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

• Lytic cycle (virulent)Lytic cycle (virulent)• Lysogenic cycle (temperate)Lysogenic cycle (temperate)

Bacteriophage propertiesBacteriophage 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 CellWhat phages do to Host Cell

Lytic Life CycleLytic Life Cycle

As lytic phage propagate, bacteria are destroyed

Discovery of Bacteria Infecting Discovery of Bacteria Infecting VirusesViruses

Frederick W. Twort given first credit Frederick W. Twort given first credit for phages: 1915for phages: 1915

Found by studying Found by studying

micrococcus coloniesmicrococcus colonies

Naming of the “Viruses”Naming of the “Viruses”

Felix D’ HerelleFelix D’ Herelle

Born in Montreal:1873Born in Montreal:1873

Medical bacteriologistMedical bacteriologist

Rediscovery of Rediscovery of

Bacteriophages: 1917Bacteriophages: 1917

First Electron MicrographFirst Electron Micrograph

Luria and Anderson Luria and Anderson 1942 first electron 1942 first electron micrograph picture micrograph picture

of a T2 phageof a T2 phage

Anderson also discovered Anderson also discovered the phages adsorbed by the phages adsorbed by the tail by the tail by

“ “critical point” techniquecritical point” technique

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

DNA injection

Bacteriophage ApplicationsBacteriophage Applications

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)

EnzymaticEnzymatic• Lysozymes• B-glucosidases• Nucleases• Proteases

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

or engineered phages or engineered phages

Administration ways:Administration ways: Orally – topically – systematicallyOrally – topically – systematically

Use of free phages or phage infected Use of free phages or phage infected bacteria (very much experimental)bacteria (very much experimental)

Usage during first step infectionUsage during first step infection

Catch infection on time before harden of Catch infection on time before harden of infection eradicationinfection eradication

Bacteriophage therapyBacteriophage therapy

Key aspects:Key aspects: 1. proper phage choice1. proper phage choice 2. quantity of delivery2. quantity of delivery 3. Timing of treatment3. Timing of treatment

Advantages:Advantages: 1. unable to modify degrade animal metabolism, highly specific1. unable to modify degrade animal metabolism, highly specific

2. self replicating -> self amplifying -> efficacy enhancement2. self replicating -> self amplifying -> efficacy enhancement

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

Bacteriophage therapyBacteriophage therapy Exponential Biotherapies (Rockville, MD)Exponential Biotherapies (Rockville, MD)

• 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

- Bioinformatics analysis- Bioinformatics analysis

- Expression analysis- Expression analysis

- 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

Antibody phage display library contains 10Antibody phage display library contains 1077 unique unique

scFV moleculesscFV molecules

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

Bacterium Phage Gene Product Phenotype

Vibrio cholerae CTX phage cholerae toxin cholera

Escherichia colilambda phage

shigalike toxinhemorrhagic

diarrhea

Clostridium botulinumclostridial

phagesbotulinum

toxinbotulism (food

poisoning)

Corynebacterium diphtheriae

corynephage beta

diphtheria toxin

diphtheria

Streptococcus pyogenes

T12erythrogenic

toxinsscarlet fever

Bacteriophage:Bacteriophage:The Flesh-Eating BacteriaThe Flesh-Eating Bacteria

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:

Scarlet Fever ToxinScarlet Fever Toxin

Streptococcal Toxic Shock SyndromeStreptococcal Toxic Shock Syndrome

Bacteriophage:Bacteriophage:Relatives of Flesh-Eating BacteriaRelatives of Flesh-Eating Bacteria

Bacteriophage has been used to fight many Bacteriophage has been used to fight many bacterial infectionsbacterial infections

Some examples of diseases treated with Some examples of diseases treated with phage therapy:phage therapy:

staphylococcal skin diseasestaphylococcal skin disease skin infections caused by Pseudomonas Klebsiella Proteus E. coli P. aeruginosa infections in cystic fibrosis patients neonatal sepsisneonatal sepsis surgical wound infections

Likewise, bacteriophage has also been used to treat animal disease.

Bacteriophage: Therapeutic UsesBacteriophage: Therapeutic Uses

Thank you for your Thank you for your AttentionAttention