biotechnology not possible without phages

Biotechnology Not possible Without phages

CONTENTS:
• What is biotechnology?
• What are phages?
• Applications of phages-
• As alternatives to antibiotics
• Phage display
• Phage display libraries
• Vaccine delivery vehicles
• Gene therapy delivery vehicles
• Detection of pathogenic bacteria
• Phage regulatory elements in gene expression technology
• Applications of phage lysis enzymes
• Clinical applications
• Conclusions
• Future directions

WHAT IS BIOTECHNOLOGY?
Biotechnology is a field of biology that involves the use of living things in engineering, technology, medicine, etc. Modern use of the term refers to genetic engineering as well as cell- and tissue culture technologies.

United Nations Convention on Biological Diversity defines biotechnology as:
"Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use."
Biotechnology is of following types:
• Blue biotechnology is a term that has been used to describe the marine and aquatic applications of biotechnology.
• Green biotechnology is biotechnology applied to agricultural processes. E.g Bt corn
• Red biotechnology is applied to medical processes. E.g. Designing of organisms to produce antibiotics, and the engineering of genetic cures through genetic manipulation.
• White biotechnology, also known as industrial biotechnology, is biotechnology applied to industrial processes.. Another example is the using of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous/polluting chemicals.
The investment and economic output of all of these types of applied biotechnologies is termed as bioeconomy.

WHAT ARE BACTERIOPHAGES?
Bacteriophages (or phages) are viruses consisting of a DNA or RNA genome contained within a protein coat. They infect bacteria and either incorporate viral DNA into the host genome, replicating as part of the host (lysogeny), or multiply inside the host cell before releasing phage particles either by budding from the membrane or by actively lysing the cell.

APPLICATIONS OF PHAGES:Field of biotechnology does not seem possible without bacteriophages as bacteriophages have several potential applications in the modern biotechnology industry:

• Alternative to antibiotics
• Phage display
• Phage display libraries
• Vaccine delivery vehicles
• Gene therapy delivery vehicles;
• Detection of pathogenic bacteria
• Phage regulatory elements in gene expression technology
• Applications of phage lysis enzymes
• Clinical applications

1. ALTERNATIVE TO ANTIBIOTICS OR PHAGE THERAPYPhage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture.
A Georgian, George Eliava, alongwith d'Hérelle, and in 1923 he founded the Eliava Institute in Tbilisi, Georgia, devoted to the development of phage therapy.
Bacteriophage treatment offers a possible alternative to conventional antibiotic treatments for bacterial infection. It is conceivable that, although bacteria can develop resistance to phage, the resistance might be easier to overcome than resistance to antibiotics. Bacteriophages are very specific, targeting only one or a few strains of bacteria. Traditional antibiotics have more wide-ranging effect, killing both harmful bacteria and useful bacteria such as those facilitating food digestion. The specificity of bacteriophages might reduce the chance that useful bacteria are killed when fighting an infection. Some evidence shows the ability of phages to travel to a required site—including the brain, where the blood brain barrier can be crossed—and multiply in the presence of an appropriate bacterial host, to combat infections such as meningitis.
• Enzobiotics are a new development at Rockefeller University that create enzymes from phage. These show potential for preventing secondary bacterial infections, e.g. pneumonia developing in patients suffering from flu, otitis etc.
• The first approval granted by the FDA and UDSA for a phage-based food additive is approval for ListShield targeted against Listeria monocytogenes) created by Intralytix. in August 2006, spraying meat with phages.
• The Southwest Regional Wound Care Centre in Texas (www.woundcarecenter.net) has been using phages (in combination with other methods) to treat antibiotic-resistant infections. To enable residents of the USA to access phage therapy more readily, the company Phage International (http://www.phageinternational.com) recently merged with the Georgian Phage Therapy Center, and opened a phage therapy centre in Tijuana, Mexico.
Example of a successful phage therapy treatment:

The patient was a 50-year-old female with venous leg ulcers. The wound was infected with a multidrug-resistant Pseudomonas, and had failed to heal after one year of conservative wound care management. It responded rapidly after the application of phage, forming a healthy, red granulating base within two weeks and later showing epithelial islands inside the wound, a characteristic of phage therapy.

