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Research Progress on Strategies for Improving the Enzyme Properties of Bacteriophage Endolysins
1Shunde Women and Children's Hospital, Guangdong Medical University, Foshan 528300, P.R. China
2Dongguan Key Laboratory of Public Health Laboratory Science, School of Public Health, Guangdong Medical University, Dongguan 523808, P.R. China
J. Microbiol. Biotechnol. 2024; 34(6): 1189-1196
Published June 28, 2024 https://doi.org/10.4014/jmb.2312.12050
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Bacteriophages are viruses that specifically invade and infect bacteria. They are divided into temperate phages and virulent phages. Virulent phages could proliferate rapidly in bacterial cells, produce endolysins to lyse the bacteria and release offspring phages [1, 2]. Phage endolysins are known as peptidoglycan-degrading proteins that could rapidly induce the lysis and death of bacteria by targeting the chemical bonds of peptidoglycan (PG) on the bacterial cell wall [3]. Because these PG hydrolases lyse ‘‘from within’’, they are referred to as ‘‘endolysins’’ or simply ‘‘lysins’’ [4].
According to the Gram-staining of host bacteria, bacteriophage endolysins are divided into two groups. One could be called GP-Lysins which are produced by bacteriophages of Gram-positive bacteria (GP-phages). The other one could be called GN-Lysins which are produced by bacteriophages of Gram-negative bacteria (GN-phages). GP-Lysins usually have a modular structure. As shown in Fig. 1A, GP-Lysins contain two domains, namely the N-terminal catalytic domain (EAD) and the C-terminal cell wall binding domain (CBD), which are connected by a short peptide. The EAD is capable of acting on most chemical bonds of the PG network in the bacterial cell wall to cause bacterial lysis [1], while the CBD is responsible for targeting the endolysin to the substrate and conferring specificity for recognizing host cells. The high specificity of lysins offers it an advantage over conventional antibiotics as endolysins do not disturb the normal microflora. Typically, these is a flexible interdomain linker sequence between the EAD and the CBD [5]. Usually, endolysins have only one EAD and one CBD, but some lysins were found to have more than one CBD or EAD arranged in different order [6-8].
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Fig. 1. Structures of bacteriophage lysins and their catalytic sites. (A) Basic structures of GP-Lysins; (B) Basic structures of GN-Lysins; (C) Catalytic sites of phage lysins (1: N-acetyl cyclase; 2: N-acetyl-β-D-aminoglucosidase; 3: Nacetylcytidyl-L-alanine amidase; 4: peptide chain endonuclease; 5: peptidase; 6: transglycosidase); where: GlcNAc: Nacetylglucosamine; MurNAc: N-acetylcytidylic acid; L-Ala: L-alanine; D-Glu: D-glutamic acid; L-Lys: L-lysine; D-Ala: Dalanine; L-Gly: L-glycine.
As shown in Fig. 1B, GN-lysins usually only have one globular structure with a single EAD [9, 10]. Gram-negative bacteria have an outer membrane (OM) composed of lipopolysaccharide (LPS) compared to Gram-positive bacteria. The OM of Gram-negative bacteria effectively prevents GN-lysins from acting on the cell wall externally. To date, only a few GN-lysins are able to lyse Gram-negative bacteria without the help of OM permeants. These GN-lysins contain amphipathic helical structures or carry positively charged groups which confer the ability to penetrate or disrupt the bacterial OM, thereby accessing and degrading the PG layer and ultimately leading to bacterial lysis and death [11, 12]. For example, the α-helical structure formed by the C-terminus of lysin AcLys enables it to penetrate the OM. Additionally, the positively charged groups present in the C-terminus enhance its ability to penetrate the OM [11, 13]. However, there are also a few of GN-Lysins owning a modular structure with a CBD at the N-terminus and a EAD at the C-terminus [14, 15]. These GN-Lysins obtain high lytic activities towards Gram-negative bacteria due to the presence of CBD which helps the lysins get close to the substrate [14, 16]. Different endolysins differ considerably in protein structure and enzymatic activity. Usually, the endolysin exhibits only one hydrolytic activity. As shown in Fig. 1C and Table 1, depending on the site of action of the EAD on the bacterial cell wall, lysins can be classified into four categories [17]. They are N-acetylmuramidase, transglycosylase, amidase and endopeptidase.
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Table 1 . GN-Lysins that can pass through the outer membrane of Gram-negative bacteria.
Host bacteria Lysins References Acinetobacter baumannii LysAB3, LysAB4 [20] PlyAB1 [21] PlyF307 [22] Lys-ABP1-01 [23] Abgp46 [15] LysPA26 [24] LysAB54 [25] Pseudomonas aeruginosa LysPA26 [24] Cytrobacter fowleri CfP1 [26] Escherichia coli T5 [27] SPN9CC [28]
Many GP-lysins exhibit rapid "contact-dependent" bactericidal activity, capable of reducing bacteria to undetectable levels within seconds [25, 26]. Comparatively, the bacterial killing kinetics of GN-lysins are in a slower manner. Endolysins contain at least one structural domain responsible for the enzymatic cleavage of PG, also known as murein, which is the major structural component of the bacterial cell wall. PGs form a vesicle-like structure that surrounds the bacterial cytoplasmic membrane and imparts the necessary mechanical resistance to avoid cell lysis [27]. Therefore, uncontrolled breakdown of the murein structure typically results in osmotic cell lysis. Significantly, exogenous addition of endolysins to a susceptible host can be exploited to produce lysis from without due to the high osmotic pressure within the cell [28]. In 2001, Nelson
Strategies for Improving Enzyme Properties of Endolysins
GP-Lysins usually show good antibacterial activities against Gram-positive bacteria. They generally have a modular structure, consisting of multiple EADs and CBD, which can be used as the basis for modifying the functional domains, such as deletion or addition or direct mutation of domains, chimeolysins and so on to improve the bactericidal activity, bactericidal spectrum, stability, solubility and adaptability to the environment.
The OM of Gram-negative bacteria effectively prevents most lysins from reaching the PG layer and acting on the cell wall externally. Though there were a few of GN-Lysins reported to have the ability to penetrate the OM by themselves, it should be noted that these lysins often suffer from limited availability and insufficient cleavage activity [11, 12, 34, 35]. Therefore, how lysins can pass through the OM of Gram-negative bacteria become a key point. Here in this review, the methods that can improve the activity of lysins against Gram-negative bacteria are summarized including fusion with the membrane-penetrating peptides (MPPs), fusion with domains targeting OM receptors or transport systems, encapsulation strategy and using OM-penetrating agents. The methods used to improve GP-Lysins are also applicable to GN-Lysins and vice versa. It is also possible to explore the development of lysins with excellent performances by interoperability of multiple methods.
Deletion of Cell Wall-Binding Domain or Changing the Net Charge of Catalytic Domain
Classical lysins require a CBD that targets the catalytic domain to the PG layer. For these classical lysins, specificity and bacteriolytic activity require the strong binding of the CBD to the cell wall. Once the CBD was deleted, the catalytic domain would lose lytic activity. Therefore, these classical lysins are CBD-dependent. However, some lysins are CBD-independent and the removal of the CBD conversely expanded the lytic spectrum and increased the lytic activity. For example, the truncated lysin PlyGBS90-1 consisting of the EAD and the last 13 amino acids at the C-terminal end of the wild lysin PlyGBS obtained a 28-fold higher lytic activity against group B
Addition of Domains
Increasing the affinity of endolysins to target cells by modifying the CBDs could improve the lytic efficacy. Schmelcher
DNA Mutagenesis
Various DNA mutagenesis methods including site-directed mutation and random mutagenesis, had been used to produce mutants of lysins with enhanced lytic activity and/or stronger thermostability. The mutation of 15 amino acids in the CBD of the pneumococcal lysin Cpl-7, resulted in the inversion of the sign of the charge of CBD, was performed to generate the mutant lysin Cpl-7S which obtained increased lytic activities and an expanded lytic spectrum against
Chimerization of Domains
The domains of endolysins can be exchanged or recombined with other domains to generate chimeolysins with desirable properties. This approach could increase the catalytic capacity of the EAD and/or the ability of the CBD to recognize substrates. Especially, the substitution of CBD is the most commonly used means. New CBDs could alter the bactericidal activity and host specificity of the chimeric lysin. Chimeolysins usually could obtain broader lytic spectrums, higher lytic activities or other improvements. To further improve the lytic activity, Ply187AN was fused with the CBD of lysin LysK to generate the chimeolysin Ply187AN-KSH3b which was more active against
Chimeolysins compensate for the deficiencies of single lysins and avoid degradation of exogenous gene expression products by the host cell protease system. It has been shown that chimerization of lysins with similar hosts may broaden their lytic spectrums and chimerization of lysins with different hosts may change and broaden their efficacy in a wide range of genera [10].
