Download Limitations of phage therapy

ANTIMICROBIAL DRUG DISCOVERY
THROUGH BACTERIOPHAGE
GENOMICS
Manoj kumar ( Ph.D SCHOLAR, DM)
N.D.R.I KARNAL
Introduction
Bacteriophages have been viewed not
only as important genetic but also as
potential
antibacterial
therapeutic
Over evolutionary time bacteriophages
have developed unique proteins that
arrest critical cellular processes to
commit bacterial host metabolism to
phage reproduction.
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
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.
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 growth–inhibitory effect of phage
polypeptides by a chemical compound , coupled
with the plethora of phages on earth, will yield
new antibiotic to combat infectious diseases.;
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 laboratory–escaped “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
Bacteriophage
from the greek phagein, meaning "to eat“
Eaters or destroyers of bacteria
First described in 1915
SEM of Phage
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
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)
Adsorption by Lytic Bacteriophage
The bacteriophage binds to specific
receptors on the bacterial cell wall.
Tail conformation changes/contracts
central core penetrates cell wall
Penetration

The bacteriophage injects its genome into
the bacterium's cytoplasm
Early Replication
-Phage-coded enzymes shut down host’s 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
Phage Release
A bacteriophage-coded enzyme break down the
peptidoglycan in the bacterial cell wall
causing osmotic lysis.
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
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%)
S.aureus infection
Treated with phage
impregnated pad
Improvement in wound healing
“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
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; d’Herelle,1930)
Limitations of phage therapy
2.The development of phage–neutralizing
antibodies-The production of neutralizing
antibodies should not be a significant obstacle
during initial or relatively short-term
therapeutic treatments at least.
Combating the limitations
Modernization of phage therapy
1. Sequencing of whole genome
2.Rapid and high –throughput, sequence –based
Screening methodologies(e.g., microarrays)
Contd……


High–throughput
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.

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.
Key steps in the genomics driven antibiotic drug
discovery process
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.
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
σ70 transcription factors.
Protein P of phage λ and B of phage P2 each bind to and
redirect the host DnaB helicase to there respective phage
origin of replication.
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 β
subunit
Essential
ORF078
71
DnaG
DNA
Primase
Essential
ORF140
101
PT-R14
Involved
in DNA
replicatio
n
Not
determined
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.<20 kbp-phage p68
2. ~40 kbp –phage 77
3. >100 kbp –phage G1
Genome sequencing of phage 77 was available from a public database
,Genbank accession no. AY508486
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
At different time intervals, aliquots of the cultures were plated onto TSA
for determination of colony –forming units
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
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-)
b.) Far-western analysis, in
which strong hybiridization
signal was detected
between immobilized Dnal
and 32P labeled 77ORF104
Far –western analysis of Dnal and 77ORF104
Dnal
[32P]-77ORF104
5 .Expression of 77ORF104 inhibits DNA Synthesis
Protein
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.
ORF67 inhibits RNA
synthesis
DNA
RNA
DNA
RNA
Protein
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
RpLLReDnal/
pMJ8426
RN4220
/pMJ8426
- IPTG
Transcompliment experiment
Strain
RpLLReDnal/pMJ8426
was
transformed with a plasmid expressing
either Dnal or DnaG of S.aureus
RpLLReDnal/
PMJ8426+Dnal
RpLLReDnal/
PMJ8426+DnaG
+IPTG
-IPTG
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 MIC≤16µg/ml
 Two compounds that were directly identified from the commercialy
available libraries are:
1.
2.
EUROPIUMCRYPTATE
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.