Phage Therapy

Phage Therapy

What comes to mind when you hear the word “bacteria”? Most people, if not all, will answer “disease,” “sickness,” or “bad for the health.” What not all people know is there are actually both good and bad bacteria and some bacterial species are probiotic – bacteria that are helpful to its host. In fact, bacterial infections can be treated with bacteriophages: viruses that have the ability to infect and fight harmful bacteria, culminating in their destruction. Bacteriophage or phage therapy is therefore very useful in various fields like medicine, veterinary science, dentistry, and even agriculture.

History of Phage Therapy

Bacteriophages were discovered by two people: the English bacteriologist Frederick Twort in 1915 and the French-Canadian microbiologist Felix d’Herelle in 1917. Immediately after their discovery, the thought of using phages to fight bacterial infections was already apparent. D’Herelle began testing the therapeutic effects that phages may have on chickens and cows first and the tests were successful. Eventually, human tests were conducted and the development of phage therapy became more extensive especially with the foundation of the Eliavia Institute in 1923; the pharmaceutical company Eli Lilly began the commercialization of phage therapy in the US during the 1940s. During the Second World War, phages were used to treat bacterial diseases among soldiers of the Soviet Union, particularly gangrene and dysentery. The development of antibiotics in the 1950s led to a temporary setback on phage therapy as the use of antibiotics became more favourable. However, there has been a renewed interest in the development and employment of phage therapy in more applications.

Advantages over Antibiotics

Viruses and bacteria evolve over time and can develop a resistance to antibiotics. In theory, this resistance can also apply to phages, but it may be less difficult to overcome compared to antibiotics.

Because phages are target specific, meaning only a one or very few bacterial strains are targeted upon, it is easier to develop new phages than new antibiotics. A time period of only a few days or weeks is needed to acquire new phages for resistant strains of bacteria, whereas it can take years to obtain new antibiotics. When resisting bacteria evolve, the assigned phages also evolve, so when super bacterium appears, an equivalent super phage fights it as long as the phage is derived from the same environment.

Compared to antibiotics, phages go deeper into the infected area. Antibiotics, on the other hand, have concentration properties that quickly decrease as they go below the surface of the infection. The replication of phages is concentrated on the infected area where they are needed the most, while antibiotics are metabolized and removed from the body. In addition, secondary resistance does not happen among phages, but happens quite often among antibiotics. Secondary resistance is acquired and occurs when there aren’t enough blood drug levels.

Certain infections in people and experimentally infected animals have been proven to be more effectively treated with phage therapy than using antibiotics. Since 1966, the average success rate of studies that used phages in various ways (systematically, topically, intravenously, or orally) is from 80 to 95%, with minimal or no allergic and/or gastrointestinal side effects. The infections studied are from E. coli, Acinetobacter, Psuedomonas, and Staphylococcus aureus. Multiple side effects like allergies, intestinal disorders, and yeast infections have been observed when using antibiotics.

Applications

Fighting and destroying bacterial infections (both in humans and animals) are the primary applications of phage therapy, but it can also be employed for other uses. It can be the key to fighting the NDM-1, a gene that can be included in the DNA of bacteria, enabling them to resist antibiotics. Waste water from sewage systems are not really considered waste because it is a rich source of phage strains for various kinds of bacteria that lead to the most up-to-date medicines. Skin grafting for extensive wounds, trauma, burns, and skin cancer can also be improved by using phage therapy to lessen the Psuedomonas aeruginosa infection. Some experiments for cells in tissue culture have also discovered antitumor agents in phages. Bacteria cause food to spoil faster, and phages have been studied for their potential to increase the freshness of food and decrease the incidents of food spoilage.

Phage therapy has many other potential benefits and giving it ample support can pave the way to a healthier future.

Phage Display

Phage Display Cycle

One of the laboratory techniques employed in studying different protein interactions is Phage Display. With this in vitro screening method, protein ligands and macromolecules can be easily identified and interactions between protein and protein, peptide and protein, & DNA and protein can be studied further.