Fusion to the Membrane-Penetrating Peptides
The main component of the OM of Gram-negative bacteria is LPS of which the stability is maintained by the ionic interactions between divalent cations and phosphate groups and the hydrophobic accumulation of lipid A. The majority of these MPP have a positive net charge, but there were also anionic antimicrobial peptides [45]. These peptides possess both hydrophilic and hydrophobic regions on their surfaces. The cationic section of the peptide interacts with the negatively charged bacterial cell surface through electrostatic interactions, while the hydrophobic section interacts with the lipids present in the bacterial membrane. As a consequence, the MPP could help the lysin to pass through the LPS layer, promote a change in the OM and reach the periplasmic space and degrade the PG leading to the eventual death of the bacteria [46, 47]. In a 2019 study, it was found that the fusion of hydrophobic amino acids at the C-terminus of the lysin Lysep3 also increased antimicrobial activity against
Therefore, MPP plays a key role in this strategy for enhancing the lysins’ activities. In an early study, seven MPPs were screened for fusions with two modular lysins (Lysin OBPgp279 from
There is no fixed pattern for the fusion of the MPP with lysins. The fusion position may be the N-terminal or C-terminal end or both the two ends of the lysin. The best fusion pattern for different lysins should be explored experimentally. Fusion with MPP is not only applicable to GN-Lysins but also to GP-Lysins.
Fusion with Domains Targeting OM Receptors or Transport Systems
Bacteriocins are antimicrobial peptides or proteins produced by bacteria that could inhibit or kill the closely related bacteria [51]. Bacteriocins can act by targeting specific receptors on the surface of susceptible bacteria, leading to the disruption of membrane integrity and subsequent cell death [52, 53]. The fusion protein of lysin and bacteriocin could exploit the delivery systems used by bacteriocins to translocate through the OM and reach the periplasm to induce PG cleavage [54]. For example, pyocin S2 (PyS2), which is a bacteriocin of
The OM transporter protein of bacteria could also help the lysin to cross the OM of the target bacteria and reach the PG layer. Pesticin is a bacteriocin produced by
Moreover, the fusion of the receptor-binding proteins (RBPs) with endolysins, coined as “Innolysin”, has recently been introduced as a novel approach to target Gram-negative bacteria [57]. Phages specifically recognize bacterial surface receptors through RBPs. The Pb5 monomer located in the tail of phage T5 is a good RBP that specifically and stably binds to the bacterial receptor FhuA [58]. The RBP Pb5 was fused with 23 phage endolysins to construct 228 novel RBP-endolysin hybrids. Among these innolysins, the innolysin Ec21 which was fused by the endolysin of phage T5 with RBP Pb5 had the highest antibacterial activity reducing
Encapsulation Strategy
In addition to the fusion engineering approach, GP-lysins can be encapsulated to enhance their stabilities and permeabilities. Liposomes have spherical structures composed of one or more phospholipid bilayers with a core of water [59]. It is safe for the human body and is therefore widely used to deliver proteins, enzymes, vitamins and antioxidants [60]. Liposomes are known to be able to penetrate bacterial membranes by membrane fusion. Depending on the surface charge of the target site, liposomes can be prepared in cationic or anionic form. Most Gram-negative bacterial membranes have an anionic surface and cationic liposomes have a higher antibacterial effect on Gram-negative bacteria than anionic liposomes because of the stronger interaction between cationic liposomes and negatively charged bacterial membranes [61-63]. The
Nanoparticles can also be used to encapsulate lysins. LysMR-5 was encapsulated by nanoparticles (Alg-Chi NPs) consisting of alginate and chitosan, resulting in enhanced bactericidal activity. The T4 lysozyme was coupled to cellulose nanocrystals (CNC) resulting in higher thermal stability and bactericidal activity against
Combination with the OM Permeabilizers
The OM of Gram-negative bacteria effectively prevents most lysins from reaching the PG layer and acting on the cell wall externally. To increase the permeability of OM is one solution to solve this problem. Divalent cations (Mg2+ and Ca2+) are known to be crucial for the integrity of the bacterial OM. There are many kinds of OM permeabilizers which are generally divided into two categories. The first category is polyvalent cationic compounds such as polymyxin and its derivatives, aminoglycosides and lysine polymers. They can compete to replace the bivalent cations for the interaction with the anionic LPS molecules, leading to disorganization of the OM [70, 71].
The second category of OM permeabilizers is chelating agent. Chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria. Among numerous chelating agents, EDTA (ethylenediaminetetraacetic acid) is the most commonly used. EDTA removes divalent cations from their binding sites, causing OM disruption. Several studies had shown that EDTA had the strongest effect on cell wall penetration for lysins [15, 72, 73]. Weak organic acids in protonated form are also used as chelating agents. In general, most lysins are inactive at low pH, but the
OM permeabilizers could not only enhance the bactericidal activity of lysins, but could also broaden the lytic spectrum. The lysin ABgp46 only had the antibacterial activity against
Conclusion
Bacteriophage endolysins are promising alternatives to antibiotics. The strategies that can be used to improve the enzyme properties (bactericidal activity, lysis spectrum, stability and targeting the substrate, etc) of bacteriophage endolysins are summarized as follows: Deletion of the cell wall-binding domain (For the CBD-independent lysins) or changing the net charge of catalytic domain; Addition of domains (Mainly addition of the cell wall-binding domain); DNA mutagenesis (Site-directed mutation and random mutagenesis); Chimerization of domains; Fusion to the MPPs (Helping lysins to reach the substrate); Fusion with domains targeting OM receptors or transport systems (Helping lysins to reach the substrate); Encapsulation strategy; Combination with the OM permeabilizers (Helping lysins to reach the substrate). Among these strategies, those related to adding other reagents in the enzyme reaction system, like encapsulation of lysins and the usage of OM permeabilizers, have to take into account the safety of those reagents, especially OM permeabilizers. The strategies used to improve GP-Lysins are also applicable to GN-Lysins and vice versa. It is also possible to explore the development of lysins with excellent performances by interoperability of multiple methods.
Acknowledgments
This work was supported by the Talents Recruitment Grant of Yangfan Plan of Guangdong Province (4YF16003G) and Dongguan Science and Technology of Social Development Program (20231800936342).
Author Contributions
Yulu Wang: Writing-original Manuscript, Writing-review and editing. Xue Wang: Editing the Manuscript. Xin Liu: Writing-review and editing the Manuscript. Bokun Lin: Writing-review and editing the Manuscript. The final version of the manuscript was read and approved by all authors.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
- Loessner MJ. 2005. Bacteriophage endolysins--current state of research and applications.
Curr. Opin. Microbiol. 8 : 480-487. - Young I, Wang I, Roof WD. 2000. Phages will out: strategies of host cell lysis.
Trends Microbiol. 8 : 120-128. - Nelson D, Loomis L, Fischetti VA. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme.
