PHAGE LYSINS

Viruses that specifically infect bacteria are called bacteriophage or phage. After replicating inside the bacterial cells, phages needs to efficiently exit the bacterium to disseminate its progeny to begin a new cycle. For this purpose, double stranded deoxyribonucleic acid phages have evolved a lytic system to weaken the bacterial cell wall resulting in hypotonic bacterial lysis or lysis from within.

Phage lytic enzymes or lysins are highly efficient molecules to release it progeny phage. These enzymes target the integrity of the cell wall, and are designed to attack one of the five major bonds in the peptidoglycan. The lysins acts along with a translocase system called holin. The holin molecules are inserted in the cytoplasmic membrane forming patches, resulting in a hole through which the lysins access the peptidoglycan to cleave the specific bonds, thereby causing immediate cell lysis and release of progeny phage. 

Structure of lysins (Endolysins)

Lysins consists of two separate functional domains , one is enzymatically catalytic domain (EAD) which is N-terminal and the second is the C-terminal cell wall binding domain (CBD). EAD is responsible for for the catalytic activity and works by cleaving various bonds in the peptidoglycan, where as the CBD recognizes and specifically binds to its target bacterial cell wall receptors. Gram positive and Gram negative lysins are structurally different. Unlike Gran positive lysins that exhibit the classical structure with both the domains, Gram negative lysins only possesses EADs , they mostly lack a modular structure.

 Mechanism of action

Several types of EADs are identified which works at different sites in the peptidoglycan layer. N-acetyl muramidases, N-acetylglucosaminidases and transglycosilases work on the sugar backbone of the peptidoglycan layer whereas the other class called endopeptidases attack on the peptide bonds of the cross-linking and interpeptide bridges, N-acetylmuramoyl-L-alanine amidases work by attacking the amide bond between N-acetylmuramic acid and L-alanine causing rapid cleavage. In addition, Lysin-Holin system seen in many Gram positive infecting phages that direct the lysins to gain easy access to the cell wall from within. Finally, the bacterial cell loses rigidity leading to cell lysis and death. 

These cell wall breaching enzymes represent a step ahead in the antibacterial campaign acting as active killing mechanisms with high specificity. Moreover, phage lysins are direct, kill instantly, lacking the issues of associated resistance with no off-target effects as peptidoglycan does not exist in mammalian tissue which is a clear advantage over antibiotics and chemical preservatives. Also the biodegradable nature of these enzymes fits perfectly into its application in/on food stuffs as well as use as therapeutics.  

Lysins as Antibacterial agents

Due to the emergence of antibiotic resistance in organisms, many infectious agents have become life threatening agents in humans. To overcome theses issues, research has focused on phage derived endolysins for both topical and systemic infections in humans.

One particular pathogen that has received significant focus is Staphylococcus aureus, due to its involvement in topical skin or tissue infections, as well as systemic blood poisoning, bone, and cardiac infections. Furthermore, the rise in multidrug resistant and methicillin resistant S. aureus (MRSA), has reduced the availability of effective antibiotics. Commercially available recombinant endolysins, Staphefekt SA.100, and XDR.300 have been implemented in patients with chronic skin disease caused by S. aureus. Another endolysin, CHAPk has the potential reduce S. aureus colonization in the skin.

In contrast, Gram negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa have considered major opportunistic pathogens in burn wounds. To treat such drug resistant pathogens, novel engineered endolysin named artilysins have been suggested.  

Use of lysins in veterinary medicine

Food animals such as cattle, poultry, and swine have been found to be a major reservoir of antibiotic resistant bacteria and its specific gene that can move to people directly or indirectly by the food chain. As a consequence, the use of antibiotics in animal feed has been banned in many countries. Therefore, there is significant need for antibiotic alternatives such as endolysins for veterinary use.

Endolysins have been recommended to treat most farm animal related pathogens such as Clostridium perfringens, Streptococcus suis, Paenibacillus larvae and Salomonella species. 

Examples include;

• Endolysin phiSM101 against Clostridium perfringens
• amidase endolysin, CP25L against Lactobacillus johnsonii
• amidase endolysin PlyG against Bacillus anthracis
• amidase endolysin PlyC Streptococcus equi

Use of lysins in Agriculture

The prevalence of antibiotic resistance in the food chain process within agriculture and crop culture has led to the cause of bacterial infection in humans. For example, Multidrug resistant leaf blight rice can cause nosocomial infections in immunocompromised individuals. Thus endolysin therapy has been suggested to ensure safety of plants. 

Examples include;

• Transgenic tomato plant with CMP1 phage to prevent infection of Clavibacter michiganensis, responsible for canker.
• Transgenic potato plant with T4 phage lysin to Erwinia carotovora. 

Use of lysins in food technology

Lysins can be safely used in the food industry as a potential alternative to the chemical preservatives and antibiotics. The advantages are;

• Phages are ubiquitous in nature and are natural commensals in of humans and animal body. Their ubiquitous nature strongly support the fact that they are part of our foods and are completely safe and harmless entities.
• High specificity in their action allows attacking target bacterial cells only while not affecting the normal microbial flora unlike most of the preservatives and antibiotics do.
• Phage treatment of foods does not lead to any change in sensory, taste, organoleptic properties which may may discourage the final consumer acceptance.

Examples include;

• LinM-Ag8- immobilized phage active against  growth of Listeria monocytogenes in cantaloupe and RTE meat
• Team1, P68,LH1-MUT- phage cocktail eradicated S. aureus load after 14 days of cheddar cheese curd ripening at 4°C
• SE07-pahge application bought significant reduction of Salmonella enteritidis population in fruit juice and fresh eggs, beef, and chicken meat samples after incubation at 4°C for 48 hour.

 Use of Endolysin in Biofilm eradication

Bacteria are universally found in nature attached to surfaces such as living tissues, medical devices, industrial equipment or food. During attachment some bacteria produce extracellular polymeric substances forming a complex cluster of bacterial cells, known as biofilm.    

In clinical and food settings, biofilms are major concerns as they form on critical locations causing contamination that affects the efficacy of the established procedures; for example, the bacterial colonization on the outer surfaces of catheters. Also they cause treatment failure in surgeries and chronic wounds due to antibiotic-resistant bacteria housed within the biofilm network.

Examples include;

• endolysin LysPA26 to eliminate Psudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Escherichia coli  in the biofilm formation.
• LySMP, to treat Streptococcus suis biofilm.