History of Phage Display

The first described instance of Phage Display occurred in 1985, when George P. Smith fused a peptide with a gene III from a filamentous phage. He filed a patent detailing the process of generating phage display libraries in the same year. Eventually, further development of Phage Display technology led by different groups from the MRC Laboratory of Molecular Biology, as well as from The Scripps Research Institute, led to the possibility of displaying proteins for the purpose of therapeutic protein engineering. The technique has been continuously improved to screen and amplify huge collections of proteins showing the connection of phenotype and genotype better.

Structure

A filamentous phage has a diameter of around 6.5 nanometers, with a length that depends on the size of its genome. It comes from a huge family of bacterial viruses that also infect other forms of bacteria. It contains a small genome with an intergenic region containing the necessary sequences for the replication and encapsidation of DNA.

A phage particle consists of five coat proteins. The particle has a hollow tube that houses so many copies of the primary coat protein. There are also binding interactions between the adjacent subunits’ hydrophobic midsections. One end of the particle is blunt, and the other is sharp. The blunt end contains plenty of copies of the two tiniest ribosomally translated proteins, while the sharp end contains around only 5 copies of the pIII and pVI genes, which are necessary for the detachment of the phage from the cell membrane.

How it works

Phage Display is a method wherein a library of phage particles that express very diverse peptides is generated. The objective is to choose those that will bind a desired target; the target can be a protein, a peptide, or a piece of DNA.

The most often used vector to build a random peptide display is the filamentous phage M13. In this display, the DNA which encodes the peptide or protein of interest is integrated into the pIII or pVI gene. To make sure that the fragments are completely inserted into the three possible reading frames, multiple cloning sites are sometimes employed, allowing the proper translation of the cDNA in its correct frame. The DNA hybrid and the phage gene are then put inside E. coli bacterial cells. Examples of these bacterial cells include XL1-Blue E. coli, SS320, TG1, and ER2738. The peptide or protein of interest is eventually expressed in either the minor or major coat protein

If another kind of vector is used, for example, a phagemid vector or simplified display construct vector, a helper phage must infect the E. coli cells; otherwise, the phage particles will not be separated from the E. coli cells. A helper phage activates the packaging of the phage DNA and assembles the mature virions with their corresponding protein fragments, which are included in the outer coating of the minor or major protein coat.

The generated phage library is then screened by addition into a microtiter plate containing immobilised target proteins or DNA sequences. Phages displaying a protein that bind to one target will remain, while the other phages can be discarded through washing. The remaining phage particles can be used to multiply the phage by infecting them into bacteria, thus increasing the diversity of the peptide display library.

Applications

The fast isolation of particular ligands through phage display has a wide variety of applications like epitope mapping, analyzing different protein interactions, vaccine development, drug design, and therapeutic target validation. Phage display is also used to pick inhibitors for the active and allosteric sites of G-protein binding modulatory peptides, enzymes, and receptor antagonists and agonists.

Determining the proper protein partners can be useful to identify the functions of various proteins. For drug discovery and design, Phage Display is employed in protein engineering or in vitro protein evolution. Therapeutic targeting with phage display is also primarily used to diagnose and determine tumour antigens, which is useful for cancer research.

Antibody Phage Display significantly improved the discovery and development of antibody drugs. Phage display for antibody libraries paved the way for rapid vaccine design and therapy. These libraries are used to learn more about the human immune system and to create human antibodies in vitro with the use of diverse synthetic substances.

Phage Display can be used in conjunction with other techniques, and with enough support and studies, more applications for it can be discovered.

Temperate Phages

Temperate Phage

A bacteriophage is a kind of virus that can infect and replicate itself inside bacterial cells. The virus has a protein-encapsulated DNA or RNA genome and can have simple or complex anatomies. There are many types of bacteriophages including M13, T phage, lambda phage, MS2, G4, and Phix174.

