LYSINS AS A NOVEL ANTIMICROBIAL TREATMENT FOR INFECTION
Jane Lim UCD School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4, Ireland
With the rise of antimicrobial resistance, healthcare professionals and scientists are searching for therapy that can both safely and effectively treat patients and combat this resistance. Lysins, also known as endolysins or murein hydrolases, are hydrolytic enzymes that lyse host cell membranes to release newly synthesised bacteriophages during the lytic cycle. These interesting enzymes are shifting into the limelight as a potential treatment for bacterial infection as advancement in antibiotic therapy begins to stagnate and resistance continues to rise. In this review article, we will discuss the function of lysins and the benefits over conventional antibiotics, whilst also reviewing the disadvantages that may limit this novel therapy which remains largely experimental.
The significance of Lysin therapy
Since the discovery of penicillin by Sir Alexander Fleming in 19281, bacterial infections have been broadly managed by antibiotics. However, due to inappropriate prescribing, excessive and unregulated agricultural use, among other reasons, there has been an alarming rise in antimicrobial resistance (AMR). According to a recent review on AMR, it has been estimated that death from bacterial infection is approximately 700,000 people per year and it is estimated to rise to 10 million by 20502. In addition to the slow developments of new antibiotics tackling AMR, clinicians are quickly running out of treatment options resulting in a global crisis.
Lysins have emerged as a candidate as potential therapy for treating infections. Similar to antibiotics, lysins have potent species-specific antibacterial activity and avoid being affected by AMR. In fact, the use of lysins as antimicrobial is not entirely novel. The study of bacteriophages has been ongoing for many centuries, beginning in the 1800s. However, it was only in the 1900s that Bacteriologist, Felix d'Herelle, who applied his findings into developing a bacteriophage therapy to treat dysentery. The name "bacteriophage" was also coined by d'Herelle, which was formed from “bacteria” and “phagein” (to eat or devour, in Greek), and was meant to imply that phages “eat” or “devour” bacteria3. However, like antimicrobials, phages can become obsolete when bacteria mutated and become resistant.
Although research and pharmacological uses of lysins have been greatly hindered due to the popularity of anitbiotics, the global interest is gradually shifting to back into its favour. Until recently, the primary treatment for infections in most countries are focused on the prescription of antibiotics. Due to the rising pandemic of antimicrobial resistance, there is now great interest in developing lysins as an alternative treatment.
Structure and mechanism of action of lysins
Bacteriophages are viruses that infect bacteria and replicate inside of bacterial host cells, hijacking their internal molecular machinery. This process may either proceed via the lytic cycle or lysogenic cycle, with the latter involving the integration of viral DNA into host cell DNA. Regardless, once newly synthesised bacteriophages are assembled and primed to infect other bacteria, an enzyme called lysin is produced which lyses the host cell to release the progenies.
To understand the mechanism of action of lysins, it is necessary to consider their structure. The structure of lysins varies between species, however all contain an N-terminal and a Cell- Binding Domain (CBD), connected by a short linker region4. The major component of bacterial cell wall is peptidoglycan or murein, providing bacteria with its unique cell shape and integrity. The N-terminal, also known as the catalytic domain, locally digests peptidoglycan to form pores, disrupting the bacterial cell wall structure and resulting in hypotonic lysis of the bacterium. The CBD of various lysins binds to different specific substrates found on the cell wall of bacteria it was produced from4. This allows lysins to exert high specificity when targeting different species and even subspecies. In addition, CBD plays a vital role in positioning the lysin such that it may be cleaved appropriately by surface enzyme to activate its catalytic abilities4.
Benefits of antibiotics
The main benefit of lysins is that bacteria do not develop resistance to them. It has been proposed that due to the evolution of bacteriophage alongside bacteria for millions of years, the CBD targets essential molecules in the cell wall, such as peptidoglycan, which unlike some surface proteins, do not change even when bacteria mutate. For example, in a study by Garcia et al., it was shown that lysins which target Streptococcus pneumoniae specifically attach to choline, an essential component of the cell wall5.
The second benefit of lysins lies in their specificity of lytic action which is unlike antibiotics that act on a wide range of bacteria. Lysins are highly specific, targeting only the desired pathogenic microbe it was intended for while leaving endogenous microflora unscathed5. This is important in preventing development of opportunistic infections such as Clostridium Difficile, a common severe hospital acquired infection in patients on long-term antibiotic treatment (such as fluoroquinolones, cephalosporins and clindamycin).
Building on the second benefit, lysins can be used more freely in prophylactic therapy as they do not pose the threat of development of AMR and disruption of commensal microflora. However, as lysins have specific action, clinicians would have to first determine the possible pathogenic strains causing infection in the patient. Patient selection would have to identify risk and stratify patients accordingly. As we do not have enough information on this topic, further research is required to determine the feasibility and effectiveness of lysins as prophylactics.
