bacteriophages

Bacteriophages

DR. KAVEH HARATIAN

MASTER EDUCATION SERIES - AUMS - 2014 1

What do you think about them ?


Some interesting factsViruses that infect bacteria.

Viruses of dsDNA(tailed dsDNA phages).easily in environmental samples

107 /mL in coastal sea-water

1031 individual tailed phage virion on planet Earth.

End to end : 200 million light years into intergalactic space

Most bacterial genomes contain 1 to 24 phage genome as prophage

Turn over every 4-5 years: 1024 productive infections/sec


What are Bacteriophages ?

Viruses that attack bacteria were observed by Twort and d'Herelle in 1915 and 1917. They

observed that broth cultures of certain intestinal bacteria could be dissolved by addition of a bacteria-free filtrate obtained from sewage


Brief historyDiscovered in 1915/1917. basis on :

◦ Clearing(cell lysis) in bacterial lawn.

◦ Propagation as infectious agents

Ideas:◦ Nature of viruses

◦ Phage therapy: using phages as an agent to combat bacterial infections

Modern era of phage research, 1940:◦ Nature of gene

◦ Using phages as experimental model system

Extra results:◦ Nature of gene

◦ Expression of genes and expression regulation

◦ Development of methods of recombinant DNA.


Bacteriophages under Electron Microscope


Bacteriophage (Phage)

Definition - Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery

Significance◦ Models for animal cell viruses

◦ Gene transfer in bacteria

◦ Medical applications◦ Identification of bacteria - phage typing

◦ Treatment and prophylaxsis???


Bacteriophages as therapeutic agents

1919: successful treatment of typhoid in chickens , dysentery in humans.

1921: using against staphylococcus in skin disease.

1920s: large-scale experiments in many countries, like india.

1939 upward:◦ Against diseases with no bacterial component: herpes, urticaria

◦ Although high specific, were used against inappropriate bacterial targets

◦ Inappropriate growth condition or preservatives could limit/prevented the inclusion of infectious Bacteriophages.

◦ No good evidence that they actually worked in the therapeutic uses.

◦ But it continued through WW2(German and soviet armies) notably against dysentery.

Antibiotic age

Renewal of interest


Bacteriophages: Definition & History

Bacteriophages are viruses that can infect and destroy bacteria.

They have been referred to as bacterial parasites, with each phage type depending on a single strain of bacteria to act as host.


BACTRIOPHAGES

Like most viruses, bacteriophages typically carry only the genetic information needed

for replication of their nucleic acid and synthesis of their protein coats.. They

require precursors, energy generation and ribosomes supplied by their bacterial host

cell.


Bacteriophages: Classification

At present, over 5000 bacteriophages have been studied by electron microscopy and can be divided into 13 virus families.


ICTV-designated phage familiesFamily Prototypes Characteristics

Siphoviridae λ dsDNA, long, noncontractile tails

Myoviridae T4 dsDNA, contractile tails

Podoviridae T7 dsDNA, short, stubby tails

Tectiviridae PRD1 dsDNA, linear 5` proteins, internal membrane

Microviridae φX174 ssDNA, circular, icosahedral

Inoviridae M13 ssDNA, filamentous

Leviviridae MS2, Qβ ssDNA, small icosahedral

Cystoviridae φ6 dsDNA, segmented, enveloped

Corticoviridae PM2 dsDNA, circular, internal membrane

Plasmaviridae L2 dsDNA, circular, enveloped


Bacteriophage

Bacteriophages make up a diverse group of viruses, some of which have complex structures, including double-stranded DNA.


Bacteriophage


BacteriophageAlso known simply as a phage; a virus that attacks and infects bacteria. The infection may or may not lead to the death of the bacterium, depending on the phage and sometimes on conditions. Each bacteriophage is specific to one form of bacteria.


Composition and StructureComposition◦Nucleic acid

◦ Genome size◦ Modified bases

◦Protein◦ Protection◦ Infection

• Structure (T4)

– Size

– Head or capsid

– Tail

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile Sheath


Phage entering a bacterial cell


Virulent vs. temperate phages• Virulent phages do not integrate their genetic material into the host cell chromosome and usually kill the host cells (lytic infection) (e.g. T-phages of E.coli).

