Transcript Thesis Proposal - Phage Ecology Research!

Bacteriophages
By Monique Hunter
Introduction

Bacteriophages or phages are viruses that infect bacteria.

In 1917, French-canadian microbiologist Felix d’Herelle observed
an unknown substance killing bacteria as did his predecessor in
1915.

Called it bacteriophage meaning bacteria-eater.

Named after the bacterial species they infect. Example:
Mycobacteriophages infect mycobacteria.

Estimated phage population is 1031 making them the most
abundant organisms in the biosphere.
Introduction

Pedulla: “If phages were the size of a beetle, they would cover the
Earth and be many miles deep”.

Have different structures, but same features: a head or capsid and
a tail.

The first mycobacteriophages were described in 1950s.
Figure1: Lambda phage or coliphage lambda
How Bacteriophages work?
 Bacteriophages are viruses
and can only replicate inside a
host.
 A phage attaches itself to
bacterial’ cell wall by binding to
specific receptors: adsorption.
 Injects its DNA or RNA inside
the host: penetration.
 Duplication of genetic material
and protein synthesis.
 Maturation and release
Figure 2: Phage adhesion to the host cell
Two alternative life cycles of Phages
Figure 3: Bacteriophages lytic and lysogenic cycles. Pierce BA. Genetics: A conceptual Approach. 3
rd edition
Two alternative life cycles (Summary)
1. The lytic cycle (T4 and T7)
 A phage attaches to a receptor on the bacterial cell wall and
injects its DNA into the cell.
 Inside the host cell, the phage will use the host cell’s chemical
energy and biosynthetic machinery to replicate its DNA,
transcribe and translate it, producing more phage DNA and
phage proteins.
 New phage particles are assembled from these components.
 The phages produce enzyme that breaks open the host cell,
releasing the new phages.
 Virulence phages use only lytic cycle, always killing their host
cells.
2. The lysogenic cycle (Lambda)
 The first 2 steps are identical to the lytic cycle.
 However, inside the host, the phage DNA integrates into the
bacterial chromosome, creating the prophage.
 When the bacterium divides, the prophage is replicated
along with the host DNA.
 Certain conditions such as exposure to UV, desiccation can
cause the prophage to enter the lytic cycle.
Phage Genomics
 Highly diverse genome
 All known mycobacteriophages are dsDNA (double strand
DNA).
 After infection, there is potentiality to recombine and
generate new genomic arrangement.
 Although L5, D29, Bxb1 and TM4 were isolated at different
places and times, they have features in common.
 Morphology, genome size (49.1-52.8kbp), arrangements of
structure and assembly genes (many of which encode
related genes products).
Figure 4: Mycobacteriophages virion morphologies. (Pedulla et al,.2003)
Mycobacteriophage (L5): first genome to be sequenced in
1993
D29 second with complete genome isolated from soil
sample in California.
“Modular or mosaic evolution”
 Postulated that phages evolve by genetic exchange at
specific intergenic sites, either through
recombination or by site-specific mechanism.
homologous
 Illegitimate recombination takes place quasi-randomly along
the recombining genomes.
 However, natural selection eliminates all but recombinants in
which biological functions are intact.
 Phages with mosaic joints that lies between genes.
 Leading to population in which the sites of recombination are
not random.
Figure 5: “Mosaicism in mycobacteriophage genomes. A segment of the Omega genome coding genes 159 to 171
is shown with homologues found in other mycobacteriophages indicated. Six of the genes (161, 165, 167, 168, 169
and 171) have no homologues. Omega gp163 and gp164 are a Clp protease and a DinG helicase, respectively, as
shown, but the functions of the other genes are not known. The genome is characteristically mosaic, with individual
genes representing modules that are present in other phage genomes”. (Cole et al, 2004)
Phage Genomics
 Group according to:

Nucleotide sequence similarities

Gene content analyses
 Arbitrary cutoff: any two genomes that have more than 50%
nucleotide sequence similarity belong to the same cluster.
Figure 6: Diversity of mycobacteriophages. Sequenced genomes for 471mycobacteriophages compared ac
cording to their shared gene contents. Colored circles are clusters (A-T), grey circles are singleton genome
s with no close relatives. 10 of the clusters are divided into subclusters.
(Hatfull et al, 2014)
Phage Genomics
 Phages acquire genes from, and contribute genes to, not
only other phages genomes but also bacterial genomes.
 Therefore are powerful forces in the evolution, physiology
and pathogenecity of their hosts.
Figure 7: Genes can be transferred from one bacterium to another through generalized transd
uction. Pierce BA. Genetics: A conceptual Approach. 3rd edition
Do phages contribute to bacterial
pathogenesis?
 They help certain hosts become more virulent.
 Examples:
Vibrio
cholerae,
Clostridium
botulinum,
Salmonella enterica serovar Typhimurium, and Escherichia
coli.
 Mycobacterium tuberculosis’s pathogenesis is not due to
mycobacteriophages, unlike other species of mycobacteria.
Potential Bacteriophage Applications
 For diagnosis of mycobacterial infection such as tuberculosis
 In the development of tools for mycobacterial genetics
 Therapeutics to cure patients with tuberculosis
 Detect drug resistant tuberculosis
 Better genetic engineering of a vaccine to prevent
tuberculosis
Potential Bacteriophage Applications
 Treat diseases: In 1921, phages were used to successfully
treat staphylococcal skin infections.
 Despite controversial results, Russia, Poland continued
using phage therapy.
 Antibiotic resistant bacteria problem rekindling the practical
applications of bacteriophages.
 Phages are highly specific against only a specific bacterial
species, and cannot infect eukaryotic cells.
Bacteriophage Applications
 In 2006 FDA approved a phage cocktail that targets Listeria
monocytogenes contaminants in meat and poultry products.
 In 2008, FDA approved first phage phase I clinical trial.
Cocktail containing 8 phages targeting Staphylococcus
aureus, Pseudomonas aeruginosa and E. coli.
 Phages usage in water treatment in USA is being evaluated
 They are abundant in the environment meaning that we are
exposed to them. So they are safe.
Limitations and concerns
 Since phages are very specific, it is important to precisely
determine the targeted pathogen before phage therapy.
 In vivo elimination of bacteriophages (several dose of
phages can be given).
 Bacteriophages neutralizing antibodies (phages persist long
enough to sustain their therapeutic effects).
 Phage-resistant mutants.
 The efficacy of phage therapy
 phages have enzymes: lysins
Applications: Mycobacteriophages
as delivery vehicles
 Since bacteriophages infect bacteria, they can be used in
the delivery of genetic material.
 Examples: use of mycobacteriophages for the delivery of
transposons and for gene replacement.
 Use for delivery of reporter genes to mycobacteria, to
monitor their physiological status.
Conclusion
 The study of bacteriophages will help understand their host.
( We still don’t understand M. tuberculosis).
 Very efficient in delivering genetic material, useful tool in
genetics.
 Phages would target pathogens without damaging beneficial
bacterial flora.
In this Lab
 Isolate phages that infect Mycobacterium smegmatis, a fastgrowing, non-pathogenic soil bacterium.
 Collect soil samples
 Follow protocol (Schwebach et al., 2006)
References
 Cole ST et al. Tuberculosis and the Tubercle Bacillus. ASM Press. 2005.
 Hatfull GF. Mycobacteriophages: Genes and Genomes. Annu. Rev. Microbiol.
2010.64:331-356.
 Hatfull GF. Mycobacteriophages: Windows into Tuberculosis. PLoS Pathog.
2014.10(3): e1003953. doi:10.1371/journal.ppat.1003953
 Phages.org/bacteriophage/
 Pedulla ML et al. Origins of Highly Mosaic Mycobacteriophages Genomes. Cell.
2003. 113: 171-182.
 Pierce BA. Genetics a conceptual approach. W.H. Freeman and Campany New
York. 3rd edition.
 Potera C. Phage Renaissance: New Hope against Antibiotic Resistance. EHP. 2015.
ehp.niehs.nih.gov/121-a48/
References
 Schwebach RJ and Jacobs RW Jr. Phage-Finding Using Mycobacteria: A Secondary
School or Undergraduate Research Module with the Potential To Gain Scientific
Authorship. The American Biology Teacher. 2006. 68:8
 Sulakvelidze A. Bacteriaphages offer opportunities for safety managing bacterial
infections. Microbe. 2011
 Travis J. All the World’s a Phage Viruses that eat bacteria abound and surprise.
Science
News
www.phschool.com/science/science_news/articles/all_words_phage.html
online.