2. PHAGE DISPLAY-
In phage display, a heterologous peptide or protein is displayed on the surface of the phage through transcriptional fusion with a coat-protein gene, producing novel phage particles that have a variety of potential uses. The most widely used phage display methods are based on the use of M13 and related filamentous phages of Escherichia coli but others, including the E. Coli phages lambda and T7, have also been used Because particulate phages are relatively easy and inexpensive to purify, phage display can also provide a means of purifying a particular protein or antibody.

Examples of some methods that have been used to fuse foreign proteins/peptides to the surface of bacteriophage. Foreign proteins can usually be displayed on more than one phage coat protein and in varying amounts. Generally, the smaller the foreign protein or peptide the more copies can be displayed, although this also depends on the phage used, the coat protein and the antigen displayed.
(a) The gene for a foreign peptide is fused directly to the coat-protein gene. All minor coat-proteins display the foreign antigen, which can limit the size of the peptide displayed. In this example, the minor coat-protein is used because it results in fewer copies of the foreign protein being displayed.
(b) Two copies of a coat protein gene can be present in the phage – one native and one fusion. As such, not all proteins display the foreign molecule, therefore larger proteins can often be displayed.
(c) Unmodified ‘helper’ phage infect cells containing a phagemid, which then expresses the coat protein fused to a foreign peptide or protein.

In a similar way to (b), this results in phage particles in which not all coat proteins display foreign antigens. Generally, a phagemid-type system is preferred because there is some control over how many copies of the foreign antigen are displayed, and manipulations can be carried out in a plasmid-like system. The exact methods used for phage display will vary, depending on the type of phage used and the antigen being displayed.


3. PHAGE-DISPLAY LIBRARIES-
can be screened in several ways to isolate displayed peptides or proteins with practical applications. For example, it is possible to isolate displayed peptides that bind target proteins with affinities similar to those of antibodies. These can then be used as therapeutics that act either as agonists or through the inhibition of receptor–ligand interactions. These high-affinity, displayed peptides also have the potential to be used for the detection of pathogens and agents posing a biological threat in the environment . In another example, directed evolution of proteins can be used to enhance enzymatic activity or binding properties. Here, the active site of an enzyme is randomly altered, and the library screened for increased activity.

A variation of phage display, which is also worthy of note, is the use of phages to display a library of Fab antibody fragments on the surface of filamentous phages . Although these libraries have generally been used for research purposes, one particularly novel use for phage-displayed antibodies was in the development of a nasally delivered treatment against cocaine addiction whole phage particles delivered nasally can enter the central nervous system where the specific phage-displayed antibody can bind to cocaine molecules and prevent their action on the brain.

4. PHAGES AS VACCINE DELIVERY VEHICLES-
Phages have been used as potential vaccine delivery vehicles in two different ways:
• by directly vaccinating with phages carrying vaccine antigens on their surface or
• by using the phage particle to deliver a DNA vaccine expression cassette that has been incorporated into the phage genome.

(i) In phage-display vaccination,- phages can be designed to display a specific antigenic peptide or protein on their surface. Alternatively, phages displaying peptide libraries can be screened with a specific antiserum to isolate novel protective antigens or mimetopes – peptides that mimic the secondary structure and antigenic properties of a protective carbohydrate, protein or lipid, despite having a different primary structure. The serum of convalescents can also be used to screen phage-display libraries to identify potential vaccines against a specific disease, without prior knowledge of protective antigens. In a few cases, whole phage particles displaying antigenic proteins have been used as vaccines in animal models. Because phage particles are naturally immunostimulatory an antigen presented on the phage coat would come ‘ready conjugated’ with a natural adjuvant activity, without the need for separate protein purification and subsequent conjugation to a carrier molecule before immunization.

