A1258689905BGTJt

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What is a Phage?
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Phage Landing and Infusion of Viral DNA
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Phage Lifecycle Lytic vs Lysogenic

• Lytic
• 1) Virus infuses DNA into host cell
• 2) Virus quickly hijacks cellular replication
  machinery
• 3) Replicates its own viral DNA
• First reverse translation if retrovirus
• 4) Cell wall bursts releasing newly synthesized
  viral phages into surrounding medium

• Lytic
• 1) Virus infuses DNA into host cell
• 2) Virus quickly hijacks cellular replication
  machinery
• 3) Replicates its own viral DNA
• First reverse translation if retrovirus
• 4) Cell wall bursts releasing newly synthesized
  viral phages into surrounding medium

• Lysogenic
• 1) Virus infuses DNA into host cell
• 2) Viral DNA incorporates itself into host DNA,
  and exists dormantly
• 3) Stress or stimulation induces virus DNA to be
  come active
• 4) Lytic life cycle begins

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Bacterial lysis via Lysin

• Lysin
• Viral enzyme that facilitate bacterial host lysis
• Hydrolyze bonds essential for peptidoglycan
  integrity
• Amidases are a class Lysins
• Well conserved N-terminal catalytic domain
• Highly divergent C-terminal binding domain
• high binding constant (KA 3.6 108, similar to
  Ab binding)
• recognizes species specific carbohydrates that
  are essential for viability
• Therefore evolution of resistance to lysins
  unlikely

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PlyG Identification

• Screened expression libraries for possible lysing
  agents of RSVF-1
• All lytic clones identified contained a 702 bp
  ORF encoding a homologue of N-acetylmuramoyl-L-ala
  nine amidase (27kDa)
• class of phage lysins
• Homology restricted to catalytic N-terminal
  portion of protein
• Table 1
• high specificity and lytic activity for all B.
  anthracis strains
• similar specificity and activity for B. cereus
  RSVF-1
• RSVF-1 chosen for remainder of experiments
  probably for safety
• note titer measurement analog to agglutination
  titer Immunology text (p.245)
• Here titer refers to lysis rather than
  agglutination

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Assay for Lytic Phage Plaque assay - Lytic phage
are enumerated by a plaque assay. A plaque is a
clear area which results from the lysis of
bacteria (Figure 4). Each plaque arises from a
single infectious phage. The infectious particle
that gives rise to a plaque is called a pfu
(plaque forming unit).
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Fig. 1

• Conserved N-terminal catalytic domain
• Divergent C-terminal binding domain

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Fig. 1
Circle represents void of bacterial cells as a
result of PlyG lysis (0.5 U of PlyG added to
plate)
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Fig. 2
20U PlyG
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Fig. 2
2U PlyG
Buffer
CFU correlation to A600
2U PlyG

• 17000 fold decrease w/in 20 sec
• near sterilization at 2 minutes

• RLU
• relative light units
• signal emitted by Luciferin in presence of ATP
  released by lysed bacterial cells
• CFU
• Colony forming units (part (b) inset due to
  strong detergent mix?)

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Fig. 3
Phase contrast microscopy
No PlyG added
1 min after 5U added
15 min after 5U added
1 min after 5U added
10 min after 5U added
Transmission Electron Micrographs (TEM)
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Fig. 4
In Vivo mouse (BALB/c) infection/survival model
No B. anthracis added
150U PlyG added
50U PlyG added
No PlyG added
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Fig. 5
PlyG mediated spore killing and detection
10U PlyG
5 min after 2U PlyG added (b) (c)
60 min after 2U PlyG added
D-alanine
L-alanine
D-alanine inhibits spore germination
L-alanine induces spore germination

• RLU
• relative light units
• signal emitted by Luciferin in presence of ATP
  released by lysed bacterial cells
• CFU
• Colony forming units (part (b) inset due to
  strong detergent mix?)

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Conclusion

• PlyG specific B. anthracis lysin identified and
  purified
• Activity and specificity verified in vitro
• Luciferin activity due to ATP release from lysed
  cells is a specific assay of PlyG activity
• PlyG rapidly kills g-sensitive bacteria in vivo
• Resistance to PlyG not observed
• even under induced mutagenesis conditions which
  caused gt1000 fold increases in acquired
  antibiotic resistance as compared to uninduced
  mutagenesis.
• g phage resistance observed to evolve
• Therefore PlyG likely to target essential
  cell-wall molecules
• And therefore intrinsic PlyG resistance is
  unlikely
• Only germinating spores are sensitive to PlyG
• RLU of only 100 RSVF-1 spores detected with
  handheld luminometer represents potential
  detection techniques of battlefield and home
  use of B. anthracis

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PlyG Lysin Specificity

• Table 1
• high specificity and lytic activity for all B.
  anthracis strains
• similar specificity and activity for B. cereus
  RSVF-1
• RSVF-1 chosen for remainder of experiments
  probably for safety
• note titer measurement analog to agglutination
  titer Immunology text (p.245)
• Here titer refers to lysis rather than
  agglutination
• Figure 1
• next slide

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PHAGE MULTIPLICATION CYCLE A. Lytic or Virulent
Phages 1. Definition - Lytic or virulent phages
are phages which can only multiply on bacteria
and kill the cell by lysis at the end of the life
cycle. 2. Life cycle - The life cycle of a lytic
phage is illustrated in Figure 3 . a. Eclipse
period - During the eclipse phase, no infectious
phage particles can be found either inside or
outside the bacterial cell. The phage nucleic
acid takes over the host biosynthetic machinery
and phage specified m-RNA's and proteins are
made. There is an orderly expression of phage
directed macromolecular synthesis, just as one
sees in animal virus infections. Early m-RNA's
code for early proteins which are needed for
phage DNA synthesis and for shutting off host
DNA, RNA and protein biosynthesis. In some cases
the early proteins actually degrade the host
chromosome. After phage DNA is made late m-RNA's
and late proteins are made. The late proteins are
the structural proteins that comprise the phage
as well as the proteins needed for lysis of the
bacterial cell. b. Intracellular Accumulation
Phase - In this phase the nucleic acid and
structural proteins that have been made are
assembled and infectious phage particles
accumulate within the cell. c. Lysis and Release
Phase - After a while the bacteria begin to lyse
due to the accumulation of the phage lysis
protein and intracellular phage are released into
the medium. The number of particles released per
infected bacteria may be as high as 1000.