chap 21 bacteriophages
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The Best for Last: Bacteriophages
Chapter 21
Bacteriophages attached to a bacterium.
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21.1 Bacteriophage Research History
Bacteriophagesviruses that infect bacteria Discovered after recognition of bacterial
hosts in the 1880s (the golden age of
microbiology) Frederick W. Twort observed glassy
transformation
Used a Chamberland filterto discover thefilterable bacteriophages that lysed cultures ofmicrococci
His work was not acknowledged for 5 years
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Felix dHerelle (1873-1849)
Credited as the sole-discoverer of bacteriophages
1917 report by dHerelledescribes the lysis ofdysentery-causing bacteria
grown in liquid medium
Tworts paper was not cited indHerelles publication
SPL/Photo Researchers, Inc. .
Figure 21.01: Felix d'Herelle (1873-1949)
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Felix dHerelles Research
2 directionsDetermining the biological nature of
bacteriophages
Exploring the use of bacteriophagesas therapy to treat bacterial infections
in a preantibiotic era
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dHerelle and Bacteriophage Therapy
1919 field trials to control an epidemic ofchicken typhoid ( ) caused by the
bacterium Salmonella gallinarum
Inoculated chickens either orally or by injectionwith bacteriophages
Flocks treated with bacteriophages suffered fewerdeaths and shorter epidemics that did not reoccur
Positive results motivated dHerelle to conducthuman trials in the 1920s
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dHerelles Human Trials
To prove that the bacteriophagepreparations were safe:
dHerelle injectedhimselfFamily membersCoworkers
No harmful effects observed
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dHerelles Bacteriophage Experiments, cont.
1925 report by dHerelle regarding experiments at theLeague of Nations quarantine station in Alexandria,Egypt
Injected 4 patients suffering from laboratory confirmedbubonic ( ) plague with bacteriophages into the
bubos present in their lymph nodes
All 4 patients recovered rapidly
After this success, dHerrelle traveled the worldcontinuing bacteriophage therapy as a means ofcontrolling cholera outbreaks
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dHerelles Career
Accepted an appointment as a professor ofprotobiology at Yale University in 1928
Played a role in establishing a bacteriophageinstitute in Tbilisi, Soviet Georgia in 1934 This institute exists today as the Georgia Eliava
Institute of Bacteriophage, Microbiology and Virology
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Bacteriophage Therapy Abandoned in the 1930s
Published reports were not consistent The therapy appeared to be hit or miss The main reason for the abandonment of
bacteriophage therapy was the development of
antibiotics
Today the Western world has a renewed interest inbacteriophage therapy to combat antibiotic-resistant
strains of pathogenic bacteria
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The History of the Georgia Eliava Institute of
Bacteriophage, Microbiology and Virology
Founded in 1923 by Professor Giorgyi Eliava Felix dHerelle visited Eliava at the Institute during
1934-1935 via invites by Josef Stalin
Stalin was interested in bacteriophage therapy for military use
Eliava was executed in 1937 dHerelle did not return to the Institute after his death
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Activities of the Georgia Eliava Institute
Continued after Eliavas death Research focus centered around bacteriological
and bacteriophage research
The Institute change names in 1952 and wasreorganized in 1988
The Institute manufactured bacteriophage sprays,salves , ointments and pills
The Institute was damaged during the Georgian civilwar.
Thousands of bacteriophage samples were lost The Institute was revitalized by energetic
entrepreneurs after a 1997 BBC Broadcast entitled TheVirus That Cures
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The American Phage Group
Formed in the 1940s Consisted of scientists from universities
throughout the U.S. that were studying
bacteria and bacteriophages
Max DelbruckSalvador LuriaAlfred Hershey
The Phage Group spent summers doingresearch experiments at Cold Spring
Harbor (Long Island, NY)
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The American Phage Group, cont.
Optimized experiments to study the biology ofbacteriophages such as:One-step growth experimentsPlaque assaysFocused on a selected group of
authorized bacteriophages such as the
Type (T) bacteriophages
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Bacteriophage Lysis and Plaque Assays
Figure 21.2a: Bacterial cells growing in
log phase (left) and being lysed bybacteriophages (right).
David B. Fankhauser, PhD, University of Cincinnati Clermont College
Figure 21.2b: Bacteriophage plaque
assays from one-step growthexperiments.