One of the characteristics of bacteriophages is their temperateness. Temperateness refers to the ability of some bacteriophages, particularly lambda phage, to choose between two cycles: lysogenic or lytic. “Temperance” generally refers to the moderation of actions, and in the case of phages, moderation is seen through the ability to not express anti-bacterial virulence.

Viral reproduction

Phage Reproduction Cycle

Viruses cannot multiply through the division of cells because they are acellular (they do not have cells). Instead, they seek a host cell in which they replicate and assemble themselves using the metabolism and machinery of the host cell. Different species of viral populations undergo different viral life cycles, but for temperate phages, as previously mentioned, they must pick between two.

The lytic-lysogeny decision

Decision making isn’t just done by people; it is also done by temperate phages as they need to choose between two different life cycles, productive (lytic) or reductive (lysogenic). There is a predominance of lytic among temperate phages, as induction can cause lysogenic to convert to lytic.

However, in most cases, temperate phages reel toward the lysogenic cycle especially when phage absorption in the infected bacteria is apparent. It is inferred that other local bacteria are undergoing the same phage infection, making the bacteria decrease in density. Because of this “crisis,” the go-to cycle is lysogenic.

On the other hand, when there is an abundance of uninfected bacteria, undergoing the lytic cycle is preferable because to increase the number of healthy bacteria, phages that have productive infections are needed.

Lysogenic cycle

In the lysogenic cycle, the genomes of temperate phages are not expressed. However, they are integrated into the genome of the bacteria and produce prophages, which are created without disrupting the bacterial cell. Moreover, because of this integration, passive replication of the bacteriophage occurs when daughter bacterial cells are produced. These prophage-containing bacteria cells are called lysogens – phages that can exist as dormant DNA within its host cell. These lysogens have the ability to stay in the lysogenic cycle for a very long time, but through induction, they can be directed to the lytic cycle at any point in time. When induction occurs, prophage DNA is cut off from the bacterial genome and coat proteins are produced via transcription and translation of the prophage DNA for the regulation of lytic growth.

Lytic cycle

The lytic cycle is similar to the lysogenic cycle in that the host DNA machinery is used to replicate the phage, but the phage is considered a separate molecule from the host DNA. When a temperate phage undergoes the lytic cycle, it becomes a virulent phage.  The infected cell and its membrane disintegrate as the viral DNA, which is considered a separate molecule from the bacterial cell, replicates separately from the DNA of the host bacteria, eventually overwhelming it.

The lytic cycle is divided into different stages. The first stage is the penetration in which the virus enters the host cell and injects its nucleic acids into it, releasing genetic material (either DNA or RNA) and infecting the cell. Viral components are then produced using the machinery of the host cell, culminating in the biosynthesis of mRNA and protein production. The host cell begins to copy the viral nucleic acids, which combine with viral proteins to produce phage particles within the cell. When there are already too many viral particles within the host, its membrane splits and the released viruses begin infecting other cells.

Applications

Temperate phages have various biological and molecular applications. They can be used to genetically manipulate eukaryotic cells, especially species that have large genomes like plants and mammals. Gene therapy, manipulation of cell lines, and construction of transgenic organisms can also employ phage enzymes. The temperate phage Mu-1 has a DNA-modifying function, which is of great importance especially in virology. Various food and biotechnology products and chemicals also employ the bacterial fermentation of phages.

In most laboratories, temperate phages are considered more of lytic phages because most lytic-lysogenic decisions result in the former. However, whether phages are lytic or lysogenic, it is apparent that even they are capable of making a decision, particularly for replication.

Bacteriophage

Bacteriophages

Bacteriophages, also known as phages, are viruses that infect bacteria. These phages also require a bacterial host in order to replicate themselves. Bacterial viruses, as these are also often called, are made up of proteins that coat an inner core of nucleic acid – either DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). Phages also vary in structure, ranging from the simple to the more elaborate and complex.