• Temperate phages may integrate into the host DNA, causing LYSOGENY.


Bacteriophage showing Lytic and lysogenic cycle


Bacteriophages: Virulence Factors Carried On Phage

Temperate phage can go through one of two life cycles upon entering a host cell.

1) Lytic:Is when growth results in lysis of the host and release of progeny phage.

2) Lysogenic:Is when growth results in integration of the phage DNA into the host chromosome or stable replication as a plasmid.

Most of the gene products of the lysogenic phage remains dormant until it is induced to enter the lytic cycle.


Bacteriophages: Lysogenic Conversion

Some lysogenic phage carry genes that can enhance the virulence of the bacterial host. For example, some phage carry genes that encode toxins.

These genes, once integrated into the bacterial chromosome, can cause the once harmless bacteria to release potent toxins that can cause disease.


Bacteriophages• Used for cloning foreign

genes among other applications

• Proteins and peptides are fused to the Capsid(surface) of the phage

• The combination of the phage and peptide is known as a Fusion Protein


Lytic and Lysogenic cycle


Bacteriophages: Lysogenic Conversion

Bacterium PhageGene

ProductPhenotype

Vibrio cholerae CTX phage cholerae toxin cholera

Escherichia colilambda

phageshigalike toxin

hemorrhagic

diarrhea

Clostridium botulinumclostridial

phages

botulinum

toxin

botulism (food

poisoning)

Corynebacterium

diphtheriae

corynephage

beta

diphtheria

toxindiphtheria

Streptococcus

pyogenesT12

erythrogenic

toxinsscarlet fever

Examples of Virulence Factors Carried by Phage


Lysogenic conversion

In some interactions between lysogenic phagesand bacteria, lysogenic conversion may occur. It is when a temperate phage induces a change in the phenotype of the bacteria infected that is not part of a usual phage cycle. Changes can often involve the external membrane of the cell by making it impervious to other phages or even by increasing the pathogenic capability of the bacteria for a host.


Assay for Lytic Phage

• Plaque assay

– Method

– Plaque forming unit (pfu)

– Measures infectious particles

Bacteria

Phage

+Phage

Plaque assay

Method

Plaque forming unit (pfu)

Measures infectious particles


Lytic vsLysogenicCycle


Transduction


Figure 13.10.1

Attachment:Phage attaches to host cell.

Penetration:Phage pnetrates host cell and injects its DNA.

Synthesis of viral compartments

1

2

3

Bacterial cell wall

Bacterial chromosome

Capsid DNA

Capsid

Sheath

Tail fiber

Base plate

Pin

Cell wall

Tail

Plasma membrane

Sheath contracted

Tail core


Figure 13.10.2

4 Maturation:Viral components

are assembled into virions.

Tail

5 Release:Host cell lyses

and new virions are released.

DNA

Capsid

Tail fibers


Examples:

* Corynebacterium diphtheria produces the toxin of diphtheria only when it is infected by the phage β. In this case, the gene that codes for the toxin is

carried by the phage, not the bacteria.

* Vibrio cholerae is a non-toxic strain that can become toxic, producing cholera toxin, when it is infected with the phage CTXφ.

* Clostridium botulinum causes botulism.

* Streptococcus pyogenes causes scarlet fever.

* Shiga toxin

* Tetanus


Medical Applications of Phages“I strongly believe phage could become an effective antibacterial tool” - Carl Merril, Chief of the Laboratory of Biochemical Genetics, National Institute of Mental Health, NIH.

“It might be another string on the bow, such that when (conventional antibiotics) fail, here’s something that has a chance of working. But it’s not going to be a panacea” -Joshua Lederberg, Sackler Foundation Scholar at The Rockefeller University


Model Organisms


Model Organisms

Fundamental problems are solved in the simplest and most accessible system in which the problem can be addressed.

These organisms are called model organisms.


Some Important Model Organisms

Escherichia coli and its phage (the T phage and phage λ)

Baker’s yeast Saccharomyces cerevisiae

The nematode Caenorhabditis elegans

The fruit fly Drosophila melanogaster

The house mouse Mus musculus


Features of Model Systems

The availability of powerful tools of traditional and molecular genetics.