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21.2 Bacteriophage Ecology
Scientific community estimates that aquaticcommunities contain 4-6 X 1030 bacteria and 1X 1031 bacteriophages
Bacteriophages recycle bacterial carbon in themarine environment
Marine microbial ecology is a rapidlydeveloping field
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21.3 The Biology of Bacteriophages
Composition and Structure
Over 5,100 bacteriophages have beenanalyzed using the transmission electron
microscope
ICTV recognizes 1 order 13 families 31 genera
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Bacteriophage Structure
4 basic shapes or symmetries Binary (head and tail structure) Icosahedral (also called cubic) Helical (filamentous) Pleomorphic ( )
The majority of bacteriophages contain a headand tail structure
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Representatives of the 13 Bacteriophage Families
Grouped by their types of nucleic acid genomes.
Figure 21-3
Adapted from H. -W. Ackermann, Arch. Virol. 146 (2001): 843-857.
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Electron Micrographs of Tailed Bacteriophages
Figure 21-4
Courtesy of Maria Schnos and used with permission of Ross Inman, Department ofMolecular Virology, Bock Laboratories, University of Wisconsin-Madisonx
Photos courtesy of ICTVdB Picture Gallery. Used with permission from Hans-W. Ackermann, Department of Medical Biology, LavalUniversity.
Bacteriophage 3/K26Bacillus licheniformis virusEnterobactervirus P2
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Bacteriophage Structure and Genomes
3 families of bacteriophages are enveloped 96% of all bacteriophages contain dsDNA genomes The remaining 4% contain ssDNA, ssRNA or dsRNA
genomes
Genomes may code for as few as 3-5 genes to asmany as 100 The genomes of bacteriophages contain unusual or
modified bases that protect them from degradation byhost nucleases during phage infection
Most famous group of bacteriophages T-Type
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Only Two Forms of DNA Genomes in
Prokaryotes
Genome
Virion
1. Lineard.s.
25~120(x106
daltons)
Complexmorphology
2. Circulars.s. 1.7~2.7(x106
daltons)
Simpleicosahedral or
helical capsids
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21.4 Overview of Bacteriophage Infection
Bacteriophages Possess Alternative Lifestyles: Lytic
vs Temperate Phages
Bacteriophages adsorb to the surface receptormolecules of bacteria during the first step of infection
Receptors may be: Pili Proteins Oligosaccharides Lipopolysaccharides (LPS)
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Adsorption and Penetration
T4 bacteriophages anchors its tail fibers in the LPSlayer of its bacterial host
This adsorption step causes a conformational change,resulting in contraction of the tail sheath and
penetration of the cell membrane and the
bacteriophage
The bacteriophage DNA genome is subsequentlyinjected into the host cell through the tail tube
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Bacteriophage T4 Adsorption and Penetration
Figure 21.5 Bacteriophage T4 penetrating through the E. coliK-12
Omp C outer membrane protein receptor.
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Other Bacteriophages
May bind to other receptors Bacteriophages that do not have tails penetrate the
host by producing polysaccharide-degrading enzymes
that digest components of the bacterial envelope or
cell wall
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Resistance to Bacteriophage Infection
Bacteria become resistant to phage infection whentheir host cell receptors are altered by mutation
There is interest in engineering new receptor-recognition elements into the tail fibers of well-
characterized bacteriophages so they can infect
genetically distant hosts
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Genome Penetration
Not an injection process Bacteriophages may package lysozyme in the base of
its tail and uses the enzyme to degrade a portion of
the peptidoglycan of the bacterial cell wall
The DNA is drawn into the cell by a process that is notwell understood for most bacteriophages
After the DNA enters the cell, it circularizes rapidly bysticky ends or termini or the linear ends are modified
and protected from bacterial nucleases
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Transcription and Translation are Coupled
Bacterial host RNA polymerase recognizes theviral DNA promoters and begins transcriptionofearly genes
Translation of the early genes is coupled withtranscription
Late genes are transcribed and translated
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Temporal Gene Expression
Class I: 0-6 min, transcribed byE. coliRNA polymerase.
Class II: 6-12 min, involvingDNA synthesis and nuclease.
Class III: 8-20 min, for viralstructural proteins.
Transcribed by T7
RNA polymerase
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Assembly and Release Process
Lytic Infection
After all bacteriophage parts are produced, newbacteriophages are assembled
A copy of the genomic DNA is reeled into apreassembled icosahedral head
A few molecules of lysozyme are packaged into thetail plate
Phage lysin, endolysins, muramidases or virolysinshydrolyze bonds in the murein or peptidoglycan of the
cell wall, allowing the viruses to escape or be released
Holin is used to create pores in the inner membrane ofthe host, allowing the lysin or other enzymes to
facilitate bacteriophage release
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Lytic Infection
Figure 21.06: E. colibacteriophage T4 lytic infection cycle.
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T7 bacteriophageDNA
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Lysogenic (Temperate) Infections
These bacteriophages infect their hosts but do not killthem.
