Department of Microbiology, Islamic Department of Microbiology, Islamic Azad University, Falavarjan Branch Azad University, Falavarjan Branch Microbial Biotechnology Microbial Biotechnology By: By: Keivan Beheshti Maal Keivan Beheshti Maal 1 IN THE NAME OF GOD
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Department of Microbiology, Islamic Azad University, Falavarjan Branch Microbial Biotechnology By: Keivan Beheshti Maal 1 IN THE NAME OF GOD.
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Department of Microbiology, Islamic Department of Microbiology, Islamic
Azad University, Falavarjan BranchAzad University, Falavarjan Branch
Microbial BiotechnologyMicrobial Biotechnology
By:By:
Keivan Beheshti MaalKeivan Beheshti Maal11
IN THE NAME OF GOD
Bacteriophage Applications and Biotechnology
http://www.seyet.com/t4phage.
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.)
Bacteriophage
What is a Bacteriophage ?What is a Bacteriophage ? Viruses that attack bacteriaViruses that attack bacteria Non-self replicatingNon-self replicating Made up of mostly proteins and DNAMade up of mostly proteins and DNA Bacterial specificBacterial specific Able to infect most group of bacteriaAble to infect most group of bacteria Isolated from soil, water, sewage and most Isolated from soil, water, sewage and most
bacterial living zonesbacterial living zones Number of progenies in a cell: 50-200Number of progenies in a cell: 50-200 Inject their genome into host cellInject their genome into host cell
Bacteriophage history in a glanceBacteriophage history in a glance 1915-1917: discovery1915-1917: discovery 1920: bacteriophage base therapy1920: bacteriophage base therapy 1940: pioneering studies of physiology1940: pioneering studies of physiology and phage-host relationshipsand phage-host relationships 1950: molecular biology techniques for studing 1950: molecular biology techniques for studing structure and genetics of bacteriophagesstructure and genetics of bacteriophages 1970: use of many phage enzymes in cloning1970: use of many phage enzymes in cloning 1990: phage displayas powerful technique in 1990: phage displayas powerful technique in identification of biomoleculesidentification of biomolecules 2000: transfer of toxin genes in invironment by 2000: transfer of toxin genes in invironment by phages (concern)phages (concern) Nowadays: bacteriophage applications in medicalNowadays: bacteriophage applications in medical biotechnology and industrial-food microbiologybiotechnology and industrial-food microbiology
Based on two major Based on two major criteriacriteria::
phage morphology and phage morphology and shape of the phage shape of the phage (electron microscopy)(electron microscopy)
nucleic acid propertiesnucleic acid properties
Bacteriophage Classification
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 phagesElectron micrographs of different phages
B. caldotenaxB. caldotenax
a:JS025a:JS025
b:JS017b:JS017
c:JS027c:JS027
B. stearothermophilusB. stearothermophilus
d:JS017d:JS017
B. anthracisB. anthracis
e:8724/25e:8724/25
St. camosusSt. camosus
f:St.cf:St.c
Double stranded DNA, Non-enveloped
Double stranded DNA,Enveloped
Single-stranded DNA
Inoviridae M13 & fd
Microviridae ΦX174
Single stranded
RNA
Leviviridae
MS2
Lipothrixviridae
TTV1
Plasmaviridae
Double stranded RNA
phi666
Cystoviridae
Myoviridae
Siphoviridae
Podoviridae
P2
T2
λ
P22
Tectiviridae PRD1
Corticoviridae PM2
FuselloviridaeSSV1
RudiviridaeSIRV 1, 2
13 Bacteriophage families
13 Bacteriophage families
Corticoviridaeicosahedral capsid with lipid layer, circular supercoiled
dsDNA
Cystoviridaeenveloped, icosahedral capsid, lipids, three molecules of
linear dsRNA
Fuselloviridaepleomorphic, 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
Plasmaviridaepleomorphic, 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
Tectiviridaeicosahedral capsid with, linear dsDNA, "tail" produced for
Bacteriophage therapyBacteriophage therapy Bacteriophage mediated microbial controlBacteriophage mediated microbial control Bacteriophage enzymesBacteriophage enzymes Bacteriophage displayBacteriophage display Baceriophage typingBaceriophage typing Bacteriophage as biological tracerBacteriophage as biological tracer Monitoring and validation toolMonitoring and validation tool Bacteriophage based diagnosticsBacteriophage based diagnostics Bacteriophage as cloning vectorBacteriophage as cloning vector Bacteriophage for biodegradationBacteriophage for biodegradation
Phage can be used biologically-based antimicrobial system
- Phage produce products that disrupt the bacterial systems Phage produce products that disrupt the bacterial systems (antimicrobial proteins)(antimicrobial proteins)
Non-enzymaticNon-enzymatic• Very effective on microbes (bacteria, viruses, fungi, etc.)Very effective on microbes (bacteria, viruses, fungi, etc.)• Some evidence effective on sporesSome evidence effective on spores• Probably not useful for toxinsProbably not useful for toxins• Bacteriocins- produced by bacteriaBacteriocins- produced by bacteria• Antimicrobial peptides (AMPs)- produced by higher Antimicrobial peptides (AMPs)- produced by higher
organismsorganisms
Bacteriophage therapyBacteriophage therapy Reducing of bacterial load by lytic phages Reducing of bacterial load by lytic phages
3. ubiquity and diversity of bacteriophages3. ubiquity and diversity of bacteriophages
