNA), RNA phage, T7, etc.

    They laid the foundation for modern molecular virology and biology. This article can't introduce all of these scientific literatures one by one, but can only mention some selected points.

    From 1947 to 1948, the use of biochemical methods to study changes in phage-infected cells during the incubation period began to prevail.

    Seymour Cohen first studied lipids and nucleic acids with Columbia University Erwin Chargaff at Columbia University. He later studied tobacco mosaic virus RNA with Wendel Stanley in Cold Spring Harbor in 1946.

    The laboratory majors in phage courses in Delbrück. He used colorimetric analisis to study the effects of DNA and RNA levels in phage-infected cells. 
    These studies show dramatic changes in macromolecular synthesis in phage-infected cells:

    (a) Net accumulation of RNA stops in these cells. [Later, this became the basis for the discovery of multiple RNAs, and for the first time proved the existence of messenger RNA].

    (b) DNA synthesis was stopped for 7 minutes, and then DNA synthesis was resumed at a speed of 5 to 10 times.

    (c) At the same time, research by Monod and Wollman showed that the synthesis of inducible β-galactosidase (galactosidase), a cellular enzyme, after phage infection was shown. These tests divide the incubation period of the virus into two phases, early (before DNA synthesis) and late. 
    Bacteriophage Image Diagram

    More importantly, the results of these studies suggest that the virus may alter the macromolecular synthesis process of infected cells.

    By the end of 1952, two trials had a significant impact on this area. First, Hershey and Chase used labeled viral proteins (SO) and nucleic acids (PO) to track phage attachment to bacteria. 
    They can use a blender to remove the protein capsids of the virus, leaving only the DNA associated with the infected cells. This allowed them to prove that the DNA had all the information needed to reproduce a large number of new viruses.

    Hershey-Chase's experiments and the new DNA structures elaborated a year later by Watson and Crick constitute the cornerstone of the molecular biology revolution.

    The second test in the field of virology was performed by G.R.Wyatt and S.S. Cohen in 1953. When they studied T-even phages, they discovered a new base, 5'hydroxymethylcytosine. 
    This newly discovered base seems to replace cytosine in bacterial DNA. This led scientists to conduct research for up to 10 years on DNA synthesis in bacteria and phage-infected cells. 

    The most critical research shows that the virus introduces genetic information into infected cells. 
    By 1964, research by Mathews et al. demonstrated that 5 & apos hydroxymethylcytosine was absent from uninfected cells and must be encoded by the virus. 

    These experiments proposed early enzymatic concepts in deoxypyrimidine biosynthesis and DNA replication, and provided clear biochemical evidence that could encode a new type of information and express it in infected cells.

    A detailed genetic analysis of these phages identified the genes encoding these phage proteins, and a genetic map was drawn to complete the concept. 
    Bacteriophage or Phage Genetic Analysis

    In fact, genetic analysis of rII and B cistrons of T-even phages has become one of the most well-studied "fine genetic structures".

    Using phage mutants and extracting viral DNA to replicate outside the object has made an important contribution to our contemporary understanding of how DNA replicates itself.

    Finally, through a detailed genetic analysis of phage assembly, the complementarity of in vitro assembly of phage mutants was used to illustrate how organisms build complex structures using the principle of self-assembly.

    Genetic and biochemical analysis of phage lysozyme helps to elucidate the molecular nature of mutations.
    Phage mutations (amber mutations) provide a clear way to study second-site suppressor mutations at the molecular level. 
    The circular arrangement of DNA and the redundant structure at the end (causing phage hybrids) can explain the circular genetic map of T even phage.

    Viral and cellular protein synthesis has undergone significant changes in phage-infected cells. This was achieved in early studies using sodium dodecyl sulfate-polyacrylamide gels. 

    It was found dramatically that the results showed a specific sequence of viral protein synthesis, divided into early proteins and late proteins. This transient basic regulatory mechanism eventually found the sigma factor that regulates RNA polymerase and conferred gene specificity.

    Almost every level of research on gene regulation (transcription, RNA stability, protein synthesis, protein processing) is revealed through raw data from phage infectivity studies.

    Although lysophage research has made such remarkable progress, no one can clearly explain lysogenic phage. This situation changed in 1949, when Andre Lwoff of the Pasteur Institute began to study Bacillus megaterium and its lysogenic phage.

    By using a micromanipulator to segment a single bacterium up to 19 times, no virus was ever released. No virus was found when lysogenic bacteria were lysed from the outside. But often a bacteria spontaneously lyses and releases many viruses. 
    Discovery of Bacteriophage or Phage Virus

    The discovery that ultraviolet light can induce the release of these viruses is an important observation that outlines the wonderful relationship between a virus and its host.

