ANTIMICROBIAL DRUG DISCOVERY ANTIMICROBIAL DRUG DISCOVERY THROUGH BACTERIOPHAGE THROUGH BACTERIOPHAGE

GENOMICSGENOMICS

Manoj kumar ( Ph.D SCHOLAR, DM)Manoj kumar ( Ph.D SCHOLAR, DM)N.D.R.I KARNALN.D.R.I KARNAL

Bacteriophages have been viewed not Bacteriophages have been viewed not only as important genetic but also as only as important genetic but also as potential antibacterial therapeuticpotential antibacterial therapeutic

Over evolutionary time bacteriophages Over evolutionary time bacteriophages have developed unique proteins that have developed unique proteins that arrest critical cellular processes to arrest critical cellular processes to commit bacterial host metabolism to commit bacterial host metabolism to phage reproduction. phage reproduction.

IntroductionIntroduction

One can exploit this concept of phage- mediated One can exploit this concept of phage- mediated bacterial growth inhibition to antibiotic discoverybacterial growth inhibition to antibiotic discovery

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

What is the need?What is the need?

There is an urgent need to develop new classes of There is an urgent need to develop new classes of antibiotics to tackle the increase in resistance in antibiotics to tackle the increase in resistance in many common bacterial pathogens.many common bacterial pathogens.

Pathogens such as Pathogens such as Staphylococcus aureus, Staphylococcus aureus, Streptococcus pneumoniaeStreptococcus pneumoniae and and Enterococcus Enterococcus faecalisfaecalis, which are each capable of causing , which are each capable of causing severe and even fatal infections , have become severe and even fatal infections , have become increasingly resistant to multiple antibiotics.increasingly resistant to multiple antibiotics.

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

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

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

BacteriophageBacteriophage

from the greek phagein, meaning "to eat“Eaters or destroyers of bacteria

First described in 1915

SEM of PhageSEM of Phage

Structure of BacteriophageStructure 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 LysogenicPhage life cycle: Lytic vs Lysogenic

Phage replicates by lytic life cyclePhage replicates by lytic life cycleNon-integration of phage genetic materialNon-integration of phage genetic material

Phage lyse host bacteriumPhage lyse host bacterium

lytic or virulent phagelytic or virulent phage

Phage replicates by lysogenic life cyclePhage replicates by lysogenic life cycleIntegration of phage genetic materialIntegration of phage genetic material

temperate phages (prophages) generally larger than lytic temperate phages (prophages) generally larger than lytic phages (carry ~40kb genetic material)phages (carry ~40kb genetic material)

AdsorptionAdsorption by Lytic Bacteriophageby Lytic Bacteriophage

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

Tail conformation changes/contracts central core penetrates cell wall

PenetrationPenetration

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

Early Replication 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 Conventional Bacteriophage Therapy in

humanshumans

Biomedical technology today is very different Biomedical technology today is very different from what it was in the early days of phage from what it was in the early days of phage therapy research therapy research

In early days bacteriophage therapy was used In early days bacteriophage therapy was used by making bacteriophage preparation and are by making bacteriophage preparation and are effective against effective against P. aeruginosa, E.coli, S.aureus, P. aeruginosa, E.coli, S.aureus, StreptococcusStreptococcus and and proteus proteus

The first reviwed report of the therapeutic The first reviwed report of the therapeutic efficacy of PhagoBioDerm (Cock tail of lytic efficacy of PhagoBioDerm (Cock tail of lytic bacteriophages) was recently published bacteriophages) was recently published (Markoishvili et al., 2002)(Markoishvili et al., 2002)

107 patients with ulcers – failed to response to 107 patients with ulcers – failed to response to conventional therapyconventional therapy

With PhagoBioDerm - Ulcers healed completely With PhagoBioDerm - Ulcers healed completely in 67(70%)in 67(70%)

S.aureusS.aureus infection infectionTreated with phage Treated with phage impregnated padimpregnated pad Improvement in wound healingImprovement in wound healing

““Pio bacteriophagum fluidum”- one of the Pio bacteriophagum fluidum”- one of the polyvalent phage preparartions produced by the polyvalent phage preparartions produced by the EIBMV.The preparation targets a variety of EIBMV.The preparation targets a variety of bacterial pathogens, including bacterial pathogens, including P.aeruginosaP.aeruginosa, , E. E. coli, S.aureus, Streptococcus coli, S.aureus, Streptococcus andand proteus proteus

Limitations of phage therapy Limitations of phage therapy

1.Emergence of bacterial strains resistant to 1.Emergence of bacterial strains resistant to

particular phages. The emergence of phage – particular phages. The emergence of phage –

resistant bacterial mutants was observed and resistant bacterial mutants was observed and

the phenomenon was suggested to be a the phenomenon was suggested to be a

potential problem of phage therapypotential problem of phage therapy (Summers, 1999; d’Herelle,1930)(Summers, 1999; d’Herelle,1930)

Limitations of phage therapy Limitations of phage therapy

2.The development of phage–neutralizing 2.The development of phage–neutralizing

antibodies-The production of neutralizing antibodies-The production of neutralizing

antibodies should not be a significant obstacle antibodies should not be a significant obstacle

during initial or relatively short-term during initial or relatively short-term

therapeutic treatments at least.therapeutic treatments at least.