Proc. Natl. Acad. Sci. USA 98 : 4107-4112. - Nelson DC, Schmelcher M, Rodriguez-Rubio L, Klumpp J, Pritchard DG, Dong S,
et al . 2012. Endolysins as antimicrobials.Adv. Virus Res. 83 : 299-365. - Korndörfer IP, Danzer J, Schmelcher M, Zimmer M, Skerra A, Loessner MJ. 2006. The crystal structure of the bacteriophage PSA endolysin reveals a unique fold responsible for specific recognition of
Listeria cell walls.J. Mol. Biol. 364 : 678-689. - Becker SC, Foster-Frey J, Donovan DM. 2008. The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA.
FEMS Microbiol. Lett. 287 : 185-191. - Pritchard DG, Dong S, Kirk MC, Cartee RT, Baker JR. 2007. LambdaSa1 and LambdaSa2 prophage lysins of
Streptococcus agalactiae .Appl. Environ. Microbiol. 73 : 7150-7154. - Oechslin F, Daraspe J, Giddey M, Moreillon P, Resch G. 2013. In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of
Streptococcus agalactiae bacteremia.Antimicrob. Agents Chemother. 57 : 6276-6283. - Shannon R, Radford DR, Balamurugan S. 2020. Impacts of food matrix on bacteriophage and endolysin antimicrobial efficacy and performance.
Crit. Rev. Food Sci. Nutr. 60 : 1631-1640. - Schmelcher M, Donovan DM, Loessner MJ. 2012. Bacteriophage endolysins as novel antimicrobials.
Future Microbiol. 7 : 1147-1171. - Sykilinda NN, Nikolaeva AY, Shneider MM, Mishkin DV, Patutin AA, Popov VO,
et al . 2018. Structure of an acinetobacter broadrange prophage endolysin reveals a C-terminal α-helix with the proposed role in activity against live bacterial cells.Viruses 10 : 309. - Raz A, Serrano A, Hernandez A, Euler CW, Fischetti VA. 2019. Isolation of phage lysins that effectively kill
Pseudomonas aeruginosa in mouse models of lung and skin infection.Antimicrob. Agents Chemother. 63 : e00024-19. - Lai MJ, Lin NT, Hu A, Soo PC, Chen LK, Chen LH,
et al . 2011. Antibacterial activity ofAcinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both gram-positive and gram-negative bacteria.Appl. Microbiol. Biotechnol. 90 : 529-539. - Walmagh M, Briers Y, dos Santos SB, Azeredo J, Lavigne R. 2012. Characterization of modular bacteriophage endolysins from Myoviridae phages OBP, 201φ2-1 and PVP-SE1.
PLoS One 7 : e36991. - Briers Y, Schmelcher M, Loessner MJ, Hendrix J, Engelborghs Y, Volckaert G,
et al . 2009. The high-affinity peptidoglycan binding domain ofPseudomonas phage endolysin KZ144.Biochem. Biophys. Res. Commun. 383 : 187-191. - Gerstmans H, Grimon D, Gutiérrez D, Lood C, Rodríguez A, van Noort V,
et al . 2020. A VersaTile-driven platform for rapid hit-tolead development of engineered lysins.Sci. Adv. 6 : eaaz1136. - Gutiérrez D, Fernández L, Rodríguez A, García P. 2018. Are phage lytic proteins the secret weapon to kill
Staphylococcus aureus ?mBio 9 : e01923-17. - García JL, García E, Arrarás A, García P, Ronda C, López R. 1987. Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin.
J. Virol. 61 : 2573-2580. - Pritchard DG, Dong S, Baker JR, Engler JA. 2004. The bifunctional peptidoglycan lysin of
Streptococcus agalactiae bacteriophage B30.Microbiology (Reading) 150 : 2079-2087. - Alrafaie AM, Stafford GP. 2023. Enterococcal bacteriophage: A survey of the tail associated lysin landscape.
Virus Res. 327 : 199073. - Paradis-Bleau C, Cloutier I, Lemieux L, Sanschagrin F, Laroche J, Auger M,
et al . 2007. Peptidoglycan lytic activity of thePseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase.FEMS Microbiol. Lett. 266 : 201-209. - Becker SC, Dong S, Baker JR, Foster-Frey J, Pritchard DG, Donovan DM. 2009. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.
FEMS Microbiol. Lett. 294 : 52-60. - Loessner MJ, Wendlinger G, Scherer S. 1995. Heterogeneous endolysins in
Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes.Mol. Microbiol. 16 : 1231-1241. - Navarre WW, Ton-That H, Faull KF, Schneewind O. 1999. Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity.
J. Biol. Chem. 274 : 15847-15856. - Pastagia M, Schuch R, Fischetti VA, Huang DB. 2013. Lysins: the arrival of pathogen-directed anti-infectives.
J. Med. Microbiol. 62 : 1506-1516. - Fischetti VA. 2008. Bacteriophage lysins as effective antibacterials.
Curr. Opin. Microbiol. 11 : 393-400. - Vollmer W, Blanot D, de Pedro MA. 2008. Peptidoglycan structure and architecture.
FEMS Microbiol. Rev. 32 : 149-167. - Fernandes S, São-José C. 2016. More than a hole: the holin lethal function may be required to fully sensitize bacteria to the lytic action of canonical endolysins.
Mol. Microbiol. 102 : 92-106. - Ferro S, Amorico T, Deo P. 2018. Role of food sanitising treatments in inducing the 'viable but nonculturablé state of microorganisms.
Food Control 91 : 321-329. - Maciejewska B, Olszak T, Drulis-Kawa Z. 2018. Applications of bacteriophages versus phage enzymes to combat and cure bacterial infections: an ambitious and also a realistic application?
Appl. Microbiol. Biotechnol. 102 : 2563-2581. - Schuch R, Nelson D, Fischetti VA. 2002. A bacteriolytic agent that detects and kills
Bacillus anthracis .Nature 418 : 884-889. - Domenech M, García E, Moscoso M. 2011. In vitro destruction of
Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases.Antimicrob. Agents Chemother. 55 : 4144-4148. - Yang H, Linden SB, Wang J, Yu J, Nelson DC, Wei H. 2015. A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method.
Sci. Rep. 5 : 17257. - Kim S, Lee DW, Jin JS, Kim J. 2020. Antimicrobial activity of LysSS, a novel phage endolysin, against
Acinetobacter baumannii andPseudomonas aeruginosa .J. Glob. Antimicrob. Resist. 22 : 32-39. - Wu M, Hu K, Xie Y, Liu Y, Mu D, Guo H,
et al . 2018. A novel phage PD-6A3, and its endolysin Ply6A3, with extended lytic activity againstAcinetobacter baumannii .Front. Microbiol. 9 : 3302. - Cheng Q, Fischetti VA. 2007. Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci.
Appl. Microbiol. Biotechnol. 74 : 1284-1291. - Mayer MJ, Garefalaki V, Spoerl R, Narbad A, Meijers R. 2011. Structure-based modification of a
Clostridium difficile -targeting endolysin affects activity and host range.J. Bacteriol. 193 : 5477-5486. - Schmelcher M, Tchang VS, Loessner MJ. 2011. Domain shuffling and module engineering of
Listeria phage endolysins for enhanced lytic activity and binding affinity.Microb. Biotechnol. 4 : 651-662. - Rodríguez-Rubio L, Martínez B, Rodríguez A, Donovan DM, García P. 2012. Enhanced staphylolytic activity of the
Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5.Appl. Environ. Microbiol. 78 : 2241-2248. - Díez-Martínez R, de Paz HD, Bustamante N, García E, Menéndez M, García P. 2013. Improving the lethal effect of cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module.
Antimicrob. Agents Chemother. 57 : 5355-5365. - Love MJ, Coombes D, Manners SH, Abeysekera GS, Billington C, Dobson RCJ. 2021. The molecular basis for
Escherichia coli O157:H7 phage FAHEc1 endolysin function and protein engineering to increase thermal stability.Viruses 13 : 1101. - Mao J, Schmelcher M, Harty WJ, Foster-Frey J, Donovan DM. 2013. Chimeric Ply187 endolysin kills
Staphylococcus aureus more effectively than the parental enzyme.FEMS Microbiol Lett. 342 : 30-36. - Dong Q, Wang J, Yang H, Wei C, Yu J, Zhang Y,
et al . 2015. Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci.Microb. Biotechnol. 8 : 210-220. - Fernandes S, Proença D, Cantante C, Silva FA, Leandro C, Lourenço S,
et al . 2012. Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistantStaphylococcus aureus .Microb. Drug Resist. 18 : 333-343. - Harris F, Dennison SR, Phoenix DA. 2009. Anionic antimicrobial peptides from eukaryotic organisms.