4. active against antibiotic resistant organisms4. active against antibiotic resistant organisms
5. used as an alternative in antibiotic-allergic persons5. used as an alternative in antibiotic-allergic persons
Bacteriophage therapyBacteriophage therapy
In eastern Europe: spraying of E.coli phages In eastern Europe: spraying of E.coli phages at room surfaces, objects, toilets in hospitals at room surfaces, objects, toilets in hospitals (very effective)(very effective)
Tretment and prophylaxis of systemic Tretment and prophylaxis of systemic E.coliE.coli
infections of human, mice and diarrhoeal infections of human, mice and diarrhoeal disease in calvesdisease in calves
Control and treatment of Ps. Aeroginosa and Control and treatment of Ps. Aeroginosa and Acintobacter baumanii in burn statesAcintobacter baumanii in burn states
• Vancomycin resistant Vancomycin resistant Enterococcus facium Enterococcus facium and and Streptococcus pneumoniaeStreptococcus pneumoniae
Phage Therapeutics (Bothell, WA)Phage Therapeutics (Bothell, WA)• Staphylococcus aureusStaphylococcus aureus and and Staphylococcus epidermidisStaphylococcus epidermidis
Intralytix, Inc. (Baltimore, MD)Intralytix, Inc. (Baltimore, MD)• SalmonellaSalmonella in meat and poultry in meat and poultry
Biopharm Ltd. (Tblisi, Georgia)Biopharm Ltd. (Tblisi, Georgia)• Infections associated with burnsInfections associated with burns
University of IdahoUniversity of Idaho• Escherichia coliEscherichia coli O157:H7 in cattle O157:H7 in cattle
Bacteriophage mediated microbial controlBacteriophage mediated microbial control Control of bacterial contamination in food industries e.g. Control of bacterial contamination in food industries e.g. Pseudomonas Pseudomonas
fragifragi in milk and in milk and Pseudomonas sp Pseudomonas sp in beef and steaksin beef and steaks
Control of bacterial contamination for water born pathogens such as Control of bacterial contamination for water born pathogens such as Vibrio choleraVibrio cholera
Control of bacterial contamination for air born pathogens in the hospital Control of bacterial contamination for air born pathogens in the hospital and environmental and environmental MycobacteriaMycobacteria
Control of bacterial contamination in poultry industries pathogens such Control of bacterial contamination in poultry industries pathogens such as Campylobacteras Campylobacter
Control of plaque forming bacteria such as Streptococcus mutans, St. Control of plaque forming bacteria such as Streptococcus mutans, St. sunguis and St. sobrinus and Lactobacillus acidophilus by addition of sunguis and St. sobrinus and Lactobacillus acidophilus by addition of bacteriophages to toothpaste, chewing gum and sweetsbacteriophages to toothpaste, chewing gum and sweets
Control of biofilm forming bacteria like Control of biofilm forming bacteria like listeria, Escherichialisteria, Escherichia and and Pseudomonas spPseudomonas sp. in different industries (compete with undiffusible . in different industries (compete with undiffusible chemicals and antibioticschemicals and antibiotics
Bacteriophage enzymesBacteriophage enzymes Use of enzymes and other products as tools for Use of enzymes and other products as tools for
molecular biology techniques specially molecular biology techniques specially thermophylic products from thermophyl phagesthermophylic products from thermophyl phages
Construction of Genomic DNA and cDNA phage libraries
Making Genomic DNA library for:Making Genomic DNA library for:- Sequencing- Sequencing
- Knock out mice production- Knock out mice production Making ESTs library for:Making ESTs library for:
- - To fined full length cDNATo fined full length cDNA
- There are more than 10- There are more than 1066 expressed sequence tags (ESTs) in expressed sequence tags (ESTs) in databases (databases (http://www.ncbi.nlm.nih.gov/dbEST/index.html))
- To focus on a known protein with interesting biological function - To focus on a known protein with interesting biological function (and, ideally, a known structure) (and, ideally, a known structure)
- To search for family member and other species gene homologue- To search for family member and other species gene homologue
Phage display technologyPhage display technology
Phage display is a powerful screening tool Phage display is a powerful screening tool permitting the discovery and permitting the discovery and characterisation of proteins that interact characterisation of proteins that interact with a desired targetwith a desired target
A protein is displayed on the surface of a A protein is displayed on the surface of a phage as a fusion with one of the coat phage as a fusion with one of the coat proteins of the virus and the DNA that proteins of the virus and the DNA that encodes this protein is housed within the encodes this protein is housed within the virion virion
A process of “biopanning” is used to A process of “biopanning” is used to rescue phage that display a protein that rescue phage that display a protein that specifically binds to a target of interestspecifically binds to a target of interest
Bacteriophage displayBacteriophage 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 capable of performing a function, typically the specific binding to a target of interestbinding to a target of interest
phenotype (binding)
pⅢtip of phage
genotype
Phage displaying a binding protein (redrawn from Viti 1999)
BiopanningBiopanning1.