Combating the limitationsCombating the limitations

Modernization of phage therapyModernization of phage therapy

1. Sequencing of whole genome1. Sequencing of whole genome

2.Rapid and high –throughput, sequence –based2.Rapid and high –throughput, sequence –based

Screening methodologies(e.g., microarrays)Screening methodologies(e.g., microarrays)

Contd……Contd……

High–throughput bacteriophage genomics High–throughput bacteriophage genomics strategy is the improvised form of conventional strategy is the improvised form of conventional phage therapy.phage therapy.

Exploitation of the Concept of phage –mediated Exploitation of the Concept of phage –mediated inhibition of bacterial growth to systematically inhibition of bacterial growth to systematically identify antimicrobial phage –encoded identify antimicrobial phage –encoded polypeptides.polypeptides.

To tackle the increase in resistance in many common To tackle the increase in resistance in many common bacterial pathogens.bacterial pathogens.

Methicillin resistance Methicillin resistance s.aureuss.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 Key steps in the genomics driven antibiotic drug discovery processdiscovery process

Key criteria to be considered in Key criteria to be considered in target selectiontarget selection The target should be present in a required The target should be present in a required

spectrum of organism.spectrum of organism. It should be absent in humans.It should be absent in humans. It should be essential for bacterial growth.It should be essential for bacterial growth. It should be expressed and relevant to be It should be expressed and relevant to be

infection process.infection process. Some thing about the function of target should Some thing about the function of target should

be known.be known.

Peptides and their targetsPeptides and their targets

Product of bacteriophage T7 gene2(gp2) binds E.coli RNA Product of bacteriophage T7 gene2(gp2) binds E.coli RNA polymerase.polymerase.

The AsiA protein of phage T4 the bacterial RNA polymerase The AsiA protein of phage T4 the bacterial RNA polymerase σσ70 transcription factors.70 transcription factors.

Protein P of phage Protein P of phage λλ and B of phage P2 each bind to and and B of phage P2 each bind to and redirect the host DnaB helicase to there respective phage redirect the host DnaB helicase to there respective phage origin of replication.origin of replication.

S.aureusS.aureus DNA replication proteins identified by DNA replication proteins identified by antimicrobial phage ORFsantimicrobial phage ORFs

Representative Representative of inhibitory of inhibitory ORF familyORF family

ORF sizeORF size

(aa)(aa)

Bacterial Bacterial target target identifiedidentified

Function Function of targetof target

Essentiality Essentiality of targetof target

77ORF10477ORF104

ORF016ORF016

5252

297297

DnaIDnaI Helicase Helicase loaderloader

EssentialEssential

ORF025ORF025

ORF168ORF168

ORF240ORF240

5858

7474

5858

DnaNDnaN DNA Pol DNA Pol III III ββ subunitsubunit

EssentialEssential

ORF078ORF078 7171 DnaGDnaG DNA DNA PrimasePrimase

EssentialEssential

ORF140ORF140 101101 PT-R14PT-R14 Involved Involved in DNA in DNA replicatioreplicationn

Not Not determineddetermined

II. . Characterization and Characterization and sequencing of sequencing of S.aureusS.aureus phage genomephage genome

150 bacteriophages that had 150 bacteriophages that had double stranded DNA double stranded DNA genomes and were capable genomes and were capable of lytic growth of S.aureus of lytic growth of S.aureus were classified aswere classified as

1.<20 kbp-phage p681.<20 kbp-phage p682.2. ~40 kbp –phage 77~40 kbp –phage 773. >100 kbp –phage G13. >100 kbp –phage G1

Genome sequencing of phage 77 was available from a public Genome sequencing of phage 77 was available from a public database ,Genbank accession no. AY508486database ,Genbank accession no. AY508486

STEPSSTEPS

2.Functional screening 2.Functional screening for antimicrobial phage for antimicrobial phage

ORFORF

Predicted phage ORFs is Predicted phage ORFs is cloned under the control of cloned under the control of an arsenite inducible an arsenite inducible promoter promoter

•The growth of The growth of S.aureusS.aureus strain RN4220 strain RN4220 transformants was transformants was compared on solid media in compared on solid media in presence or absence of presence or absence of sodium arsenite sodium arsenite