    By 1954, Jacob and Wollman of the Pasteur Institute reached important research results, that is, a lysogenic strain (Hfr, λ) and non-lysogenic receptors genetic crosses following binding lead to induction of the virus. They call this process zygotic induction.

    In fact, the location of the lysogenic phage, or prophage in the chromosome of its host E. coli can be mapped after genetic hybridization using standard interrupted mating experiments
    This is one of the most critical tests for conceptually understanding a lysogenic virus for the following reasons:

    (a) The virus behaves like a bacterial gene on the chromosome of a bacterium.

    (b) It indicates that the viral genetic material is negative due to One of the results of a test that regulates while remaining still in the virus. The genetic material of the virus is lost when chromosomes are passed from a lysogenic donor bacterium to a non-lysogenic recipient host.

    (c) This helps explain the enzymes recognized by Jacob and Walman as early as 1954 Synthesis and induction of phage production are manifestations of the same phenomenon.

    "These experiments laid the foundation for the nature of the operon model and coordinate gene regulation.

    Although the structure of DNA was described in 1953, and zygote induction was described in 1954, the relationship between bacterial chromosomes and viral chromosomes in the lysogen phenomenon is still called the attachment site, which can only be considered from these perspectives.

    Later, Campbell proposed a model of lambda integration between DNA and bacterial chromosomes based on the fact that the order of phage markers in the integrated state is different from the replication or growth state.

    At this point, the true close relationship between the virus and its host has been recognized. This led to the isolation of negatively regulated genes or repressors of lambda phages, which is a clear understanding of the immune characteristics of lysogens and one of the early examples of how genes can be coordinated.

    Genetic analysis of the lambda phage life cycle is a major academic exploration in the field of microbial genetics. 
    It deserves detailed research by all molecular virology and biologists.

    A lysogenic phage such as Salmonella typhimurium P22 is the first example of general transduction, and lambda phage is the first example of special transduction.

    Viruses may carry cellular genes and transfer such genes from one cell to another, which not only provides a method for accurate genetic mapping, but also a new concept in virology.

    As the genetic factors of bacteria are studied in more detail, it can be clearly seen that the development of lysogenic phages has progressed to episomes, transposons, retrotransposons and insertions.

    Element (insertion element), retrovirus (retrovirus), hepadnovirus (hepadnovirus), virus-like (viroid), virusoid (viroid-like) refers to a type of virus enclosed in plant virus particles, 
    Translator's note and prion research, all of which have blurred the definition and classification of genetic information between viruses and their hosts. 

    Genetic and biochemical concepts derived from phage research make possible further development of virology.

    The experience and lessons of lysogen and lysogenic phage research are often re-learned and modified with the study of animal viruses.

    What are the Bacterial Defense Methods against Phage?

    Bacteria's main defense against phage is to synthesize enzymes that can degrade foreign DNA. 
    These enzymes are called restriction endonucleases, and they cut the viral DNA that phages inject into bacterial cells. 

    Bacteria also contain another defense system, which uses CRISPR sequences to retain genome fragments of viruses they have encountered in the past, allowing them to block virus replication by RNA interference. 
    This genetic system provides bacteria with a mechanism similar to adaptive immunity to fight viral infections.
    Application of Bacteriophage or Phage

    What is the Application of Phage?

    Bacteriophage Application: As an experimental tool for molecular biology research Bacteriophages are important materials or tools in basic biological research and genetic engineering in genetic regulation, replication, transcription and translation. 

    The role of transduction in genetics is to use bacteriophage as a vector to transfer genetic material between two strains of bacteria.

    What is the norm for identification and typing of bacteria?

    Bacteriophages can only infect the corresponding bacteria, and are highly specific and can be used for bacterial identification. At the same time, phages have type specificity, which can be used to type and identify bacteria.
    Bacteriophage can be used to type Salmonella, E. coli and typhoid.

    Phage display technology and phage antibody library

    Phage display technology is a powerful gene expression screening technology, which was first described by American scientist SmithMl in the journal (Science) in 1985. 

    The basic principle of phage display technology is to clone the gene of a foreign protein into the genomic DNA of the phage, thereby expressing a specific foreign protein on the surface of the phage.

     Ellis SEp et al. pointed out that the use of phage display peptide library can screen and determine the antigen of nematode vaccine, which is a new method for vaccine antigen identification. 
    With the gradual increase of diseases caused by epidemic viruses in recent years, antiviral peptides are considered as a very promising method for preventing and treating diseases.

    Castel G et al. pointed out that recombinant peptides specifically expressed by phage display technology can be applied to antiviral research and drug development.

    Based on phage display technology and PCR cloning technology, British scientist Winter et al.