Curr. Protein Pept. Sci. 10 : 585-606. - Mahlapuu M, Håkansson J, Ringstad L, Björn C. 2016. Antimicrobial peptides: An emerging category of therapeutic agents.
Front. Cell Infect. Microbiol. 6 : 194. - Bechinger B. 2015. The SMART model: Soft membranes adapt and respond, also transiently, in the presence of antimicrobial peptides.
J. Pept. Sci. 21 : 346-355. - Briers Y, Walmagh M, Van Puyenbroeck V, Cornelissen A, Cenens W, Aertsen A,
et al . 2014. Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens.mBio 5 : e01379-01314. - Yan G, Yang R, Fan K, Dong H, Gao C, Wang S,
et al . 2019. External lysis ofEscherichia coli by a bacteriophage endolysin modified with hydrophobic amino acids.AMB Express. 9 : 106. - Mancoš M, Šramková Z, Peterková D, Vidová B, Godány AJB. 2020. Functional expression and purification of tailor-made chimeric endolysin with the broad antibacterial spectrum.
Biologia 75 : 2031-2043. - Cotter PD, Ross RP, Hill C. 2013. Bacteriocins - a viable alternative to antibiotics?
Nat. Rev. Microbiol. 11 : 95-105. - Vincent PA, Morero RD. 2009. The structure and biological aspects of peptide antibiotic microcin J25.
Curr. Med. Chem. 16 : 538-549. - Parks WM, Bottrill AR, Pierrat OA, Durrant MC, Maxwell A. 2007. The action of the bacterial toxin, microcin B17, on DNA gyrase.
Biochimie 89 : 500-507. - Heselpoth RD, Euler CW, Schuch R, Fischetti VA. 2019. Lysocins: Bioengineered antimicrobials that deliver lysins across the outer membrane of Gram-negative bacteria.
Antimicrob. Agents Chemother. 63 : e00342-19. - Lukacik P, Barnard TJ, Keller PW, Chaturvedi KS, Seddiki N, Fairman JW,
et al . 2012. Structural engineering of a phage lysin that targets gram-negative pathogens.Proc. Natl. Acad. Sci. USA 109 : 9857-9862. - Yan G, Liu J, Ma Q, Zhu R, Guo Z, Gao C,
et al . 2017. The N-terminal and central domain of colicin A enables phage lysin to lyseEscherichia coli extracellularly.Antonie Van Leeuwenhoek 110 : 1627-1635. - Zampara A, Sørensen MCH, Grimon D, Antenucci F, Vitt AR, Bortolaia V,
et al . 2020. Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria.Sci. Rep. 10 : 12087. - Plançon L, Janmot C, le Maire M, Desmadril M, Bonhivers M, Letellier L,
et al . 2002. Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.J. Mol. Biol. 318 : 557-569. - Mozafari MR, Johnson C, Hatziantoniou S, Demetzos C. 2008. Nanoliposomes and their applications in food nanotechnology.
J. Liposome Res. 18 : 309-327. - Mozafari MR, Flanagan J, Matia-Merino L, Awati A, Singh H. 2010. Recent trends in the lipid-based nanoencapsulation of antioxidants and their role in foods.
J. Sci. Food Agric. 86 : 2038-2045. - Alhajlan M, Alhariri M, Omri A. 2013. Efficacy and safety of liposomal clarithromycin and its effect on
Pseudomonas aeruginosa virulence factors.Antimicrob. Agents Chemother. 57 : 2694-2704. - Solleti VS, Alhariri M, Halwani M, Omri A. 2015. Antimicrobial properties of liposomal azithromycin for
Pseudomonas infections in cystic fibrosis patients.J. Antimicrob. Chemother. 70 : 784-796. - Rajendran V, Rohra S, Raza M, Hasan GM, Dutt S, Ghosh PC. 2015. Stearylamine liposomal delivery of monensin in combination with free artemisinin eliminates blood stages of
Plasmodium falciparum in culture andP. berghei infection in murine malaria.Antimicrob Agents Chemother. 60 : 1304-1318. - Bai J, Yang E, Chang PS, Ryu S. 2019. Preparation and characterization of endolysin-containing liposomes and evaluation of their antimicrobial activities against gram-negative bacteria.
Enzyme Microb Technol. 128 : 40-48. - Kaur J, Kour A, Panda JJ, Harjai K, Chhibber S. 2020. Exploring endolysin-loaded alginate-chitosan nanoparticles as future remedy for staphylococcal infections.
AAPS PharmSciTech. 21 : 233. - Abouhmad A, Dishisha T, Amin MA, Hatti-Kaul R. 2017. Immobilization to positively charged cellulose nanocrystals enhances the antibacterial activity and stability of hen egg white and T4 lysozyme.
Biomacromolecules 18 : 1600-1608. - Agnihotri SA, Mallikarjuna NN, Aminabhavi TM. 2004. Recent advances on chitosan-based micro- and nanoparticles in drug delivery.
J. Control Release 100 : 5-28. - Ragelle H, Vandermeulen G, Préat V. 2013. Chitosan-based siRNA delivery systems.
J. Control Release. 172 : 207-218. - Gondil VS, Dube T, Panda JJ, Yennamalli RM, Harjai K, Chhibber S. 2020. Comprehensive evaluation of chitosan nanoparticle based phage lysin delivery system; a novel approach to counter
S. pneumoniae infections.Int. J. Pharm. 573 : 118850. - Vaara, M. 1992. Agents that increase the permeability of the outer membrane.
Microbiol. Rev. 56 : 395-411. - Briers Y, Walmagh M, Lavigne R. 2011. Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against
Pseudomonas aeruginosa .J. Appl. Microbiol. 110 : 778-785. - Lim JA, Shin H, Heu S, Ryu S. 2014. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria.
J. Microbiol. Biotechnol. 24 : 803-811. - Walmagh M, Boczkowska B, Grymonprez B, Briers Y, Drulis-Kawa Z, Lavigne R. 2013. Characterization of five novel endolysins from Gram-negative infecting bacteriophages.
Appl. Microbiol. Biotechnol. 97 : 4369-4375. - Oliveira H, Thiagarajan V, Walmagh M, Sillankorva S, Lavigne R, Neves-Petersen MT,
et al . 2014. A thermostableSalmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.PLoS One 9 : e108376. - Oliveira H, Vilas Boas D, Mesnage S, Kluskens LD, Lavigne R, Sillankorva S,
et al . 2016. Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity.Front. Microbiol. 7 : 208.
Related articles in JMB

Article
Review
J. Microbiol. Biotechnol. 2024; 34(6): 1189-1196
Published online June 28, 2024 https://doi.org/10.4014/jmb.2312.12050
Copyright © The Korean Society for Microbiology and Biotechnology.
Research Progress on Strategies for Improving the Enzyme Properties of Bacteriophage Endolysins
Yulu Wang1,2, Xue Wang2, Xin Liu2*, and Bokun Lin1,2*
1Shunde Women and Children's Hospital, Guangdong Medical University, Foshan 528300, P.R. China
2Dongguan Key Laboratory of Public Health Laboratory Science, School of Public Health, Guangdong Medical University, Dongguan 523808, P.R. China
Correspondence to:Xin Liu, lx@gdmu.edu.cn
Bokun Lin, bklin@gdmu.edu.cn
Abstract
Bacterial resistance to commonly used antibiotics is one of the major challenges to be solved today. Bacteriophage endolysins (Lysins) have become a hot research topic as a new class of antibacterial agents. They have promising applications in bacterial infection prevention and control in multiple fields, such as livestock and poultry farming, food safety, clinical medicine and pathogen detection. However, many phage endolysins display low bactericidal activities, short half-life and narrow lytic spectrums. Therefore, some methods have been used to improve the enzyme properties (bactericidal activity, lysis spectrum, stability and targeting the substrate, etc) of bacteriophage endolysins, including deletion or addition of domains, DNA mutagenesis, chimerization of domains, fusion to the membrane-penetrating peptides, fusion with domains targeting outer membrane transport systems, encapsulation, the usage of outer membrane permeabilizers. In this review, research progress on the strategies for improving their enzyme properties are systematically presented, with a view to provide references for the development of lysins with excellent performances.