Bacterial viruses are specific to one or a limited number of bacteria; thus, they are named after the bacteria group, strain, or species that they infect. For example, the phages that infect the bacterium Escherichia coli are called coliphages.

History

Before bacteriophages were officially discovered, several bacteriologists in the 1890s had already observed an unidentified substance that seemed to be responsible for limiting bacterial activity. Although they had noted such a phenomenon, none of them further explored these findings. Thus, it wasn’t until 1915 when Frederick Twort, a bacteriologist from England, observed the same phenomenon (the killing of bacteria by an unknown agent) and advanced the hypothesis that it might be a virus. Unfortunately, lack of funding as well as the start of World War I interrupted Twort’s work. Two years later, a French-Canadian microbiologist named Félix d’Hérelle made a similar observation and succeeded in discovering what he called “an invisible, antagonistic microbe of the dysentery bacillus”.  Unlike the previous investigators, d’Hérelle was certain that the unknown substance he had discovered was a virus that was able to parasitize bacteria. He called it a “bacteria-eater” or “bacteriophage”, from the word bacteria and the Greek word “phagein” which means to eat or devour.

Structure

Bacteriophages come in different sizes and shapes but most of them have the same basic features: a head or capsid and a tail. A bacteriophage’s head structure, regardless of its size or shape, is made up of one or more proteins which protectively coats the nucleic acid. Though there are some phages that don’t have a tail, most of them do have one attached to its head structure. It is a hollow tube through which the nucleic acid passes through when the bacteriophage infects a host cell. Some of the more complex phages such as T4 have a tail with a base plate as well as one or more tail fibers that aid the phage in attaching itself to a bacterial cell.

How Bacteriophages Work

In order to infect a host cell, the bacteriophage attaches itself to the bacteria’s cell wall, specifically on a receptor found on the bacteria’s surface. Once it becomes tightly bound to the cell, the bacterial virus injects its genetic material (its nucleic acid) into the host cell. Depending on the type of phage, one of two cycles will occur – the lytic or the lysogenic cycle. During a lytic cycle, the phage will make use of the host cell’s chemical energy as well as its biosynthetic machinery in order to produce phage nucleic acids (phage DNA and phage mRNA) and phage proteins. Once the production phase is finished, the phage nucleic acids and structural proteins are then assembled. After a while, certain proteins produced within the cell will cause the cell wall to lyse, allowing the assembled phages within to be released and to infect other bacterial cells.

Viral reproduction can also occur through the lysogenic cycle. The main difference between the two types of cycles is that during lysogeny, the host cell is not destroyed or does not undergo lysis. Once the host cell is infected, the phage DNA integrates or combines with the bacterial chromosome, creating the prophage. When the bacterium reproduces, the prophage is replicated along with the host chromosomes. Thus, the daughter cells also contain the prophage which carries the potential of producing phages. The lysogenic cycle can continue indefinitely (daughter cells with prophage present within continuing to replicate) unless exposed to adverse conditions which can trigger the termination of the lysogenic state and cause the expression of the phage DNA and the start of the lytic cycle. These adverse conditions include exposure to UV or mutagenic chemicals and desiccation.

Applications

Bacteriophages have several applications. In some countries such as Russia and other Eastern European nations, phages are used therapeutically for the treatment of pathogenic bacterial infections that are resistant to antibiotics.  Also known as phage therapy, this method involves the use of a phage to destroy the infective bacteria such as E. coli or salmonella. Bacteriophage is also used in identifying pathogenic bacteria (also called phage typing) in diagnostic laboratories. One other use for bacteriophages is for killing specific bacteria found in food. For example, LISTEX by Micreos is made up of bacteriophages that can kill the L. monocytogenes bacteria in cheese.

Lambda Phages

Lambda Phage

The lambda phage, also called Enterobacteria phage λ and colphage λ, is a type of temperate bacteriophage or bacterial virus that infects the Escherichia coli (E. coli) species of bacteria. The virus may be housed in the genome of its host via lysogeny.

