ANTIMICROBIAL DRUG DISCOVERY THROUGH BACTERIOPHAGE GENOMICS

1. ANTIMICROBIAL DRUG DISCOVERY THROUGH BACTERIOPHAGE GENOMICS Manojkumar ( Ph.D SCHOLAR, DM) N.D.R.I KARNAL

2. Introduction 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.

3. 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

4. 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.

5. 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.;

6. 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

7. Bacteriophage from the greek phagein, meaning "to eat“ Eaters or destroyers of bacteria First described in 1915

8. SEM of Phage

9. 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

10. 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)

11. Adsorptionby Lytic Bacteriophage The bacteriophage binds to specific receptors on the bacterial cell wall. Tail conformation changes/contracts central core penetrates cell wall

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

13. 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

14. Phage Release A bacteriophage-coded enzyme break down the peptidoglycan in the bacterial cell wall causing osmotic lysis.

16. 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

17. 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%)

18. Treated with phage impregnated pad S.aureus infection Improvement in wound healing

19. “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

20. 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)

21. 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.

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

23. 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.

24. 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.

25. Key steps in the genomics driven antibiotic drug discovery process

26. 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.

27. 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.

28. S.aureus DNA replication proteins identified by antimicrobial phage ORFs

29. 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 • ~40 kbp –phage 77 • 3. >100 kbp –phage G1 Genome sequencing of phage 77 was available from a public database ,Genbank accession no. AY508486

30. 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

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

32. 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

33. 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-)

34. b.) Far-western analysis, in which strong hybiridization signal was detected between immobilized Dnal and 32P labeled 77ORF104

35. Far –western analysis of Dnal and 77ORF104 [32P]-77ORF104 Dnal

36. 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

37. 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

38. Transcompliment experiment Strain RpLLReDnal/pMJ8426 was transformed with a plasmid expressing either Dnal or DnaG of S.aureus +IPTG -IPTG RpLLReDnal/ PMJ8426+Dnal RpLLReDnal/ PMJ8426+DnaG

39. 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. 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.