Most lysins are still in the pre-clinical phase of research. Thus far, some potential has been realised. Cheng et al. report administration of LysEF-P10 to mice provided protection against lethal vancomycin-resistant Enterococcus Faecalis (VREF)6. It also further reduced the number of colonies in the mice’s gut and prevents microbiota imbalances caused by VREF6. By using modern molecular techniques such as polymerase chaing reaction and subcloning, it is possible to swap CBD and N-terminal domains to synthesise novel lysins that target specific microbes and have different lytic properties. Of note, lysins may contain more than one N-terminal domain to a single CBD. The catalytic function of a lysin is not limited to a single catalytic function, but rather may be manipulated to contain multiple catalytic action to manage difficult strains. As bacteria evolve and become more multidrug resistant, we too can synthesise novel lysins whose functions are rarely affected by mutating strains.
Hospitalised patients are at risk of bacterial infection. Hospital-acquired infections occur frequently resulting in complications that prolong inpatient admission and increase morbidity and mortality rates7. In addition to the rising AMR, some antimicrobials are also ineffective in managing pathogens that produces biofilms. Biofilms are a layer of extracellular polymeric substance matrix generated by pathogenic microorganism that serve to act as a protective barrier. Pseudomonas aeruginosa is a prominent example and accounts for ~10% of all hospital-acquired infection7. Interestingly, lysins have shown efficacy in managing biofilm-producing bacteria; Sass et al. showed the successful use of recombinant phage lysin from phi11 to effectively hydrolyse Staphylococcus and its biofilm8.
Furthermore, lysins can be used in synergy with other types of lysins or antibiotics to target multiple resistant strains. In one study, a novel lysin MV-L has effectively lysed several Staphylococcus aureus strains, including methicillin-resistant and vancomycin-resistant species, and a subset of vancomycin-intermediate Staphyloccous aureus in growing conditions9. Furthermore, synergistic effects have been noted when co-administered with glycopeptide antibiotics. Thus, using a combination of lysins and specific antibiotics, multidrug-resistant bacterial infections may be effectively treated. As mentioned, administration of appropriate lysin-antibiotic treatment would first require identify the causative strains of infection. This may not be feasible in mixed infections that compromise of many different strains.
Lysins can also be applied in the agricultural industry. Almost all classes of antibiotics used by humans are also used on animals in agriculture and has been estimated to exceed human usage worldwide10. This widespread use of antibiotics in prevention of infectious disease has resulted in a staggering rise of AMR which is then transmitted to humans through many different routes, namely the food-borne route and potentially direct contact. Using lysins which have greater specificity than antimicrobials, specific common pathogens can be eliminated without risking further development of AMR. The development of lysin Ply3626 which has shown lytic activity against several strains of Clostridium perfringens, a common causative organism of food poisoning11. This lysin, which can be easily introduced to the animals through food or feed, can effectively manage infectious disease in animals and prevent development and spread of resistant zoonotic AMR strains.
As lysins are proteins, they may invoke a reactionary immune response when introduced systemically. Effectiveness of repeated usage may be dampened as lysins are targeted and removed by immune cells. Loeffler et al. however report that despite the presence of immune response following administration, effectiveness of lytic activity of Cpl-1 was slowed but not blocked when introduced repeatedly into rabbits13. Similar results were achieved when experiments were done on Bacillus anthracis and an Streptococcus pyogenes-specific lysins13.
Natural lysins generally have poor activity due to the presence of an outer membrane that prevents it from effectively crossing over and binding to species such as gram-negative bacteria. Lysins can only interact with cell wall either by disrupting the outer membrane or manipulating channels found on outer membrane surface. There has been progress in developing lysins that can tackle gram-negative pathogens such as artilysins which are natural lysins fused to peptides or other proteins. Yang et al. describe an artilysin effective against Pseudomonas aeruginosa synthesised by fusing sheep myeloid antimicrobial peptide to endolysin KZ14414. The additional antimicrobial peptide can facilitate the crossing of the artilysin across the outer membrane and effective eliminate the gram-negative pathogen without eliciting inflammatory response14. Limited research on artilysin has shown potential, however further research is required before it can be truly recognised as an alternate treatment.
Despite the current potential of lysins, many questions remain unanswered. Future research is necessary to confirm efficacy and benefits over the use of antibiotics. There are also many unknown variables and undocumented side effects that may accompany the use of lysins. Going forward, more evidence will emerge with randomised controlled trials and it may inform new avenues for investigations of lysins as a novel antimicrobial therapy or additional streams of research.
In conclusion, considering the potential benefits of lysins, they stand as a strong contender in the future antimicrobial treatment for infection.
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