At different time intervals, aliquots of the cultures were plated onto TSA At different time intervals, aliquots of the cultures were plated onto TSA for determination of colony –forming unitsfor determination of colony –forming units

3 S.aureus Dnal is the cellular target 3 S.aureus Dnal is the cellular target of phage 77ORF104of phage 77ORF104

The bacterial targets of phage ORF –The bacterial targets of phage ORF –induced growth inhibitionwere identified induced growth inhibitionwere identified by affinity chromatography of by affinity chromatography of S.aureus S.aureus lysates and visualization of phage lysates and visualization of phage associated proteins on polyacrylamide associated proteins on polyacrylamide gelgel

4.Validation of the interaction 4.Validation of the interaction between Dna l and 77 between Dna l and 77 ORF104ORF104

(a).Matchmaker Two Hybrid (a).Matchmaker Two Hybrid System 3System 3

Association between Association between 77ORF104 and Dnal was 77ORF104 and Dnal was confirmed in a yeast two –confirmed in a yeast two –hybrid assay in which only hybrid assay in which only co-expression of the two co-expression of the two protein allowed specific protein allowed specific growth of growth of saccharomyces saccharomyces cerevisiaecerevisiae on selective on selective medium(THAL-) medium(THAL-)

b.) Far-western analysis, in b.) Far-western analysis, in which strong hybiridization which strong hybiridization signal was detected signal was detected between immobilized Dnal between immobilized Dnal and and 3232P labeled 77ORF104 P labeled 77ORF104

Far –western analysis of Dnal and 77ORF104Far –western analysis of Dnal and 77ORF104

[[3232P]-77ORF104P]-77ORF104DnalDnal

DNADNA

RNARNA

ProteinProtein

DNADNA

RNARNA ProteinProtein

5 .Expression of 77ORF104 inhibits DNA Synthesis5 .Expression of 77ORF104 inhibits DNA Synthesis

Exponentially growing Exponentially growing s.aureuss.aureus RN4200 cells RN4200 cells containing cloned phage containing cloned phage ORFs under induced and ORFs under induced and uninduced conditions were uninduced conditions were labeled with labeled with 33H-thymidine H-thymidine (DNA),(DNA),33H-uridine (RNA) or H-uridine (RNA) or 3535S-methionine(protein) for S-methionine(protein) for 15 min.15 min.

ORF67 inhibits RNA ORF67 inhibits RNA synthesis synthesis

6.Dnal is an essential protein in 6.Dnal is an essential protein in S.aureusS.aureus

RpLLRe Dnal genetically RpLLRe Dnal genetically modified modified S.aureusS.aureus strain strain in which the expression of in which the expression of dnal is under the control dnal is under the control of the IPTG inducible spac of the IPTG inducible spac promoter promoter

+IPTG+IPTG - IPTG- IPTG

RpLLReDnalRpLLReDnal//pMJ8426pMJ8426

RN4220RN4220//pMJ8426pMJ8426

+IPTG+IPTG -IPTG-IPTG

Transcompliment experimentTranscompliment experiment

Strain RpLLReDnal/pMJ8426 was Strain RpLLReDnal/pMJ8426 was transformed with a plasmid expressing transformed with a plasmid expressing either Dnal or DnaG of either Dnal or DnaG of S.aureusS.aureus

RpLLReDnal/RpLLReDnal/PMJ8426+DnalPMJ8426+Dnal

RpLLReDnal/RpLLReDnal/PMJ8426+DnaGPMJ8426+DnaG

7.Mimicking the screened polypeptide by a chemical compound7.Mimicking the screened polypeptide by a chemical compound

The ability of these compound (from the commercially available libraries) The ability of these compound (from the commercially available libraries) to inhibit bacterial growth expressed as minimum inhibitory concentration to inhibit bacterial growth expressed as minimum inhibitory concentration (MIC), and there effect on DNA and RNA synthesis were determined. (MIC), and there effect on DNA and RNA synthesis were determined.

Among the 36 compounds, 11 were found to have MICAmong the 36 compounds, 11 were found to have MIC≤≤1616µg/mlµg/ml

Two compounds that were directly identified from the commercialy Two compounds that were directly identified from the commercialy available libraries are:available libraries are:

1. EUROPIUMCRYPTATE1. EUROPIUMCRYPTATE2. ALLOPHYCOCYANIN2. ALLOPHYCOCYANIN

Both compounds were found to inhibit DNA synthesis more than RNA Both compounds were found to inhibit DNA synthesis more than RNA synthesis in synthesis in s.aureuss.aureus. .

Neither compound was significantly cytotoxic to human primary Neither compound was significantly cytotoxic to human primary hepatocytes or to the cell lines HepG2 and HeLa. hepatocytes or to the cell lines HepG2 and HeLa.