VIEWS: 363 PAGES: 40 POSTED ON: 1/17/2011
IN THE NAME OF GOD Esfahan University Department of Biology Advanced Virology Professor Bouzari Bacteriophage Applications and Biotechnology http://www.seyet.com/t4phage. Keivan Beheshti Maal April 2008 Bacteriophage Definition: Bacteriophage (phage) are obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria.) What is a Bacteriophage ? Viruses that attack bacteria Non-self replicating Made up of mostly proteins and DNA Bacterial specific Able to infect most group of bacteria Isolated from soil, water, sewage and most bacterial living zones Number of progenies in a cell: 50-200 Inject their genome into host cell • Lytic cycle (virulent) • Lysogenic cycle (temperate) Bacteriophage properties Phages are ubiquitous and can be found in all reservoirs populated by bacterial hosts, e.g., soil or animal intestine. One of the densest natural sources for phages & other viruses is sea water, where up to 109 virions/ml found at the surface, and up to 70% of marine bacteria may be infected The dsDNA tailed phages, or Caudovirales, account for 95% of all the phages reported in the scientific literature What phages do to Host Cell Lytic Life Cycle As lytic phage propagate, bacteria are destroyed Discovery of Bacteria Infecting Viruses Frederick W. Twort given first credit for phages: 1915 Found by studying micrococcus colonies Naming of the “Viruses” Felix D’ Herelle Born in Montreal:1873 Medical bacteriologist Rediscovery of Bacteriophages: 1917 First Electron Micrograph Luria and Anderson 1942 first electron micrograph picture of a T2 phage Anderson also discovered the phages adsorbed by the tail by “critical point” technique Bacteriophage history in a glance 1915-1917: discovery 1920: bacteriophage base therapy 1940: pioneering studies of physiology and phage-host relationships 1950: molecular biology techniques for studing structure and genetics of bacteriophages 1970: use of many phage enzymes in cloning 1990: phage displayas powerful technique in identification of biomolecules 2000: transfer of toxin genes in invironment by phages (concern) Nowadays: bacteriophage applications in medical biotechnology and industrial-food microbiology Bacteriophage Classification Based on two major criteria: phage morphology and shape of the phage (electron microscopy) nucleic acid properties http://www.seyet.com/t4phage. How many kinds of Bacteriophage? Over 5000 bacteriophages have been studied by electron microscopy which can be divided into 13 virus families Electron micrographs of different phages B. caldotenax a:JS025 b:JS017 c:JS027 B. stearothermophilus d:JS017 B. anthracis e:8724/25 St. camosus f:St.c 13 Bacteriophage families Double stranded DNA, Double stranded Enveloped DNA, Non-enveloped P2 SIRV 1, 2 Rudiviridae Myoviridae T2 Plasmaviridae Fuselloviridae SSV1 λ Tectiviridae TTV1 PRD1 Siphoviridae Lipothrixviridae P22 Corticoviridae PM2 Podoviridae Single Double Single-stranded DNA stranded stranded Inoviridae M13 & fd RNA RNA MS2 phi666 Microviridae ΦX174 Leviviridae Cystoviridae 13 Bacteriophage families icosahedral capsid with lipid layer, circular supercoiled Corticoviridae dsDNA enveloped, icosahedral capsid, lipids, three molecules of Cystoviridae linear dsRNA pleomorphic, envelope, lipids, no capsid, circular Fuselloviridae supercoiled dsDNA Inoviridae genus long filaments/short rods with helical symmetry, circular (Inovirus/Plectrovirus) ssDNA Leviviridae quasi-icosahedral capsid, one molecule of linear ssRNA Lipothrixviridae enveloped filaments, lipids, linear dsDNA Microviridae icosahedral capsid, circular ssDNA Myoviridae (A-1,2,3) tail contractile, head isometric pleomorphic, envelope, lipids, no capsid, circular Plasmaviridae supercoiled dsDNA Podoviridae (C-1,2,3) tail short and noncontractile, head isometric Rudiviridae helical rods, linear dsDNA Siphoviridae (B-1,2,3) tail long and noncontractile, head isometric icosahedral capsid with, linear dsDNA, "tail" produced for Tectiviridae DNA injection Bacteriophage Applications Bacteriophage therapy Bacteriophage mediated microbial control Bacteriophage enzymes Bacteriophage display Baceriophage typing Bacteriophage as biological tracer Monitoring and validation tool Bacteriophage based diagnostics Bacteriophage as cloning vector Bacteriophage for biodegradation Phage can be used biologically-based antimicrobial system - Phage produce products that disrupt the bacterial systems (antimicrobial proteins) Enzymatic • Lysozymes • B-glucosidases • Nucleases • Proteases Non-enzymatic • Very effective on microbes (bacteria, viruses, fungi, etc.) • Some evidence effective on spores • Probably not useful for toxins • Bacteriocins- produced by bacteria • Antimicrobial peptides (AMPs)- produced by higher organisms Bacteriophage therapy Reducing of bacterial load by lytic phages or engineered phages Administration ways: Orally – topically – systematically Use of free phages or phage infected bacteria (very much experimental) Usage during first step infection Catch infection on time before harden of infection eradication Bacteriophage therapy Key aspects: 1. proper phage choice 2. quantity of delivery 3. Timing of treatment Advantages: 1. unable to modify degrade animal metabolism, highly specific 2. self replicating -> self amplifying -> efficacy enhancement 3. ubiquity and diversity of bacteriophages 4. active against antibiotic resistant organisms 5. used as an alternative in antibiotic-allergic persons Bacteriophage therapy In eastern Europe: spraying of E.coli phages at room surfaces, objects, toilets in hospitals (very effective) Tretment and prophylaxis of systemic E.coli infections of human, mice and diarrhoeal disease in calves Control and treatment of Ps. Aeroginosa and Acintobacter baumanii in burn states Bacteriophage therapy Exponential Biotherapies (Rockville, MD) • Vancomycin resistant Enterococcus facium and Streptococcus pneumoniae Phage Therapeutics (Bothell, WA) • Staphylococcus aureus and Staphylococcus epidermidis Intralytix, Inc. (Baltimore, MD) • Salmonella in meat and poultry Biopharm Ltd. (Tblisi, Georgia) • Infections associated with burns University of Idaho • Escherichia coli O157:H7 in cattle Bacteriophage mediated microbial control Control of bacterial contamination in food industries e.g. Pseudomonas fragi in milk and Pseudomonas sp in beef and steaks Control of bacterial contamination for water born pathogens such as Vibrio cholera Control of bacterial contamination for air born pathogens in the hospital and environmental Mycobacteria Control of bacterial contamination in poultry industries pathogens such as Campylobacter Control of plaque forming bacteria such as Streptococcus mutans, St. sunguis and St. sobrinus and Lactobacillus acidophilus by addition of bacteriophages to toothpaste, chewing gum and sweets Control of biofilm forming bacteria like listeria, Escherichia and Pseudomonas sp. in different industries (compete with undiffusible chemicals and antibiotics Bacteriophage enzymes Use of enzymes and other products as tools for molecular biology techniques specially thermophylic products from thermophyl phages Construction of Genomic DNA and cDNA phage libraries Making Genomic DNA library for: - Sequencing - Knock out mice production Making ESTs library for: - To fined full length cDNA - Bioinformatics analysis - Expression analysis - There are more than 106 expressed sequence tags (ESTs) in databases (http://www.ncbi.nlm.nih.gov/dbEST/index.html) - To focus on a known protein with interesting biological function (and, ideally, a known structure) - To search for family member and other species gene homologue Phage display technology Phage display is a powerful screening tool permitting the discovery and characterisation of proteins that interact with a desired target A protein is displayed on the surface of a phage as a fusion with one of the coat proteins of the virus and the DNA that encodes this protein is housed within the virion A process of “biopanning” is used to rescue phage that display a protein that specifically binds to a target of interest Bacteriophage display A polypeptide can be displayed on the phage surface by inserting the gene coding for the polypeptide in the phage genome capable of performing a function, typically the specific binding to a target of interest phenotype (binding) pⅢ tip of phage genotype Phage displaying a binding protein (redrawn from Viti 1999) Biopanning 1. 5. 2. Phage Binding Amplified Phage 3. Binders Eluted 4. Infect E.coli Construction and application of phage antibody libraries Display of antibody fragments on bacteriophage the favored format of antibody fragment is single-chain FV (scFV) antigen VH Fab binding (50 kD) site CH1 VL CL CH2 whole Ab (150 kD) CH3 FV (25 kD) scFV (27 kD) Schematic representation of different antibody formats (redrawn from Viti 1999) scFV Antibody Phage Display Antibodies have been exploited for therapeutics and targeting Traditionally relied on long process of generation and screening Antibody phage display library contains 107 unique scFV molecules Affinity binding allows rapid selection of scFV which bind target of interest Bacteriophage typing First practical applications of bacteriophages Very spesific technique for identification of bacterial strains according to their phage sensitivity Has been stablished for detecting bacteria such as Staphylococccus, Salmonella, Escherichia, Mycobacterium, Listeria, Campylobacter Bacteriophage as biological tracer For tracing air born and water (ground waters) movement Coli phage T4 was successfully used to trace ground water flow for 1.6 km (Southern Missouri, U.S.A) Advantages: Small size, negligible impact on water quality, detectable in low number, adaptable to filtration recovery method Use of T4 for detection of contamination of sewage in water wells (New Zeland) Other phages: MS2, PRD1, f2 Monitoring and validation tool Use of bacteriophage as a model for evaluating and testing of filtration systems in removing dangerous viral particles such as HIV and SARS, HBV Seratia marcescens active phage and coliphage MS2 Bacteriophage based diagnostic Rapid and accurate detection tool for targeted bacteria Phages vs Abs: 1.Simple and economical 2.Producible in large amounts at low cost 3. Use of luciferase gene (lux) in phage expression in bacterium light emission -have been used to detect enteric bacteria in food, L.monocytogenes in foods and environmental samples Lysogenic Bacteriophages: Examples of Virulence Factors Carried by Phage Bacterium Phage Gene Product Phenotype Vibrio cholerae CTX phage cholerae toxin cholera lambda hemorrhagic Escherichia coli shigalike toxin phage diarrhea clostridial botulinum botulism (food Clostridium botulinum phages toxin poisoning) Corynebacterium corynephage diphtheria diphtheria diphtheriae beta toxin Streptococcus erythrogenic T12 scarlet fever pyogenes toxins Bacteriophage: The Flesh-Eating Bacteria Then it rapidly kills tissues causing gangrene conditions. If treat early with antibiotics and removal of infected tissue then amputation and death can be averted. There are between 500-1500 case in the U.S.A. each year Flesh-eating bacteria has a death rate of 20-50% Bacteriophage: Relatives of Flesh-Eating Bacteria Other Group A Streptococci which have acquired virulence factors: Scarlet Fever Toxin Streptococcal Toxic Shock Syndrome Bacteriophage: Therapeutic Uses Bacteriophage has been used to fight many bacterial infections Some examples of diseases treated with phage therapy: staphylococcal skin disease skin infections caused by Pseudomonas Klebsiella Proteus E. coli P. aeruginosa infections in cystic fibrosis patients neonatal sepsis surgical wound infections Likewise, bacteriophage has also been used to treat animal disease. Thank you for your Attention
"Bacteriophage Applications in Biotechnology Slide diphtheria"