2. Phage Binding
3. Binders Eluted
4. Infect E.coli
Amplified Phage
5.
Construction and application of phage antibody libraries
Display of antibody fragments on Display of antibody fragments on bacteriophagebacteriophage
the favored format of antibody fragment is the favored format of antibody fragment is single-chain Fsingle-chain FVV (scF (scFVV))
Schematic representation of different antibody formats (redrawn from Viti 1999)
whole Ab (150 kD) scFV (27 kD)FV (25 kD)
Fab (50 kD)
CH2
CH3
CH1
VL
CL
VHantigenbinding
site
scFV Antibody Phage Display Antibodies have been exploited for therapeutics and Antibodies have been exploited for therapeutics and
targetingtargeting
Traditionally relied on long process of generation and Traditionally relied on long process of generation and screeningscreening
Affinity binding allows rapid selection of scFV which Affinity binding allows rapid selection of scFV which bind target of interestbind target of interest
Bacteriophage typingBacteriophage typing First practical applications of First practical applications of
bacteriophagesbacteriophages
Very spesific technique for identification of Very spesific technique for identification of bacterial strains according to their phage bacterial strains according to their phage sensitivitysensitivity
Has been stablished for detecting bacteria Has been stablished for detecting bacteria such as such as Staphylococccus, Salmonella, Escherichia, Mycobacterium, Listeria, Campylobacter
Bacteriophage as biological tracer For tracing air born and water (ground waters) For tracing air born and water (ground waters)
movementmovement
Coli phage T4 was successfully used to trace ground Coli phage T4 was successfully used to trace ground water flow for 1.6 km (Southern Missouri, U.S.A)water flow for 1.6 km (Southern Missouri, U.S.A)
Advantages:Advantages: Small size, negligible impact on water quality, Small size, negligible impact on water quality, detectable in low number, adaptable to filtration detectable in low number, adaptable to filtration recovery methodrecovery method
Use of T4 for detection of contamination of sewage in Use of T4 for detection of contamination of sewage in water wells (New Zeland)water wells (New Zeland)
Other phages:Other phages: MS2, PRD1, f2 MS2, PRD1, f2
Monitoring and validation toolMonitoring and validation tool
Use of bacteriophage as a model for Use of bacteriophage as a model for evaluating and testing of filtration evaluating and testing of filtration systems in removing dangerous viral systems in removing dangerous viral particles such asparticles such as HIVHIV andand SARSSARS,, HBV HBV
Seratia marcescens active phage and active phage and coliphage MS2coliphage MS2
Bacteriophage based diagnosticBacteriophage based diagnostic
Rapid and accurate detection tool for targeted Rapid and accurate detection tool for targeted bacteriabacteria
Phages vs Abs:Phages vs Abs:1.Simple and economical1.Simple and economical2.Producible in large amounts at low cost2.Producible in large amounts at low cost3. Use of luciferase gene (3. Use of luciferase gene (luxx) in phage) in phage
expression in bacteriumexpression in bacterium light emissionlight emission
--have been used to detect enteric bacteria in have been used to detect enteric bacteria in food, food, L.monocytogenes in foods and in foods and environmental samplesenvironmental samples
Lysogenic Bacteriophages: Examples of Virulence Factors Carried by Phage
Then it rapidly kills tissues causing gangrene Then it rapidly kills tissues causing gangrene conditions.conditions.
If treat early with antibiotics and removal of If treat early with antibiotics and removal of infected tissue then amputation and death can be infected tissue then amputation and death can be averted. averted.
There are between 500-1500 case in the U.S.A. There are between 500-1500 case in the U.S.A. each yeareach year
Flesh-eating bacteria has a death rate of 20-50%Flesh-eating bacteria has a death rate of 20-50%
Other Group A Streptococci which have acquired virulence Other Group A Streptococci which have acquired virulence factors:factors: