ANTIMICROBIAL DRUG DISCOVERY THROUGH BACTERIOPHAGE GENOMICS

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• ANTIMICROBIAL DRUG DISCOVERY THROUGH
  BACTERIOPHAGE GENOMICS

Manoj kumar ( Ph.D SCHOLAR, DM) N.D.R.I KARNAL
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Bacteriophages have been viewed not only as
important genetic but also as potential
antibacterial therapeuticOver evolutionary time
bacteriophages have developed unique proteins
that arrest critical cellular processes to commit
bacterial host metabolism to phage reproduction.
Introduction
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One can exploit this concept of phage- mediated
bacterial growth inhibition to antibiotic
discovery

• So far many phages have been sequenced and
  identified several novel polypeptide families
  that inhibited growth upon expression in bacteria

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What is the need?

• There is an urgent need to develop new classes of
  antibiotics to tackle the increase in resistance
  in many common bacterial pathogens.
• Pathogens such as Staphylococcus aureus,
  Streptococcus pneumoniae and Enterococcus
  faecalis, which are each capable of causing
  severe and even fatal infections , have become
  increasingly resistant to multiple antibiotics.

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The cellular targets for some of these
polypeptides were identified and several were
shown to be essential components of the host DNA
replication and transcription machineries

• Mimicking the growthinhibitory effect of phage
  polypeptides by a chemical compound , coupled
  with the plethora of phages on earth, will yield
  new antibiotic to combat infectious diseases.

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• Phages are recently resurfaced as the saviors of
  humankind in the best selling novel-Prey by
  Michael Crichton(2002) in which phages are used
  to destroy laboratoryescaped bacterial
  nanoparticles threatening life on earth.This
  reflects the potential of bacteriophages to be
  used as a powerful tool in dealing with
  infectious diseases of bacterial etiology

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Bacteriophage
from the greek phagein, meaning "to eat Eaters
or destroyers of bacteria First described in
1915
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SEM of Phage
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Structure of Bacteriophage

• Phage head composed of coat protein and genome
  in the core
• Genome DNA codes for enzymes and proteins
  necessary to replicate more viruses
• Tail Sheath DNA travels from head to bacteria
  through sheath
• Tail fiber helps anchor the phage on the cell
  membrane

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Phage life cycle Lytic vs Lysogenic

• Phage replicates by lytic life cycle
• Non-integration of phage genetic material
• Phage lyse host bacterium
• lytic or virulent phage
• Phage replicates by lysogenic life cycle
• Integration of phage genetic material
• temperate phages (prophages) generally larger
  than lytic phages (carry 40kb genetic material)
  

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Adsorption by Lytic Bacteriophage

• The bacteriophage binds to specific receptors on
  the bacterial cell wall.

Tail conformation changes/contracts
?central core penetrates cell wall
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Penetration

• The bacteriophage injects its genome into the
  bacterium's cytoplasm

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• Early Replication

-Phage-coded enzymes shut down hosts
DNA,RNA,protein synthesis -Early function
inovolve the takeover of the host cell and the
synthesis of early viral mRNA -Late functions
include the subsequent synthesis of other
proteins and assembly of the nucleocapsid.
-Replication phage DNA protected from host
restriction endonucleaes
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Phage Release                                 
        A bacteriophage-coded enzyme
break down the peptidoglycan in the bacterial
cell wall causing osmotic lysis.
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• Conventional Bacteriophage Therapy in
• humans
• Biomedical technology today is very different
  from what it was in the early days of phage
  therapy research
• In early days bacteriophage therapy was used by
  making bacteriophage preparation and are
  effective against P. aeruginosa, E.coli,
  S.aureus, Streptococcus and proteus

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• The first reviwed report of the therapeutic
  efficacy of PhagoBioDerm (Cock tail of lytic
  bacteriophages) was recently published
  (Markoishvili et al., 2002)
• 107 patients with ulcers failed to response to
  conventional therapy
• With PhagoBioDerm - Ulcers healed completely in
  67(70)

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Treated with phage impregnated pad
S.aureus infection
Improvement in wound healing
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• Pio bacteriophagum fluidum- one of the
  polyvalent phage preparartions produced by the
  EIBMV.The preparation targets a variety of
  bacterial pathogens, including P.aeruginosa, E.
  coli, S.aureus, Streptococcus and proteus

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Limitations of phage therapy

• 1.Emergence of bacterial strains resistant to
• particular phages. The emergence of phage
• resistant bacterial mutants was observed and
  
• the phenomenon was suggested to be a
• potential problem of phage therapy
• 
  (Summers, 1999 dHerelle,1930)

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Limitations of phage therapy

• 2.The development of phageneutralizing
• antibodies-The production of neutralizing
  
• antibodies should not be a significant
  obstacle
• during initial or relatively short-term
• therapeutic treatments at least.