(ii) DNA vaccine expression cassette- More recently, it has also been shown that unmodified phages can be used to deliver DNA vaccines more efficiently than standard plasmid DNA vaccination. The vaccine gene, under the control of a eukaryotic expression cassette, is cloned into a standard lambda bacteriophage, and purified whole phage particles are injected into the host. The phage coat protects the DNA from degradation and, because it is a virus-like particle, it should target the vaccine to the antigen-presenting cells. When compared with standard DNA vaccination, superior antibody responses have been shown in mice and rabbits. We have also recently shown that, similar to phage display, genome libraries in phage (such as Lambda ZAP expresstm containing both a prokaryotic and eukaryotic promoter) can be expressed in E. Coli, screened with convalescent serum and potential vaccines identified. These can then be used, directly, for vaccination.

5. PHAGES FOR TARGETED GENE-DELIVERY-
Phages have also been proposed as potential therapeutic-gene delivery vectors. Although conceptually different, the rationale for using phages for this purpose is similar to that for using phages for DNA vaccine delivery – the phage coat protects the DNA from degradation after injection, and the ability to display foreign molecules on the phage coat also enables targeting of specific cell types, a prerequisite for effective gene therapy.

Both artificial covalent conjugation and phage display have been used to display targeting and/or processing molecules on the surface of the phages. Targeting sequences, such as fibroblast growth factor, have been used to deliver phages to cells bearing the appropriate receptors and, whereas protein sequences such as the penton base of adenovirus, which mediates viral attachment, entry and endosomal release, have been used to enhance phage uptake and endosomal release. Similarly, the protein transduction domain of the HIV tat protein and the nuclear localization signal derived from the SV40 T antigen have been used to enhance uptake and nuclear targeting of modified phage lambda. Other examples of displayed peptides, which might facilitate phage-mediated gene delivery, include integrin-binding peptides (to enhance binding and uptake) and dnase II inhibitors (to reduce DNA degradation).

6. PHAGES FOR THE DETECTION AND TYPING OF BACTERIA-
For many years the specificity of phages for their bacterial hosts has enabled them to be used for the typing of bacterial strains and for the detection of low numbers of pathogenic bacteria. Phages bound to bacteria can be detected by specific, labeled antibodies, thereby increasing the sensitivity of detection. For specific typing, different species of phage can be plated out onto a lawn comprising an unidentified bacterial strain, and the presence of clear areas (plaques) where an individual phage particle has grown and lysed the surrounding cells enables identification of the specific bacteria. Other methods that have been used to detect pathogenic bacteria include: using phages specifically to deliver reporter genes (e.g. Lux or green fluorescent protein), which are expressed after infection of target bacteria; using phages that have a fluorescent dye covalently attached to the phage coat, and detecting the specific adsorption; the detection of released cellular components, such as adenylate kinase, after specific lysis; and using phages displaying peptides or antibody fragments that will bind specific bacterial pathogens or toxins.

7. PHAGE REGULATORY ELEMENTS IN GENE EXPRESSION TECHNOLOGY-
Several cloning vectors are either derivatives of bacteriophages (e.g., phages λ and M13 of E. Coli) or contain some elements of phage origin (e.g., cosmids and phagemid vectors). Site specific integration elements (attachment sites) in the genomes of temperate phages and host bacterium (attp and attb, respectively) can be used for stable insertion of cloned genes into the bacterial genome. Such integration vectors have been developed for a variety of organisms. Phages are a potential source of genetic elements for vector construction.