Keywords: Phage, endolysin, bactericidal activity, lysis spectrum, outer membrane permeabilizers
Introduction
Bacteriophages are viruses that specifically invade and infect bacteria. They are divided into temperate phages and virulent phages. Virulent phages could proliferate rapidly in bacterial cells, produce endolysins to lyse the bacteria and release offspring phages [1, 2]. Phage endolysins are known as peptidoglycan-degrading proteins that could rapidly induce the lysis and death of bacteria by targeting the chemical bonds of peptidoglycan (PG) on the bacterial cell wall [3]. Because these PG hydrolases lyse ‘‘from within’’, they are referred to as ‘‘endolysins’’ or simply ‘‘lysins’’ [4].
According to the Gram-staining of host bacteria, bacteriophage endolysins are divided into two groups. One could be called GP-Lysins which are produced by bacteriophages of Gram-positive bacteria (GP-phages). The other one could be called GN-Lysins which are produced by bacteriophages of Gram-negative bacteria (GN-phages). GP-Lysins usually have a modular structure. As shown in Fig. 1A, GP-Lysins contain two domains, namely the N-terminal catalytic domain (EAD) and the C-terminal cell wall binding domain (CBD), which are connected by a short peptide. The EAD is capable of acting on most chemical bonds of the PG network in the bacterial cell wall to cause bacterial lysis [1], while the CBD is responsible for targeting the endolysin to the substrate and conferring specificity for recognizing host cells. The high specificity of lysins offers it an advantage over conventional antibiotics as endolysins do not disturb the normal microflora. Typically, these is a flexible interdomain linker sequence between the EAD and the CBD [5]. Usually, endolysins have only one EAD and one CBD, but some lysins were found to have more than one CBD or EAD arranged in different order [6-8].
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Figure 1. Structures of bacteriophage lysins and their catalytic sites. (A) Basic structures of GP-Lysins; (B) Basic structures of GN-Lysins; (C) Catalytic sites of phage lysins (1: N-acetyl cyclase; 2: N-acetyl-β-D-aminoglucosidase; 3: Nacetylcytidyl-L-alanine amidase; 4: peptide chain endonuclease; 5: peptidase; 6: transglycosidase); where: GlcNAc: Nacetylglucosamine; MurNAc: N-acetylcytidylic acid; L-Ala: L-alanine; D-Glu: D-glutamic acid; L-Lys: L-lysine; D-Ala: Dalanine; L-Gly: L-glycine.
As shown in Fig. 1B, GN-lysins usually only have one globular structure with a single EAD [9, 10]. Gram-negative bacteria have an outer membrane (OM) composed of lipopolysaccharide (LPS) compared to Gram-positive bacteria. The OM of Gram-negative bacteria effectively prevents GN-lysins from acting on the cell wall externally. To date, only a few GN-lysins are able to lyse Gram-negative bacteria without the help of OM permeants. These GN-lysins contain amphipathic helical structures or carry positively charged groups which confer the ability to penetrate or disrupt the bacterial OM, thereby accessing and degrading the PG layer and ultimately leading to bacterial lysis and death [11, 12]. For example, the α-helical structure formed by the C-terminus of lysin AcLys enables it to penetrate the OM. Additionally, the positively charged groups present in the C-terminus enhance its ability to penetrate the OM [11, 13]. However, there are also a few of GN-Lysins owning a modular structure with a CBD at the N-terminus and a EAD at the C-terminus [14, 15]. These GN-Lysins obtain high lytic activities towards Gram-negative bacteria due to the presence of CBD which helps the lysins get close to the substrate [14, 16]. Different endolysins differ considerably in protein structure and enzymatic activity. Usually, the endolysin exhibits only one hydrolytic activity. As shown in Fig. 1C and Table 1, depending on the site of action of the EAD on the bacterial cell wall, lysins can be classified into four categories [17]. They are N-acetylmuramidase, transglycosylase, amidase and endopeptidase.
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Table 1 . GN-Lysins that can pass through the outer membrane of Gram-negative bacteria..
Host bacteria Lysins References Acinetobacter baumannii LysAB3, LysAB4 [20] PlyAB1 [21] PlyF307 [22] Lys-ABP1-01 [23] Abgp46 [15] LysPA26 [24] LysAB54 [25] Pseudomonas aeruginosa LysPA26 [24] Cytrobacter fowleri CfP1 [26] Escherichia coli T5 [27] SPN9CC [28]
Many GP-lysins exhibit rapid "contact-dependent" bactericidal activity, capable of reducing bacteria to undetectable levels within seconds [25, 26]. Comparatively, the bacterial killing kinetics of GN-lysins are in a slower manner. Endolysins contain at least one structural domain responsible for the enzymatic cleavage of PG, also known as murein, which is the major structural component of the bacterial cell wall. PGs form a vesicle-like structure that surrounds the bacterial cytoplasmic membrane and imparts the necessary mechanical resistance to avoid cell lysis [27]. Therefore, uncontrolled breakdown of the murein structure typically results in osmotic cell lysis. Significantly, exogenous addition of endolysins to a susceptible host can be exploited to produce lysis from without due to the high osmotic pressure within the cell [28]. In 2001, Nelson
Strategies for Improving Enzyme Properties of Endolysins
GP-Lysins usually show good antibacterial activities against Gram-positive bacteria. They generally have a modular structure, consisting of multiple EADs and CBD, which can be used as the basis for modifying the functional domains, such as deletion or addition or direct mutation of domains, chimeolysins and so on to improve the bactericidal activity, bactericidal spectrum, stability, solubility and adaptability to the environment.
The OM of Gram-negative bacteria effectively prevents most lysins from reaching the PG layer and acting on the cell wall externally. Though there were a few of GN-Lysins reported to have the ability to penetrate the OM by themselves, it should be noted that these lysins often suffer from limited availability and insufficient cleavage activity [11, 12, 34, 35]. Therefore, how lysins can pass through the OM of Gram-negative bacteria become a key point. Here in this review, the methods that can improve the activity of lysins against Gram-negative bacteria are summarized including fusion with the membrane-penetrating peptides (MPPs), fusion with domains targeting OM receptors or transport systems, encapsulation strategy and using OM-penetrating agents. The methods used to improve GP-Lysins are also applicable to GN-Lysins and vice versa. It is also possible to explore the development of lysins with excellent performances by interoperability of multiple methods.
Deletion of Cell Wall-Binding Domain or Changing the Net Charge of Catalytic Domain
Classical lysins require a CBD that targets the catalytic domain to the PG layer. For these classical lysins, specificity and bacteriolytic activity require the strong binding of the CBD to the cell wall. Once the CBD was deleted, the catalytic domain would lose lytic activity. Therefore, these classical lysins are CBD-dependent. However, some lysins are CBD-independent and the removal of the CBD conversely expanded the lytic spectrum and increased the lytic activity. For example, the truncated lysin PlyGBS90-1 consisting of the EAD and the last 13 amino acids at the C-terminal end of the wild lysin PlyGBS obtained a 28-fold higher lytic activity against group B
Addition of Domains
Increasing the affinity of endolysins to target cells by modifying the CBDs could improve the lytic efficacy. Schmelcher
DNA Mutagenesis
Various DNA mutagenesis methods including site-directed mutation and random mutagenesis, had been used to produce mutants of lysins with enhanced lytic activity and/or stronger thermostability. The mutation of 15 amino acids in the CBD of the pneumococcal lysin Cpl-7, resulted in the inversion of the sign of the charge of CBD, was performed to generate the mutant lysin Cpl-7S which obtained increased lytic activities and an expanded lytic spectrum against
Chimerization of Domains
The domains of endolysins can be exchanged or recombined with other domains to generate chimeolysins with desirable properties. This approach could increase the catalytic capacity of the EAD and/or the ability of the CBD to recognize substrates. Especially, the substitution of CBD is the most commonly used means. New CBDs could alter the bactericidal activity and host specificity of the chimeric lysin. Chimeolysins usually could obtain broader lytic spectrums, higher lytic activities or other improvements. To further improve the lytic activity, Ply187AN was fused with the CBD of lysin LysK to generate the chimeolysin Ply187AN-KSH3b which was more active against
Chimeolysins compensate for the deficiencies of single lysins and avoid degradation of exogenous gene expression products by the host cell protease system. It has been shown that chimerization of lysins with similar hosts may broaden their lytic spectrums and chimerization of lysins with different hosts may change and broaden their efficacy in a wide range of genera [10].