The study of each model system attracted a critical mass of investigators. (Ideas,methods, tools and strains could be shared)


HOW to choose a model organism?

It depends on what question is being asked. When studying fundamental issues of molecular biology, simpler unicellular organisms or viruses are convenient. For developmental questions, more complicated organisms should be used.


Model 1: BACTERIOPHAGE


Bacteriophage (Viruses)

The simplest system

Their genomes are replicated only after being injected into a host cell.

The genomes can recombine during these infections.


Figure BacteriophageMASTER EDUCATION SERIES - AUMS - 2014 46

Each phage attaches to a specific cell surface molecule (usually a protein) and so only cells bearing that “receptor” can be infected by a given phage.


Two Basic Types

1. Lytic phage: eg. T phage

infect a bacterial cell

DNA replication

coat proteins expression

host cell lysed to release the new phage


Figure 21-1

The lytic growth cycle


2. Temperate phage:

eg. Phage λ

Lysogeny—the phage genome integrated into the bacterial genome and replicated passively as part of the host chromosome, coat protein genes not expressed.

•The phage is called a prophage.

•Daughter cells are lysogens.


Figure 21-2 The lysogenic

cycle of a bacteriophage


The lysogenic state can switch to lytic growth, called induction.

Excision of the prophage DNA

DNA replication

Coat proteins expression

Lytic growth


Figure 16-24 Growth and induction of λlysogen


Assays of Phage Growth

Progagate phage:

by growth on a suitable bacterial host in liquid culture.

Quantify phage:

plaque assay

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Progagate phage

Find a suitable host cell that supports the growth of the virus.

The mixture of viruses and bacteria are filtered through a bacterial-proof filter.


Quantify phage

Phage are mixed with and adsorb to bacterial cells.

Dilute the mix.

Add dilutions to “soft agar” (contain many uninfected bacterial cells).

Poured onto a hard agar base.

Incubated to allow bacterial growth and phage infection.


Soft

agarHard agar

a petri dish


This circle-of-death produces a hole orPLAQUE in a lawn of living cells. These plaques can be easily seen and counted so that the numbers of virus can be quantitated.

As the viruses replicate and are released, they spreadand infect the nearby cells.


The Single-Step Growth Curve

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Figure 21-4

Latent period-the time lapse between infection and release of progeny.

Burst size-the number of phage released


The Single-Step Growth Curve

It reveals the life cycle of a typical lytic phage.

It reveals the length of time it takes a phage to undergo one round of lytic growth, and also the number of progeny phage produced per infected cell.


Method1. Phage were mixed with bacterial cells for

10 minutes. (Long enough for adsorption but too short for further infection progress.)

2. The mixture is diluted by 10,000. (Only those cells that bound phage in the initial incubation will contribute to the infected population; progeny phage produced from those infections will not find host cells to infect.)


3. Incubate the dilution. At intervals, a sample can be removed from the mixture and the number of free phage counted using a plaque assay.


Phage Crosses and

Complementation Tests

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Mixed infection: a single cell is infected with two phage particles at once.


Mixed infection (co-infection)

1. It allows one to perform phage crosses.

If two different mutants of the same phage co-infect a cell, recombination can occur between the genomes. The frequency of this genetic exchange can be used to order genes on the genome.


2. It allows one to assign mutations to complementation groups.

If two different mutant phage co-infect the same cell and as a result each provides the function that the other was lacking, the two mutations must be in different genes (complementation groups). If not, the two mutations are likely located in the same gene.


Transduction and Recombinant DNA

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During infection, a phage might pick up a piece of bacterial DNA (mostly happens when a prophage excises form the bacterial chromosome).

The resulting recombinant phage can transfer the bacterial DNA from one host to another, known as specialized transduction.

eg. Phage λMASTER EDUCATION SERIES - AUMS - 2014 66

This series Created by Dr.Kaveh Haratian Ph.D. for Medical and Master learning.

[email protected]


bacteriophages

Health & Medicine