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Bacteriophage Applications in Biotechnology Slide diphtheria

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Bacteriophage Applications in Biotechnology Slide  diphtheria Powered By Docstoc
					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

				
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