Fusion to the Membrane-Penetrating Peptides
The main component of the OM of Gram-negative bacteria is LPS of which the stability is maintained by the ionic interactions between divalent cations and phosphate groups and the hydrophobic accumulation of lipid A. The majority of these MPP have a positive net charge, but there were also anionic antimicrobial peptides [45]. These peptides possess both hydrophilic and hydrophobic regions on their surfaces. The cationic section of the peptide interacts with the negatively charged bacterial cell surface through electrostatic interactions, while the hydrophobic section interacts with the lipids present in the bacterial membrane. As a consequence, the MPP could help the lysin to pass through the LPS layer, promote a change in the OM and reach the periplasmic space and degrade the PG leading to the eventual death of the bacteria [46, 47]. In a 2019 study, it was found that the fusion of hydrophobic amino acids at the C-terminus of the lysin Lysep3 also increased antimicrobial activity against
Therefore, MPP plays a key role in this strategy for enhancing the lysins’ activities. In an early study, seven MPPs were screened for fusions with two modular lysins (Lysin OBPgp279 from
There is no fixed pattern for the fusion of the MPP with lysins. The fusion position may be the N-terminal or C-terminal end or both the two ends of the lysin. The best fusion pattern for different lysins should be explored experimentally. Fusion with MPP is not only applicable to GN-Lysins but also to GP-Lysins.
Fusion with Domains Targeting OM Receptors or Transport Systems
Bacteriocins are antimicrobial peptides or proteins produced by bacteria that could inhibit or kill the closely related bacteria [51]. Bacteriocins can act by targeting specific receptors on the surface of susceptible bacteria, leading to the disruption of membrane integrity and subsequent cell death [52, 53]. The fusion protein of lysin and bacteriocin could exploit the delivery systems used by bacteriocins to translocate through the OM and reach the periplasm to induce PG cleavage [54]. For example, pyocin S2 (PyS2), which is a bacteriocin of
The OM transporter protein of bacteria could also help the lysin to cross the OM of the target bacteria and reach the PG layer. Pesticin is a bacteriocin produced by
Moreover, the fusion of the receptor-binding proteins (RBPs) with endolysins, coined as “Innolysin”, has recently been introduced as a novel approach to target Gram-negative bacteria [57]. Phages specifically recognize bacterial surface receptors through RBPs. The Pb5 monomer located in the tail of phage T5 is a good RBP that specifically and stably binds to the bacterial receptor FhuA [58]. The RBP Pb5 was fused with 23 phage endolysins to construct 228 novel RBP-endolysin hybrids. Among these innolysins, the innolysin Ec21 which was fused by the endolysin of phage T5 with RBP Pb5 had the highest antibacterial activity reducing
Encapsulation Strategy
In addition to the fusion engineering approach, GP-lysins can be encapsulated to enhance their stabilities and permeabilities. Liposomes have spherical structures composed of one or more phospholipid bilayers with a core of water [59]. It is safe for the human body and is therefore widely used to deliver proteins, enzymes, vitamins and antioxidants [60]. Liposomes are known to be able to penetrate bacterial membranes by membrane fusion. Depending on the surface charge of the target site, liposomes can be prepared in cationic or anionic form. Most Gram-negative bacterial membranes have an anionic surface and cationic liposomes have a higher antibacterial effect on Gram-negative bacteria than anionic liposomes because of the stronger interaction between cationic liposomes and negatively charged bacterial membranes [61-63]. The
Nanoparticles can also be used to encapsulate lysins. LysMR-5 was encapsulated by nanoparticles (Alg-Chi NPs) consisting of alginate and chitosan, resulting in enhanced bactericidal activity. The T4 lysozyme was coupled to cellulose nanocrystals (CNC) resulting in higher thermal stability and bactericidal activity against
Combination with the OM Permeabilizers
The OM of Gram-negative bacteria effectively prevents most lysins from reaching the PG layer and acting on the cell wall externally. To increase the permeability of OM is one solution to solve this problem. Divalent cations (Mg2+ and Ca2+) are known to be crucial for the integrity of the bacterial OM. There are many kinds of OM permeabilizers which are generally divided into two categories. The first category is polyvalent cationic compounds such as polymyxin and its derivatives, aminoglycosides and lysine polymers. They can compete to replace the bivalent cations for the interaction with the anionic LPS molecules, leading to disorganization of the OM [70, 71].
The second category of OM permeabilizers is chelating agent. Chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria. Among numerous chelating agents, EDTA (ethylenediaminetetraacetic acid) is the most commonly used. EDTA removes divalent cations from their binding sites, causing OM disruption. Several studies had shown that EDTA had the strongest effect on cell wall penetration for lysins [15, 72, 73]. Weak organic acids in protonated form are also used as chelating agents. In general, most lysins are inactive at low pH, but the
OM permeabilizers could not only enhance the bactericidal activity of lysins, but could also broaden the lytic spectrum. The lysin ABgp46 only had the antibacterial activity against
Conclusion
Bacteriophage endolysins are promising alternatives to antibiotics. The strategies that can be used to improve the enzyme properties (bactericidal activity, lysis spectrum, stability and targeting the substrate, etc) of bacteriophage endolysins are summarized as follows: Deletion of the cell wall-binding domain (For the CBD-independent lysins) or changing the net charge of catalytic domain; Addition of domains (Mainly addition of the cell wall-binding domain); DNA mutagenesis (Site-directed mutation and random mutagenesis); Chimerization of domains; Fusion to the MPPs (Helping lysins to reach the substrate); Fusion with domains targeting OM receptors or transport systems (Helping lysins to reach the substrate); Encapsulation strategy; Combination with the OM permeabilizers (Helping lysins to reach the substrate). Among these strategies, those related to adding other reagents in the enzyme reaction system, like encapsulation of lysins and the usage of OM permeabilizers, have to take into account the safety of those reagents, especially OM permeabilizers. The strategies used to improve GP-Lysins are also applicable to GN-Lysins and vice versa. It is also possible to explore the development of lysins with excellent performances by interoperability of multiple methods.
Acknowledgments
This work was supported by the Talents Recruitment Grant of Yangfan Plan of Guangdong Province (4YF16003G) and Dongguan Science and Technology of Social Development Program (20231800936342).
Author Contributions
Yulu Wang: Writing-original Manuscript, Writing-review and editing. Xue Wang: Editing the Manuscript. Xin Liu: Writing-review and editing the Manuscript. Bokun Lin: Writing-review and editing the Manuscript. The final version of the manuscript was read and approved by all authors.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

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Table 1 . GN-Lysins that can pass through the outer membrane of Gram-negative bacteria..
Host bacteria Lysins References Acinetobacter baumannii LysAB3, LysAB4 [20] PlyAB1 [21] PlyF307 [22] Lys-ABP1-01 [23] Abgp46 [15] LysPA26 [24] LysAB54 [25] Pseudomonas aeruginosa LysPA26 [24] Cytrobacter fowleri CfP1 [26] Escherichia coli T5 [27] SPN9CC [28]
References
- Loessner MJ. 2005. Bacteriophage endolysins--current state of research and applications.