Instead their genome becomes integrated into aspecific region of the host chromosome
The integrated bacteriophage genome is called aprophage
The viral DNA replicates every time the cell copies itschromosomal DNA during cell division
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Lysogenic (Temperate) Infections, cont
Some temperate phages encode transposase whichallows the bacteriophage to insert randomly into the
chromosome
Other bacteriophages integrate into site specificlocations within the chromosome
All bacteriophage gene expression is repressed by arepressor protein
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Infection Cycle of a Temperate Bacteriophage
Figure 21.07: Infection cycle of a temperate bacteriophage.
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Derepression or Induction
If the repressorloses function (becomesinactivated), viral DNA is excised from thebacterial chromosome and the excised DNA
acts like a lytic virus
Spontaneous derepression orinductionhappens about 1 in every 10,000 cell divisions
Temperate bacteriophages can carry hostgenes from one bacterial cell to another in aprocess called transduction
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Phage
Icosahedral head (55 nm in diameter) Tail 15x135 nm long Linear double-stranded DNA About 50 kb with 12 unpaired bases at both
ends called cohesive ends.
Biological active form is circular rather thanlinear.
Lytic and lysogenic life cycle. Rolling circle replication mechanism.
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Lytic cycle and lysogenic cycle
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Regulatory elements that control
lysogenic and lytic development
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Repression of the lytic genes in a lysogenic cells
CI: OR1 > OR2 > OR3
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repressor
1. The CI gene of phage encodes a repressor.2. CI: 27 kD and form dimmer.3. Binds to OL (for transcription in a left direction)4. Binds to OR (for transcription in a right direction)
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Autogenous regulation of phage repressor synthesis
1. CIO
L1,O
R1
(1>2>3)
2. OR1PR, OL1PL, OR3PRM
3. PR:cro
4. PL :N5. CIbind
OL1,L2,R1, R2,
6.RNA polymerasebind PRPL(cro, N)
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Autogenous regulation of phage repressor synthesis
7. CI Bind OR2PRM,CI
8. PRM: Repressor Maintenance9. Repressor,bind OR1,R210.Repressor,bind
OR1,R2,R311. Repressor bind OR3 Block
PRM (self regulation)
12.OR2, CI repressor
13.U.V.activate proteaseCI repressor
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Lytic Cycle (45 mins)
1. Immediately early genes:Cro: control of repressor and other thingsN: antiterminator, prevent termination at TL1
and TR1.
2. Delayed-early genes:O and P proteins: for circular DNA replication.Q: 2nd antiterminator, prevent termination at TR4.
3. Late genes:Block early genes, rolling-circle replication.
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Genes and recognition sites involved in the
lytic regulatory cascade
1. Cro, N, Q: regulatory proteins required for lytic development.2. O
R
PR
, OL
PL
: leftward and rightward transcription.
3. OR1, OR2, OR3: three repressor binding sites.4. nutL, nutR: N protein binding site prevent transcription
termination at tL1 and tR1.
Express delayedearly genes
Express
late genes
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Functions ofcro at early and late stages of lytic cycle
Cro: OR3 > OR2 > OR1
CI: OR1 > OR2 > OR3
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N protein functions as an antiterminator at tR1 and tL1
N, RNA polymerasetR1
N: antiterminator
1. N, RNA polymerasereadthrough stop site (tR1)
2. nut: N utilization3. Nus: N utilization substance4. RNA polymeraseN,
NusB/S10, NusA,pass
tR1,
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lysogenic or lytic cycle?
1. [cro] [CI]OR, OL2. CIICIII3. PRE: promoter for repressor establishment,CI croantisense mRNA
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lysogenic or lytic cycle?
Poor growth condition CIIICII lysogenyGood growth condition CIII (less active)CII lytic
RNA PolCII
bind PRE,CII,CIII
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Regulatory elements that control lysogenic and lytic
development
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Phage
rolling circlereplication
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21.5 Bacteriophages Create
Pathogenic Bacteria in Nature
Lysogenic conversiona prophage may carrygenes that alter the phenotype of a lysogenicbacterium.
e.g. Corynebacterium diphtheriae ( )produces a toxin responsible for diphtheria only ifit carries the temperate bacteriophage calledphage.
The toxgene is located near one end of the phagegenome
1883 Klebsdiphtheriae 1884 Loeffer
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Other Examples of Lysogenic Conversion
IfStreptococcus pyogenes( contains atemperate bacteriophage T2,the bacterium changes to apyrogenic orerythrogenicexotoxinproducing bacterium
This exotoxin causes the rashofscarlet fever ( )
Figure 21.08: This child has the Scarlet Fever rash caused
by a lysogenic Streptococcus pyogenes bacterium.
