1. ENZYBIOTICS Review: Enzybiotics:New Weapon in the Army of Antimicrobials Reference: Asian Journal of Animal and Veterinary Advances,(Ruchi et al., 2014) Muhammad Haseeb Malik PhD Scholar (Biotechnology) BCH- 751 (2 nd ppt)

2. Sequence Introduction Antimicrobial resistance Enzybiotics & major classes Mechanism of action Beneficial applications Conclusion

3. Introduction Enzybiotic = enzyme + antibiotic Consists of lytic enzymes that are naturally present in viruses, bacteria, body fluids (tears, saliva, mucous) Mainly belong to the class of peptidoglycan hydrolases Bacteriophage enzyme with bacterial cell wall degrading capacity Help to fight bacterial or fungal diseases by using viruses or viral derived lysins and antimicrobial peptides Phage encoded endolysins attack and lyse bacteria when added exogenously

4. Introduction Novel mode of antibacterial action Highly active with explicit specificity Capacity to kill antibiotic resistant bacteria Less chances of developing bacterial resistance Main Characteristics

5. Antimicrobial Resistance

6. Bacterial Cell Wall

7. Enzybiotics

8. Enzybiotic Classes Bacteriocins Lysins Lysozymes Autolysins

9. Bacteriocins Bacterial toxins from non pathogenic bacteriaProteinaceous in natureNarrow spectrum antibioticsColicins from gram +ve bacteriaMicrocins from Archea

10. Lysins Known as EndolysinsEncoded by bacteriophages Basic enzymes cleaving peptidoglycans covalent bonds N-acetylmuramoyl-L-alanine amidases Endopeptidases N-acetylmuramidases (lysozymes) Endo-β-N-acetylglucosaminidases Lytic transglycosylases Five major classes

11. Lysozymes Catalysis of 1,4-β-linkage hydrolysis between N-actylmuramic and N-acetyl D-glucosamine is done by lysozyme by damaging bacterial cell wall Natural antibiotic as found in number of secretions (egg white) Against gram +ve pathogens (Bacillus, Streptococcus) Antibacterial, antiviral, anti inflammatory activities

12. Autolysins An enzyme hydrolyzing a biological cell or tissue’s component in which it is produced Similar in function to a lysozyme Atl – major autolysin of Staphylococcus epidermidis, S.aureus

13. Sources  Ply genes encoded proteins from Bacillus cereus bacteriophage  Bastille  TP21  TP12  All three enzymes rapidly and specifically lyse several Bacillus spps  Enzymatic activity is due to N-terminal region while C-termini are responsible for specific recognition and binding with the peptidoglycan of Bacillus spp

14. Sources  Bacillus anthracis prophage Ba02 endolysin  PlyL encoded by Bacillus anthracis genome  Capable of cleaving cell wall of several Bacillus spp  PlyC bacteriophage lysine  Exert murine hydrolase action against Streptococcus pneumoniae cell wall  Prevent colonization of group A streptococci in the URT and leads to bacterial exclusion by killing microorganisms  Mur-LH endolysin  Broad spectrum endolysin  Obtained from Lactobacillus helveticus bacteriophage

15. Mechanism of action Through bacterial cell walls without killing host (Filamentous phages) Use specific lysine enzymes (Non filamentous phages) to either:- Inhibit synthesis of peptidoglycan in cell wall of bacteria OR Hydrolyze the built peptidoglycan by Holin-endolysin system Bacteriophages progeny virions are released by

16. Phage Cycle

17. Holin-endolysin System Phage requires Holin protein and endolysin for host cell lysis Holin helps endolysin to find its substrate molecule in host cell wall Holin forms pores in inner membrane of infected host cell It results in access of lysin to the peptidoglycan causing rapid cell lysis thus releasing mature phage progeny Recombinant lysin enzymes when exogenously applied to gram +ve bacteria cause lipid lysis as no outer membrane is present to limit their access to the cell wall Due to narrow host range phage lysins selectively target specific pathogenic bacteria without affecting commensal microflora

18. Holin-endolysin System

19. Therapy

20. Lyz endolysin  Lysis of the host cell with Lyz endolysin of bacteriophage P1 is mediated by an N-terminal Trans-memberane Domain (TMD), without holin  TMD is capable of exporting endolysin to the membrane but also facilitates its release into the periplasm  N -Terminal Domain acts as a signal-arrest-release sequence  First, it directs endolysin to periplasm in membrane tethered form and then allows it to be released as a soluble active enzyme in the periplasm

21. Cell Binding Domains CBDs of Phage encoded peptidoglycan hydrolases (endolysins) have affinity for host bacterial cell wall Exclusively recognize specific ligands on the gram +ve cell wall (Bacillus cereus, L. monocytogenes, C. perfringens) CBD based Magnetic Separation (CBD – MS) Paramagnetic beads coated with recombinant Listeria phage endolysin derived CBD molecules could capture and detect more than 90% of the viable L.monocytogenes; from contaminated food samples within 20 to 40 mins

22. Beneficial Applications Role of Bacteriocin in food industry Addition of nisin and lacticin as prophylactic measure against mastitis Role of Endolysins in food industry Bio-control tool Use of Enzybiotics in farm animals

23. Conclusion

24. Q &A

25. THANKS