Curr. Opin. Microbiol. 8 : 480-487. - Young I, Wang I, Roof WD. 2000. Phages will out: strategies of host cell lysis.
Trends Microbiol. 8 : 120-128. - Nelson D, Loomis L, Fischetti VA. 2001. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme.
Proc. Natl. Acad. Sci. USA 98 : 4107-4112. - Nelson DC, Schmelcher M, Rodriguez-Rubio L, Klumpp J, Pritchard DG, Dong S,
et al . 2012. Endolysins as antimicrobials.Adv. Virus Res. 83 : 299-365. - Korndörfer IP, Danzer J, Schmelcher M, Zimmer M, Skerra A, Loessner MJ. 2006. The crystal structure of the bacteriophage PSA endolysin reveals a unique fold responsible for specific recognition of
Listeria cell walls.J. Mol. Biol. 364 : 678-689. - Becker SC, Foster-Frey J, Donovan DM. 2008. The phage K lytic enzyme LysK and lysostaphin act synergistically to kill MRSA.
FEMS Microbiol. Lett. 287 : 185-191. - Pritchard DG, Dong S, Kirk MC, Cartee RT, Baker JR. 2007. LambdaSa1 and LambdaSa2 prophage lysins of
Streptococcus agalactiae .Appl. Environ. Microbiol. 73 : 7150-7154. - Oechslin F, Daraspe J, Giddey M, Moreillon P, Resch G. 2013. In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of
Streptococcus agalactiae bacteremia.Antimicrob. Agents Chemother. 57 : 6276-6283. - Shannon R, Radford DR, Balamurugan S. 2020. Impacts of food matrix on bacteriophage and endolysin antimicrobial efficacy and performance.
Crit. Rev. Food Sci. Nutr. 60 : 1631-1640. - Schmelcher M, Donovan DM, Loessner MJ. 2012. Bacteriophage endolysins as novel antimicrobials.
Future Microbiol. 7 : 1147-1171. - Sykilinda NN, Nikolaeva AY, Shneider MM, Mishkin DV, Patutin AA, Popov VO,
et al . 2018. Structure of an acinetobacter broadrange prophage endolysin reveals a C-terminal α-helix with the proposed role in activity against live bacterial cells.Viruses 10 : 309. - Raz A, Serrano A, Hernandez A, Euler CW, Fischetti VA. 2019. Isolation of phage lysins that effectively kill
Pseudomonas aeruginosa in mouse models of lung and skin infection.Antimicrob. Agents Chemother. 63 : e00024-19. - Lai MJ, Lin NT, Hu A, Soo PC, Chen LK, Chen LH,
et al . 2011. Antibacterial activity ofAcinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both gram-positive and gram-negative bacteria.Appl. Microbiol. Biotechnol. 90 : 529-539. - Walmagh M, Briers Y, dos Santos SB, Azeredo J, Lavigne R. 2012. Characterization of modular bacteriophage endolysins from Myoviridae phages OBP, 201φ2-1 and PVP-SE1.
PLoS One 7 : e36991. - Briers Y, Schmelcher M, Loessner MJ, Hendrix J, Engelborghs Y, Volckaert G,
et al . 2009. The high-affinity peptidoglycan binding domain ofPseudomonas phage endolysin KZ144.Biochem. Biophys. Res. Commun. 383 : 187-191. - Gerstmans H, Grimon D, Gutiérrez D, Lood C, Rodríguez A, van Noort V,
et al . 2020. A VersaTile-driven platform for rapid hit-tolead development of engineered lysins.Sci. Adv. 6 : eaaz1136. - Gutiérrez D, Fernández L, Rodríguez A, García P. 2018. Are phage lytic proteins the secret weapon to kill
Staphylococcus aureus ?mBio 9 : e01923-17. - García JL, García E, Arrarás A, García P, Ronda C, López R. 1987. Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin.
J. Virol. 61 : 2573-2580. - Pritchard DG, Dong S, Baker JR, Engler JA. 2004. The bifunctional peptidoglycan lysin of
Streptococcus agalactiae bacteriophage B30.Microbiology (Reading) 150 : 2079-2087. - Alrafaie AM, Stafford GP. 2023. Enterococcal bacteriophage: A survey of the tail associated lysin landscape.
Virus Res. 327 : 199073. - Paradis-Bleau C, Cloutier I, Lemieux L, Sanschagrin F, Laroche J, Auger M,
et al . 2007. Peptidoglycan lytic activity of thePseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase.FEMS Microbiol. Lett. 266 : 201-209. - Becker SC, Dong S, Baker JR, Foster-Frey J, Pritchard DG, Donovan DM. 2009. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.
FEMS Microbiol. Lett. 294 : 52-60. - Loessner MJ, Wendlinger G, Scherer S. 1995. Heterogeneous endolysins in
Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes.Mol. Microbiol. 16 : 1231-1241. - Navarre WW, Ton-That H, Faull KF, Schneewind O. 1999. Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity.
J. Biol. Chem. 274 : 15847-15856. - Pastagia M, Schuch R, Fischetti VA, Huang DB. 2013. Lysins: the arrival of pathogen-directed anti-infectives.
J. Med. Microbiol. 62 : 1506-1516. - Fischetti VA. 2008. Bacteriophage lysins as effective antibacterials.
Curr. Opin. Microbiol. 11 : 393-400. - Vollmer W, Blanot D, de Pedro MA. 2008. Peptidoglycan structure and architecture.
FEMS Microbiol. Rev. 32 : 149-167. - Fernandes S, São-José C. 2016. More than a hole: the holin lethal function may be required to fully sensitize bacteria to the lytic action of canonical endolysins.
Mol. Microbiol. 102 : 92-106. - Ferro S, Amorico T, Deo P. 2018. Role of food sanitising treatments in inducing the 'viable but nonculturablé state of microorganisms.
Food Control 91 : 321-329. - Maciejewska B, Olszak T, Drulis-Kawa Z. 2018. Applications of bacteriophages versus phage enzymes to combat and cure bacterial infections: an ambitious and also a realistic application?
Appl. Microbiol. Biotechnol. 102 : 2563-2581. - Schuch R, Nelson D, Fischetti VA. 2002. A bacteriolytic agent that detects and kills
Bacillus anthracis .Nature 418 : 884-889. - Domenech M, García E, Moscoso M. 2011. In vitro destruction of
Streptococcus pneumoniae biofilms with bacterial and phage peptidoglycan hydrolases.Antimicrob. Agents Chemother. 55 : 4144-4148. - Yang H, Linden SB, Wang J, Yu J, Nelson DC, Wei H. 2015. A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method.
Sci. Rep. 5 : 17257. - Kim S, Lee DW, Jin JS, Kim J. 2020. Antimicrobial activity of LysSS, a novel phage endolysin, against
Acinetobacter baumannii andPseudomonas aeruginosa .J. Glob. Antimicrob. Resist. 22 : 32-39. - Wu M, Hu K, Xie Y, Liu Y, Mu D, Guo H,
et al . 2018. A novel phage PD-6A3, and its endolysin Ply6A3, with extended lytic activity againstAcinetobacter baumannii .Front. Microbiol. 9 : 3302. - Cheng Q, Fischetti VA. 2007. Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci.
Appl. Microbiol. Biotechnol. 74 : 1284-1291. - Mayer MJ, Garefalaki V, Spoerl R, Narbad A, Meijers R. 2011. Structure-based modification of a
Clostridium difficile -targeting endolysin affects activity and host range.J. Bacteriol. 193 : 5477-5486. - Schmelcher M, Tchang VS, Loessner MJ. 2011. Domain shuffling and module engineering of
Listeria phage endolysins for enhanced lytic activity and binding affinity.Microb. Biotechnol. 4 : 651-662. - Rodríguez-Rubio L, Martínez B, Rodríguez A, Donovan DM, García P. 2012. Enhanced staphylolytic activity of the
Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5.Appl. Environ. Microbiol. 78 : 2241-2248. - Díez-Martínez R, de Paz HD, Bustamante N, García E, Menéndez M, García P. 2013. Improving the lethal effect of cpl-7, a pneumococcal phage lysozyme with broad bactericidal activity, by inverting the net charge of its cell wall-binding module.