    First published an article in the journal Nature to explain the phage antibody library technology. 
    The technology is to recombine the heavy chain and light chain variable region genes of the antibody and the bacteriophage coat protein gene, and display the antibody fragment Fab or scFv and the bacteriophage coat protein as a fusion protein on the surface of the phage particle. Then quickly and efficiently screen and enrich specific antibodies directed against a certain antigen have fundamentally changed the traditional monoclonal antibody preparation process.

    Krishnaswamy et al. used the phage antibody library technology to screen scFv-C1-positive phage antibodies against Candida albicans HM-1 killer toxin.
    The phage antibodies are 60 times more specific than the monoclonal antibodies.

    Tong Youshang et al. reported the application of phage antibody library technology in the fields of biological parasite detection, virus detection, genetically modified product detection, drug residue detection, etc. 
    He pointed out that this technology has a natural fit advantage and bright prospects in the field of inspection and quarantine.

    The phage antibody library technology will definitely become the main technology for antibody production, which will bring extremely broad prospects for humans in disease diagnosis, tumor research, autoimmune disease research, gene therapy, disease prevention and pathogenesis.

    Detection and control of pathogenic bacteria

    There are many pathogenic bacteria in food and environment. Studies have shown that phage can detect and control the growth of pathogenic and spoilage bacteria in food and environment.

    Bmvko LYu et al. discussed the advantages and disadvantages of phage detection of pathogenic bacteria, and pointed out that the use of phage to detect pathogenic bacteria in food safety and processing and manufacturing has great application prospects.

    Jiang Qin et al. pointed out that the use of bacteriophages can detect Salmonella in food in real time, quickly and accurately, and has important significance in public and food hygiene, animal husbandry and veterinary medicine, and quarantine on shore.

    Xinyan Liu believes that phages can be used not only to detect foodborne pathogens, but also to kill pathogens during raw material collection, disinfect equipment during production or processing, extend food storage periods, and disinfect fresh fruits and vegetables.
    Aplications of Bacteriophage or Phage Therapy

    What are the Application of Phage Therapy in various fields?

    Phage Therapy Applications: Phage growth and reproduction in host cells can cause the lysis of pathogenic bacteria, reduce the density of pathogenic bacteria, thereby reducing or avoiding the chance of pathogenic infection or disease, and achieving the purpose of treating and preventing diseases, that is, phage therapy".
    This therapy has been widely used in veterinary, agricultural and food microbiology fields.

    (1) Application of phage therapy in animal husbandry

    Domestic aquaculture, especially chicken industry, is often plagued by intestinal diarrhea of ​​livestock and poultry, which is mainly caused by pathogenic microorganisms such as E. coli and Salmonella.

    With the emergence of a large number of drug-resistant bacteria, the treatment of bacterial diseases with relevant phages that have the advantages of specificity and difficulty in generating resistance has been valued. 
    Smith and Barrow et al. used phage therapy to reduce the risk of E. coli intestinal disease in lambs, piglets and chicks.  

    (2) Application of phage therapy in aquaculture

    Frequent outbreaks of diseases have caused huge economic losses to the aquaculture industry. Phage therapy for bacterial diseases has a good application prospect in the aquaculture industry.
    In treating bacterial blood group ascites infection caused by bacteria such as Pseudomonas plecoghssicida, Park et al. can effectively remove pathogenic bacteria by feeding food containing phage.  


    (3) Application of phage therapy in the treatment of human diseases

    Phage therapy was first applied in the treatment of human diseases.
    In 1921, Bruynoghe and Maisin were the first to treat staphylococcal skin infections with phage preparations.
    Since then phages have been widely used in the treatment of otolaryngology, stomatology, ophthalmology, dermatology, pediatrics and lung diseases.

    With the advent of antibiotics, phage therapy was gradually neglected. 
    Kutter et al reported that phage therapy has great potential for the treatment or prevention of human diseases, pointing out that the ultimate commercialization of phage therapy through methods such as practice and experimentation is the key to avoiding this therapy being ignored.

    With the widespread existence of antibiotic resistance in bacteria, phages are used in many areas to control the growth and expansion of pathogenic bacteria.

    Phage therapy can avoid intestinal flora imbalance and maintain the body's normal immunity. It is considered to be a safe, effective and promising micro-ecological agent to replace antibiotics.

    Author's Bio

    Doctor Shawna Reason, Virologist
    Dr. Shawna Reason
    Name: Shawna Reason

    Education: MBBS, MD

    Occupation: Medical Doctor / Virologist 

    Specialization: Medical Science, Micro Biology / Virology, Natural Treatment

    Experience: 15 Years as a Medical Practitioner

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