ENZYBIOTICS

Review: Enzybiotics:New Weapon in the Army of AntimicrobialsReference: Asian Journal of Animal and Veterinary

Advances ,(Ruchi et al., 2014)

Muhammad Haseeb MalikPhD Scholar (Biotechnology)

BCH- 751(2nd ppt)

Sequence

• Introduction• Antimicrobial resistance • Enzybiotics & major classes• Mechanism of action• Beneficial applications• Conclusion

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

• Novel mode of antibacterial action• Highly active with explicit specificity• Capacity to kill antibiotic resistant

bacteria• Less chances of developing

bacterial resistance

Main Characteristics

Bacteriocins

Lysins

Lysozymes

Autolysins

Bacteriocins

Bacterial toxins from non pathogenic bacteria

Proteinaceous in nature

Narrow spectrum antibiotics

Colicins from gram +ve bacteria

Microcins from Archea

Lysins

Known as Endolysins

Encoded by bacteriophages

Basic enzymes cleaving peptidoglycans covalent bonds

Five major classes• N-acetylmuramoyl-L-alanine amidases• Endopeptidases• N-acetylmuramidases (lysozymes)• Endo-β-N-acetylglucosaminidases• Lytic transglycosylases

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

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

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

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

Mechanism of action

Bacteriophages progeny virions are released by

• 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

Phage Cycle

Holin-endolysin SystemPhage 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 progenyRecombinant 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 wallDue to narrow host range phage lysins selectively target specific pathogenic bacteria without affecting commensal microflora

Holin-endolysin System

Therapy

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

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

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

Conclusion

Q

&A

THANKS