History of Lambda Phage

In 1950, Esther Lederberg, an American microbiologist, was performing experiments on E. coli mixtures. She happened to observe streaks of mixtures of two types of E. coli strains that seemed to have been nibbled on and had viral plaque. One E. coli strain had been treated with ultraviolet light, so the damage was more apparent. It was later determined that this was caused by bacterial viruses, which replicated and spread resulting in cell destruction. The discovery led to the employment of Lambda phage as a model organism in microbial genetics as well as in molecular genetics.

Structure

A lambda phage has a head measuring around 50-60 nanometers in diameter and a flexible tail that is around 150 nanometers long and may contain tail fibers.

The head consists of various proteins and over a thousand protein molecules including X1, X2, B, B*, E, D, and W. The head functions as a capsid that contains its genome, which contains 48,490 base pairs of double-stranded linear DNA. This number also includes 12-base single-stranded parts at its 5’ ends. The single-stranded parts are known as sticky sites and are also called the cos site, which encircles the DNA in the host cytoplasm. Hence, when in circular form, the phage genome is comprised of 48,502 pairs in length. The weight of the genome is estimated to be 32×106 Da, which is around half of the weight of the phage.

The tail has a 135 nanometer-tube that is hollow and contains a conical cap which is around 15 nanometers. The tail’s inner diameter is around 3 nanometers, while on the outside, it is around 9-18 nanometers depending on the knob-like structures that give the tail a rough appearance.

Life cycle

When E. coli is infected with a lambda phage, two cycles may happen: lytic or lysogenic.

The lytic cycle happens when progeny phage particles are produced. The lytic cycle is the more common life cycle that comes after most infections. The first step of this cycle is the attachment of the phage to the host cell, injecting its DNA into the cell. Nucleic acid from the phage is replicated, and the phage’s genes are expressed, allowing the production of phage proteins. The phage proteins are assembled into phage particles, which are released when the host cell undergoes lysis (it breaks down). The lysis is mediated by lysis genes S, R, Rz, and Rz1 which, upon expression, yield proteins that work together to break down the host bacterium’s cell wall. This mode of lambda replication typically yields many phage particles.

The lysogenic cycle, in contrast, does not produce a huge number of progeny phage or break down the host cell. Instead, the λ DNA recombines with its host’s genome to produce a prophage. This typically is the favoured pathway when unfavourable environmental conditions prevent intense replication of the bacterial cells. Like the lytic cycle, the first step of the lysogenic cycle is also the attachment of the phage and the injection of its DNA into the host cell. The phage DNA then integrates with the host chromosome, producing an integrated DNA combination called the prophage DNA. Host cells that carry this DNA are said to be in the lysogenic state. The prophage DNA is replicated along each time the host bacterial cell replicates itself, producing more cells, each with a copy of the prophage DNA. When these cells are exposed to certain chemicals or to ultraviolet light, phage induction happens; the prophage DNA is then cut out of the host genome and proceeds to the lytic cycle.

Applications

The lambda phage has different applications, most of which are related to DNA cloning. This is because lambda phage can be used as a vector for generating recombinant DNA, which are combined DNA sequences that result from using laboratory techniques like molecular cloning to assemble genetic material from several sources. The site-specific recombinase of lambda phage can be used for shuffling cloned DNAs via the gateway cloning system, a molecular biology technique that ensures the effective transfer of DNA fragments between plasmids.

The lambda phage’s ability to mediate genetic recombincation is due to its red operon, which is a functioning unit of genomic DNA that has a cluster of genes controlled by a promoter or a single regulatory signal. This red operon can be expressed to yield the proteins red alpha (or exo), beta, and gamma, which can be used in recombination-mediated genetic engineering, a method commonly employed in bacterial genetics, generation of target vectors, and DNA modification.

Undoubtedly, the lambda phage is a powerful genetic tool that can be used in many important studies.