Transcript Slide 1

IN THE NAME OF GOD
Department of Microbiology, Islamic
Azad University, Falavarjan Branch
Microbial Biotechnology
By:
Keivan Beheshti Maal
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Bacteriophage Applications and
Biotechnology
http://www.seyet.com/t4phage.
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 ?
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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
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Phages are ubiquitous and can
be found in all reservoirs
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populated by bacterial hosts,
e.g., soil or animal intestine .
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One of the densest natural
sources for phages & other
viruses is sea water, where
up to 109 virions/ml
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found at the surface, and up to
70% of marine bacteria may be
infected
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The dsDNA tailed phages, or
Caudovirales95% rof tnuocca ,
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
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Frederick W. Twort given first credit
for phages: 1915
Found by studying
micrococcus colonies
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Naming of the “Viruses”
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Felix D’ Herelle
Born in Montreal:1873
Medical bacteriologist
Rediscovery of
Bacteriophages: 1917
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First Electron Micrograph
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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
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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
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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?
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Over 5000
bacteriophages
have been
studied by
electron
microscopy
which can be
divided into 13
virus families
Electron micrographs of different phages
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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, Non-enveloped
Double stranded DNA,
Enveloped
P2
Rudiviridae
Myoviridae
T2
Siphoviridae
Fuselloviridae SSV1
λ
Tectiviridae
Plasmaviridae
TTV1
PRD1
Lipothrixviridae
Corticoviridae PM2
P22
Podoviridae
Single-stranded DNA
Inoviridae
SIRV 1, 2
M13 & fd
Single
stranded
RNA
Double
stranded
RNA
MS2
Microviridae ΦX174
phi666
Leviviridae
Cystoviridae
13 Bacteriophage families
Corticoviridae
icosahedral capsid with lipid layer, circular supercoiled
dsDNA
Cystoviridae
enveloped, icosahedral capsid, lipids, three molecules of
linear dsRNA
Fuselloviridae
pleomorphic, envelope, lipids, no capsid, circular
supercoiled dsDNA
Inoviridae genus
(Inovirus/Plectrovirus)
long filaments/short rods with helical symmetry, circular
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
Plasmaviridae
pleomorphic, envelope, lipids, no capsid, circular
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
Tectiviridae
icosahedral capsid with, linear dsDNA, "tail" produced for
DNA injection
Bacteriophage Applications
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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
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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
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Reducing of bacterial load by lytic phages
or engineered phages
Administration ways:
Orally – topically – systematically
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Use of free phages or phage infected
bacteria (very much experimental)
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Usage during first step infection
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Catch infection on time before harden of
infection eradication
Bacteriophage therapy
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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
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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
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Exponential Biotherapies (Rockville, MD)
• Vancomycin resistant Enterococcus facium and
Streptococcus pneumoniae
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Phage Therapeutics (Bothell, WA)
• Staphylococcus aureus and Staphylococcus epidermidis
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Intralytix, Inc. (Baltimore, MD)
• Salmonella in meat and poultry
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Biopharm Ltd. (Tblisi, Georgia)
• Infections associated with burns
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University of Idaho
• Escherichia coli O157:H7 in cattle
Bacteriophage mediated microbial control
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Control of bacterial contamination in food industries e.g. Pseudomonas
fragi in milk and Pseudomonas sp in beef and steaks
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Control of bacterial contamination for water born pathogens such as
Vibrio cholera
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Control of bacterial contamination for air born pathogens in the hospital
and environmental Mycobacteria
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Control of bacterial contamination in poultry industries pathogens such
as Campylobacter
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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
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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
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Making Genomic DNA library for:
- Sequencing
- Knock out mice production
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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
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Phage display is a powerful screening tool
permitting the discovery and
characterisation of proteins that interact
with a desired target
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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
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A process of “biopanning” is used to
rescue phage that display a protein that
specifically binds to a target of interest
Bacteriophage display
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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
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2. Phage Binding
Amplified Phage
3. Binders Eluted
4. Infect E.coli
Construction and application of
phage antibody libraries
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Display of antibody fragments on
bacteriophage
the favored format of antibody fragment is
single-chain FV (scFV)
antigen
binding
site
VH
CH1
CH2
whole Ab
(150 kD)
CH3
Fab
(50 kD)
VL
CL
FV (25 kD)
scFV (27 kD)
Schematic representation of different antibody formats
(redrawn from Viti 1999)
scFV Antibody Phage Display
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Antibodies have been exploited for therapeutics
and targeting
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Traditionally relied on long process of
generation and screening
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Antibody phage display library contains 107
unique scFV molecules
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Affinity binding allows rapid selection of scFV
which bind target of interest
Bacteriophage typing
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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
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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
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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
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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
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-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
Escherichia coli
lambda
phage
shigalike toxin
hemorrhagic
diarrhea
Clostridium botulinum
clostridial
phages
botulinum
toxin
botulism (food
poisoning)
Corynebacterium
diphtheriae
corynephage
beta
diphtheria
toxin
diphtheria
Streptococcus
pyogenes
T12
erythrogenic
toxins
scarlet fever
Bacteriophage:
The Flesh-Eating Bacteria
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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
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Streptococcal Toxic Shock Syndrome
Bacteriophage: Therapeutic Uses
Bacteriophage has been used to fight many bacterial
infections
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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
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Likewise, bacteriophage has also been used to treat
animal disease.
Thank you for your
Attention