Antimicrob. Agents Chemother. 57 : 5355-5365. - Love MJ, Coombes D, Manners SH, Abeysekera GS, Billington C, Dobson RCJ. 2021. The molecular basis for
Escherichia coli O157:H7 phage FAHEc1 endolysin function and protein engineering to increase thermal stability.Viruses 13 : 1101. - Mao J, Schmelcher M, Harty WJ, Foster-Frey J, Donovan DM. 2013. Chimeric Ply187 endolysin kills
Staphylococcus aureus more effectively than the parental enzyme.FEMS Microbiol Lett. 342 : 30-36. - Dong Q, Wang J, Yang H, Wei C, Yu J, Zhang Y,
et al . 2015. Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci.Microb. Biotechnol. 8 : 210-220. - Fernandes S, Proença D, Cantante C, Silva FA, Leandro C, Lourenço S,
et al . 2012. Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistantStaphylococcus aureus .Microb. Drug Resist. 18 : 333-343. - Harris F, Dennison SR, Phoenix DA. 2009. Anionic antimicrobial peptides from eukaryotic organisms.
Curr. Protein Pept. Sci. 10 : 585-606. - Mahlapuu M, Håkansson J, Ringstad L, Björn C. 2016. Antimicrobial peptides: An emerging category of therapeutic agents.
Front. Cell Infect. Microbiol. 6 : 194. - Bechinger B. 2015. The SMART model: Soft membranes adapt and respond, also transiently, in the presence of antimicrobial peptides.
J. Pept. Sci. 21 : 346-355. - Briers Y, Walmagh M, Van Puyenbroeck V, Cornelissen A, Cenens W, Aertsen A,
et al . 2014. Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens.mBio 5 : e01379-01314. - Yan G, Yang R, Fan K, Dong H, Gao C, Wang S,
et al . 2019. External lysis ofEscherichia coli by a bacteriophage endolysin modified with hydrophobic amino acids.AMB Express. 9 : 106. - Mancoš M, Šramková Z, Peterková D, Vidová B, Godány AJB. 2020. Functional expression and purification of tailor-made chimeric endolysin with the broad antibacterial spectrum.
Biologia 75 : 2031-2043. - Cotter PD, Ross RP, Hill C. 2013. Bacteriocins - a viable alternative to antibiotics?
Nat. Rev. Microbiol. 11 : 95-105. - Vincent PA, Morero RD. 2009. The structure and biological aspects of peptide antibiotic microcin J25.
Curr. Med. Chem. 16 : 538-549. - Parks WM, Bottrill AR, Pierrat OA, Durrant MC, Maxwell A. 2007. The action of the bacterial toxin, microcin B17, on DNA gyrase.
Biochimie 89 : 500-507. - Heselpoth RD, Euler CW, Schuch R, Fischetti VA. 2019. Lysocins: Bioengineered antimicrobials that deliver lysins across the outer membrane of Gram-negative bacteria.
Antimicrob. Agents Chemother. 63 : e00342-19. - Lukacik P, Barnard TJ, Keller PW, Chaturvedi KS, Seddiki N, Fairman JW,
et al . 2012. Structural engineering of a phage lysin that targets gram-negative pathogens.Proc. Natl. Acad. Sci. USA 109 : 9857-9862. - Yan G, Liu J, Ma Q, Zhu R, Guo Z, Gao C,
et al . 2017. The N-terminal and central domain of colicin A enables phage lysin to lyseEscherichia coli extracellularly.Antonie Van Leeuwenhoek 110 : 1627-1635. - Zampara A, Sørensen MCH, Grimon D, Antenucci F, Vitt AR, Bortolaia V,
et al . 2020. Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria.Sci. Rep. 10 : 12087. - Plançon L, Janmot C, le Maire M, Desmadril M, Bonhivers M, Letellier L,
et al . 2002. Characterization of a high-affinity complex between the bacterial outer membrane protein FhuA and the phage T5 protein pb5.J. Mol. Biol. 318 : 557-569. - Mozafari MR, Johnson C, Hatziantoniou S, Demetzos C. 2008. Nanoliposomes and their applications in food nanotechnology.
J. Liposome Res. 18 : 309-327. - Mozafari MR, Flanagan J, Matia-Merino L, Awati A, Singh H. 2010. Recent trends in the lipid-based nanoencapsulation of antioxidants and their role in foods.
J. Sci. Food Agric. 86 : 2038-2045. - Alhajlan M, Alhariri M, Omri A. 2013. Efficacy and safety of liposomal clarithromycin and its effect on
Pseudomonas aeruginosa virulence factors.Antimicrob. Agents Chemother. 57 : 2694-2704. - Solleti VS, Alhariri M, Halwani M, Omri A. 2015. Antimicrobial properties of liposomal azithromycin for
Pseudomonas infections in cystic fibrosis patients.J. Antimicrob. Chemother. 70 : 784-796. - Rajendran V, Rohra S, Raza M, Hasan GM, Dutt S, Ghosh PC. 2015. Stearylamine liposomal delivery of monensin in combination with free artemisinin eliminates blood stages of
Plasmodium falciparum in culture andP. berghei infection in murine malaria.Antimicrob Agents Chemother. 60 : 1304-1318. - Bai J, Yang E, Chang PS, Ryu S. 2019. Preparation and characterization of endolysin-containing liposomes and evaluation of their antimicrobial activities against gram-negative bacteria.
Enzyme Microb Technol. 128 : 40-48. - Kaur J, Kour A, Panda JJ, Harjai K, Chhibber S. 2020. Exploring endolysin-loaded alginate-chitosan nanoparticles as future remedy for staphylococcal infections.
AAPS PharmSciTech. 21 : 233. - Abouhmad A, Dishisha T, Amin MA, Hatti-Kaul R. 2017. Immobilization to positively charged cellulose nanocrystals enhances the antibacterial activity and stability of hen egg white and T4 lysozyme.
Biomacromolecules 18 : 1600-1608. - Agnihotri SA, Mallikarjuna NN, Aminabhavi TM. 2004. Recent advances on chitosan-based micro- and nanoparticles in drug delivery.
J. Control Release 100 : 5-28. - Ragelle H, Vandermeulen G, Préat V. 2013. Chitosan-based siRNA delivery systems.
J. Control Release. 172 : 207-218. - Gondil VS, Dube T, Panda JJ, Yennamalli RM, Harjai K, Chhibber S. 2020. Comprehensive evaluation of chitosan nanoparticle based phage lysin delivery system; a novel approach to counter
S. pneumoniae infections.Int. J. Pharm. 573 : 118850. - Vaara, M. 1992. Agents that increase the permeability of the outer membrane.
Microbiol. Rev. 56 : 395-411. - Briers Y, Walmagh M, Lavigne R. 2011. Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against
Pseudomonas aeruginosa .J. Appl. Microbiol. 110 : 778-785. - Lim JA, Shin H, Heu S, Ryu S. 2014. Exogenous lytic activity of SPN9CC endolysin against gram-negative bacteria.
J. Microbiol. Biotechnol. 24 : 803-811. - Walmagh M, Boczkowska B, Grymonprez B, Briers Y, Drulis-Kawa Z, Lavigne R. 2013. Characterization of five novel endolysins from Gram-negative infecting bacteriophages.
Appl. Microbiol. Biotechnol. 97 : 4369-4375. - Oliveira H, Thiagarajan V, Walmagh M, Sillankorva S, Lavigne R, Neves-Petersen MT,
et al . 2014. A thermostableSalmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.PLoS One 9 : e108376. - Oliveira H, Vilas Boas D, Mesnage S, Kluskens LD, Lavigne R, Sillankorva S,
et al . 2016. Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity.Front. Microbiol. 7 : 208.