8. APPLICABILITY OF LYSIS GENES FOR LYSIS OF BACTERIA-
Recent advances in the purification technologies (like fluidisized beds and expanded bed adsorption methodology) permit the adsorption of proteins directly from the culture medium (Chase 1994). Such purification techniques are especially suitable for proteins secreted into culture medium. In order to make the intracellular or periplasmic located biomolecules accessible for purification, the bacterial cells must be disrupted.
The first suggestion for the use of phage genes to obtain lysates containing intracellular enzymes was from Sher and Mallette (1952), who purified L-lysine decarboxylase and L-arginine decarboxylase from a phage lysate after infection of E. Coli with phage T2. Auerbach and Rosenberg (1987) have patented the use of an E. Coli strain containing defective temperature sensitive lambda lysogens as a method for cell disruption. The prophage lacks the genes for replication or structural protein assembly, and functional phages can not therefore be produced. The lysis genes are under temperature sensitive control through the use of the lambda pl promoter and the ci857 repressor. The lysis could be induced in mid-log phase by a temperature shift to 42-44C. The phage lambda lysis genes S (holin), R (transglycosylase), and Rz cloned under control of the lac promoter cause rapid lysis within 40 minutes after induction of the gene expression with IPTG. In the absence of the functional holin gene S, lysis does not occur. E. Coli can tolerate relatively high amount of intracellularly accumulated phage lysin (up to 2 % in the case of the phage T4 lysin) without lysis (Perry et al. 1985). Injuries in the cytoplasmic membrane by phage holin, freeze-thawing, osmotic shock or chemicals (like chloroform or toluene) yield rapid degradation of the cell wall and lysis of bacteria. Phage T7 lysin (amidase) has been used for construction of E. Coli strains with increased susceptibility for lysis. Such strains can be conveniently used for externalization of intracellular gene products by osmotic shock treatment . The T7 lysin has a dual function. Beside its cell wall hydrolyzing activity it downregulates phage T7 RNA polymerase (and thus expression of the genes cloned under control of T7 promoter)
Recently, de Ruyter et al. (1997) cloned the holin and lysin genes of phage into Lactococcus lactis under control of a nisin inducible promoter. They were able to obtain nisin-inducible lysis of bacteria and acceleration of ripening of experimental cheeses. These very promising results wait for exploitation for larger scale cheese manufacturing.

9. CLINICAL APPLICATIONS –
Because S. sanguis is the first colonizer of newly cleaned teeth and because other bacteria then attach to it, the formation of dental plaque is reduced on newly cleaned teeth by introducing into the mouth bacteriophages which are parasitic to S. sanguis. Because S. sanguis is the means of attachment of plaque forming bacterial colonies to tooth surfaces and forms 10-15% of the organisms in plaque, destruction of S. Sanguis by introduction of its parasitic bacteriophages will remove plaque from teeth surfaces. And removal of plaque containing acid forming bacteria and other harmful bacteria reduces the incidence of dental caries and other disease.

10. OTHER APPLICATIONS:
• used as model viruses in a number of contexts:
• as indicators of the presence of human viruses in natural Waters and wastewater,
• in particle adsorption studies,
• as model viruses in treatment studies i.e. coagulation, filtration, chlorination,
• Ultraviolet [UV] disinfection studies


CONCLUSION-
These are an indication of the large range of biotechnology and/or medical applications of phages, ranging from disease prevention (phage vaccines) through diagnosis (detection and typing of bacteria) to actual treatment (anti-bacterial phage therapy, phage-display antibodies or phage-delivered gene therapy).
This versatility raises two interesting points:
• The possibility of engineering a ‘jack-of-all-trades’ bacteriophage, or phage mixture, to treat different stages of disease (i.e. Both prophylactic and therapeutic),
• The commonality of procedures and techniques across a wide range of potential applications.

FUTURE DIRECTIONS-
Conferences attempting to bridge the gap, such as the American Society for Microbiology Conference on New Phage Biology in Florida, USA in 2004, a Bacteriophage Group Session at the 156th Society for General Microbiology Meeting in production and operation Washington State, USA, which deserves a special mention for successfully bringing together practical and applied phage biologists. Although biotechnologists at these meetings are still the exception rather than the rule, this situation can hopefully only improve as the remarkable versatility and potential applications of these organisms becomes more widely known and developed.