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( )
( )
( )( )
( )
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21.6 Control of Bacteriophages in
Industrial Fermentations
Bacteria are used in a variety of foodfermentation processes:
Yogurt Cheese Sauerkraut Soy sauce
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Pharmaceutical and biotechnology industriesuse bacteria on a large scale to produce
products such as: Alcohols Vitamins Amino acids Enzymes Hormones Biopolymers Antibiotics Other therapeutics
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Bacteriophages are a Threat to Fermentation
and Pharmaceutical Industries
Attacks by bacteriophages can results in considerableeconomic losses
About 1-10% of dairy product fermentation batches arelost to bacteriophage infection
Often happens because the raw starting materials arecontaminated with undetectable numbers of bacteriophages
Laboratory staff are trained to expect and respond tobacteriophage outbreaks
Bacteriophages are ubiquitous Bacteriophage seasons are January to March and October to
November
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If a bacteriophage were the size of a
cockroach- these cockroaches would
cover the surface of the earth in a layerthat is 31,071 miles or 50,000 km deep!
Bacteriophages are ubiquitous
- Bacteriophage researcher Roger Hendrix
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Microbe Fermentation Vessel Used to Make
Enzymes for Medical and Industrial Use
Figure 21-9Courtesy of Shawn Walshaw-Wertz, Cherokee Pharmaceuticals LLC
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21.7 Biofilms and Bacteriophages
Bacteria form complex communities called biofilms onsolid wet surfaces in natures such as
River rocks Walls of limestone caves Pipes Industrial equipment Hulls of ships Teeth Lining of the colon Sutures Lungs of Cystic Fibrosis Patients Medical devices such as catheters ( ), heart valves Surrounding tissues of the heart Orthopedic devices Facial implants
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Biofilms, cont.
Biofilms may consist of a single bacterial species tohundreds or thousands of bacterial species
The bacteria produce extracellular polysaccharidepolymers that surround and encase the microbes,
facilitating their adhesion to surfaces
Biofilms can cause environmental problems
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Biofilms Can Cause Chronic Bacterial
Infections in Humans
Biofilms are a challenge to medical care Bacteria commonly isolated from medical devices:
Enterococcus faecalis
S. aureus
S. epidermidis Streptococcus viridans E. coli Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa
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Biofilms Can Cause Chronic Bacterial
Infections in Humans, cont.
These bacteria originate from the skin of the patient orthe healthcare workers, entry ports of catheters etc.
Preventative strategies to treat biofilms on medicaldevices:
Application of silver-impregnated catheters Coating devices with antibiotics Disinfection at the surface Adding inline filters into the ports of catheters
Bacteriophages studies to pretreat catheter surfacesto control biofilms are promising
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EM ofStaphylococcus aureus Bacteria Found
on a Patients Catheter
Figure 21.10: EM ofStaphylococcus aureus bacteria found on the surface
of a patient's catheter. Magnified 2363X.
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21.8 FDA-Approved Listeria-Specific
Bacteriophage Preparation on Ready-to-Eat Meats
Listeriosis ( ) is a foodborne illnesscaused by the bacterium Listeriamonocytogenes
Listeriosis occurs in pregnant women (cancause a miscarriage, stillbirth or prematuredelivery), newborns and people with weakened
immune systems
2006FDA approved LISTEX P100which is acocktail of 6 bacteriophages in the form of an
application spray
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LISTEX P100
LISTEX P100 will be used in clean rooms ofcheese, meat, and poultry processing plantsto inhibit the growth of Listeria on products
such as lunch meat and hot dogs
LISTEX P100 will not be declared as aningredient on the label of a treated product
p 592Vi Fil 21 1
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Bacteriophage Therapy
Makes a Comeback
3 Georgia woodsman suffered from burnscaused by Soviet-era makeshift heaters
packed with radiation (Strontium 90 )
Their burns became infected withStaphylococcus aureus
Antibiotics failed to prevent the bacteria frominvading deeply into the wounds
p.592Virus File 21-1
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Bacteriophage Therapy
Makes a Comeback, cont.
The men were rushed to a hospital in Tbilisiwhere doctors placed bacteriophage patches
on the wounds
The infections cleared in a few weeks The men were stable enough to undergo skin
grafting abroad
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Figure 21.VF01: These wounds were treated
with the bacteriophage powder PhagoBioDerm.
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Bacteriophage Therapy Hurdles Being Addressed
Contamination of bacteriophage stocks withbacterial toxins and debris that causes sideeffects in patients
Narrow host range of bacteriophages The elimination of bacteriophages by the
hosts immune system (antibodies)
Bacterial host resistance to phage infection Lytic bacteriophages are ideal for therapy
50% of all bacteriophages are temperate
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