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Combating the limitations

• Modernization of phage therapy
• 1. Sequencing of whole genome
• 2.Rapid and high throughput, sequence based
• Screening methodologies(e.g., microarrays)

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Contd

• Highthroughput bacteriophage genomics strategy
  is the improvised form of conventional phage
  therapy.
• Exploitation of the Concept of phage mediated
  inhibition of bacterial growth to systematically
  identify antimicrobial phage encoded
  polypeptides.

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• To tackle the increase in resistance in many
  common bacterial pathogens.
• Methicillin resistance s.aureus
• Vancomycin resistant enterococci.
• Genomic is providing a new strategy by revealing
  new
• molecular targets and peptides that are giving
  rise to novel antimicrobialdrug.

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Key steps in the genomics driven antibiotic drug
discovery process
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Key criteria to be considered in target selection

• The target should be present in a required
  spectrum of organism.
• It should be absent in humans.
• It should be essential for bacterial growth.
• It should be expressed and relevant to be
  infection process.
• Some thing about the function of target should be
  known.

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Peptides and their targets

• Product of bacteriophage T7 gene2(gp2) binds
  E.coli RNA polymerase.
• The AsiA protein of phage T4 the bacterial RNA
  polymerase s70 transcription factors.
• Protein P of phage ? and B of phage P2 each bind
  to and redirect the host DnaB helicase to there
  respective phage origin of replication.

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S.aureus DNA replication proteins identified by
antimicrobial phage ORFs
Representative of inhibitory ORF family ORF size (aa) Bacterial target identified Function of target Essentiality of target
77ORF104 ORF016 52 297 DnaI Helicase loader Essential
ORF025 ORF168 ORF240 58 74 58 DnaN DNA Pol III ß subunit Essential
ORF078 71 DnaG DNA Primase Essential
ORF140 101 PT-R14 Involved in DNA replication Not determined
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STEPS

• I. Characterization and sequencing of S.aureus
  phage genome
• 150 bacteriophages that had double stranded
  DNA genomes and were capable of lytic growth of
  S.aureus were classified as
• 1.lt20 kbp-phage p68
• 40 kbp phage 77
• 3. gt100 kbp phage G1

Genome sequencing of phage 77 was available from
a public database ,Genbank accession no. AY508486
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• 2.Functional screening for antimicrobial phage
  ORF
• Predicted phage ORFs is cloned under the control
  of an arsenite inducible promoter
• The growth of S.aureus strain RN4220
  transformants was compared on solid media in
  presence or absence of sodium arsenite

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At different time intervals, aliquots of the
cultures were plated onto TSA for determination
of colony forming units
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3 S.aureus Dnal is the cellular target of phage
77ORF104 The bacterial targets of phage ORF
induced growth inhibitionwere identified by
affinity chromatography of S.aureus lysates and
visualization of phage associated proteins on
polyacrylamide gel
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4.Validation of the interaction between Dna l and
77 ORF104 (a).Matchmaker Two Hybrid System
3 Association between 77ORF104 and Dnal was
confirmed in a yeast two hybrid assay in which
only co-expression of the two protein allowed
specific growth of saccharomyces cerevisiae on
selective medium(THAL-)
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b.) Far-western analysis, in which strong
hybiridization signal was detected between
immobilized Dnal and 32P labeled 77ORF104
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Far western analysis of Dnal and 77ORF104
32P-77ORF104
Dnal
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5 .Expression of 77ORF104 inhibits DNA Synthesis
Protein
DNA
Exponentially growing s.aureus RN4200 cells
containing cloned phage ORFs under induced and
uninduced conditions were labeled with
3H-thymidine (DNA),3H-uridine (RNA) or
35S-methionine(protein) for 15 min.
RNA
DNA

• ORF67 inhibits RNA synthesis

Protein
RNA
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6.Dnal is an essential protein in S.aureus
RpLLRe Dnal genetically modified S.aureus strain
in which the expression of dnal is under the
control of the IPTG inducible spac promoter
IPTG
- IPTG
RpLLReDnal/ pMJ8426
RN4220 /pMJ8426
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Transcompliment experiment Strain
RpLLReDnal/pMJ8426 was transformed with a plasmid
expressing either Dnal or DnaG of S.aureus
IPTG
-IPTG
RpLLReDnal/ PMJ8426Dnal
RpLLReDnal/ PMJ8426DnaG
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7.Mimicking the screened polypeptide by a
chemical compound

• The ability of these compound (from the
  commercially available libraries)
• to inhibit bacterial growth expressed as
  minimum inhibitory concentration (MIC),
  and there effect on DNA and RNA synthesis were
  determined.
• Among the 36 compounds, 11 were found to have
  MIC16µg/ml
• Two compounds that were directly identified
  from the commercialy
• available libraries are
• 1. EUROPIUMCRYPTATE
• 2. ALLOPHYCOCYANIN
• Both compounds were found to inhibit DNA
  synthesis more than RNA
• synthesis in s.aureus.
• Neither compound was significantly cytotoxic
  to human primary
• hepatocytes or to the cell lines HepG2
  and HeLa.