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Lysozyme-like phage lysin
Crystal structure of the modular CPL-1 endolysin from Streptococcus phage Cp-1 complexed with a peptidoglycan analogue. PDB entry 2j8g.[1]
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EC no.3.2.1.17
CAS no.9001-63-2
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Lysins, also known as endolysins or murein hydrolases, are hydrolytic enzymes produced by host bacteria when infected with bacteriophages in order to cleave the cell wall during the final stage of the lytic cycle to release the viral particles. Lysins are highly evolved enzymes that are able to target one of the five bonds in peptidoglycan (murein), the main component of bacterial cell walls, which allows the release of progeny virions from the lysed cell. Cell-wall-containing Archaea are also lysed by specialized pseudomurein-cleaving lysins,[2] while most archaeal viruses employ alternative mechanisms.[3] Similarly, not all bacteriophages synthesize lysins: some small single-stranded DNA and RNA phages produce membrane proteins that activate the host's autolytic mechanisms such as autolysins.[4]

Lysins were first used therapeutically in 2001 by the Fischetti lab (see below) and are now being used as antibacterial agents due to their high effectiveness and specificity in comparison with antibiotics, which are susceptible to bacterial resistance.[5] Because lysins are essential for bacteriophage survival, resistance to lysins is an extremely rare event. Over the >20 years of lysin development as therapeutics, resistance has not been observed, even when resistance is forced by mutagenesis experiments.

Structure

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Double-stranded DNA phage lysins tend to lie within the 25 to 40 kDa range in terms of size. A notable exception is the streptococcal PlyC endolysin, which is 114 kDa. PlyC is not only the biggest and most potent lysin, but also structurally unique since it is composed of two different gene products, PlyCA and PlyCB, with a ratio of eight PlyCB subunits for each PlyCA in its active conformation.[6]

All other lysins are monomeric and comprise two domains separated by a short linker region. For gram positive bacteria lysins, the N-terminal domain catalyses the hydrolysis of peptidoglycan whereas the C-terminal domain binds to the cell wall substrate.

Catalytic domain

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The catalytic domain is responsible for the cleavage of peptidoglycan bonds. Functionally, five types of lysin catalytic domain can be distinguished:

Peptidoglycan consists of cross-linked amino acids and sugars which form alternating amino sugars: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Endo-β-N-acetylglucosaminidase lysins cleave NAGs while N-acetylmuramidase lysins (lysozyme-like lysins) cleave NAMs. Endopeptidase lysins cleave any of the peptide bonds between amino acids, whereas N-acetylmuramoyl-l-alanine amidase lysins (or simply amidase lysins) hydrolyze the amide bond between the sugar and the amino acid moieties. Finally, the recently discovered γ-d-glutaminyl-l-lysine endopeptidase lysins cleave the gamma bond between D-glutamine and L-lysine residues. As is the case for autolysins, early confusion around the cleavage specificity of these individual enzymes has led to some misattributions of the name "lysozyme" to proteins without this activity.[7]

Usually, two or more different catalytic domains are linked to a single cell-binding domain. This is typical in many staphylococcal lysins as well as the streptococcal PlyC holoenzyme, which contains two catalytic domains.[6][8] Catalytic domains are highly conserved in phage lysins of the same class.[5]

Cell-binding domain

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The cell-binding domain (CBD) binds to a specific substrate found in the host bacterium's cell wall, usually a carbohydrate. In contrast to the catalytic domain, the cell-binding domain is variable, which allows a great specificity and decreases bacterial resistance.[9] Binding affinity to the cell wall substrate tends to be high, possibly so as to sequester onto cell wall fragments any free enzyme, which could compete with phage progeny from infecting adjacent host bacteria.[10]

Evolution

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It has been proposed that the main mechanism of evolution in phage lysins is the exchange of modular units, a process by which different catalytic and cell-binding domains have been swapped between lysins, which would have resulted in new combinations of both bacterial binding and catalytic specificities.[11]

Mode of action

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The lysin catalytic domain digests peptidoglycan locally at a high rate, which causes holes in the cell wall. Since the cross-linked peptidoglycan cell wall is the only mechanism that prevents the spontaneous burst of bacterial cells due to the high internal pressure (3 to 5 atmospheres), enzymatic digestion by lysins irreversibly causes hypotonic lysis. Theoretically, due to the catalytic properties of phage lysins, a single enzyme would be sufficient to kill the host bacterium by cleaving the necessary number of bonds, even though this has yet to be proven.[5] The work by Loessner et al suggests that cleavage is typically achieved by the joint action of multiple lysin molecules at a local region of the host's cell wall.[10] The high binding affinity to the cell wall substrate (close to that of IgG for its substrate) of each lysin appear to be reason why multiple molecules are required, since every lysin binds so tightly to the cell wall that it can't break enough bonds to cause lysis by itself.[10]

In order to reach the cell wall, phage lysins have to cross the cell membrane. However, they generally lack a signal peptide that would allow them to do so. In order to solve such a problem, phage viruses synthesize another protein called holin which binds to the cell membrane and makes holes in it (hence its name), allowing lysins to reach the peptidoglycan matrix. The prototypical holin is the lambda phage S protein, which assists the lambda phage R protein (lysin). All holins embed themselves in the cell membrane and contain at least two transmembrane helical domains. The hole making process is thought to be achieved by holin oligomerization at a specific moment when progeny virions are set to be released.[4][12]

Efficacy

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Phage lysins are generally species or subspecies specific, which means that they are only effective against bacteria from which they were produced. While some lysins only act upon the cell walls of several bacterial phylotypes, some broad-spectrum lysins have been found.[13] Similarly, some thermostable lysins are known, which makes them easier to use in biotechnology.[14] Regarding their use as antibacterial agents, lysins have been found effective mainly against Gram-positive bacteria, since Gram-negative bacteria possess an outer membrane that prevents extracellular lysin molecules from digesting peptidoglycan.[5] However, lysins with activity against Gram-negative bacteria, such as OBPgp279, have garnered interest as potential therapeutics.[15]

Immune response

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One of the most problematic aspects of the use of phage lysins as antimicrobial agents is the potential immunogenicity of these enzymes. Unlike most antibiotics, proteins are prone to antibody recognition and binding, which means that lysins could be ineffective when treating bacterial infections or even dangerous, potentially leading to a systemic immune response or a cytokine storm. Nonetheless, experimental data from immunologically rich rabbit serum showed that hyperimmune serum slows down but does not block the activity of pneumococcal lysin Cpl-1.[16]

Antimicrobial use

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Phage lysins have been successfully tested in animal models to control pathogenic antibiotic-resistant bacteria found on mucous membranes and in blood. The main advantage of lysins compared to antibiotics is not only the low bacterial resistance but also the high specificity towards the target pathogen, and low activity towards the host's normal bacterial flora.[5]

Lysins were first used therapeutically in animals in 2001, in a publication in which mice orally colonized with Streptococcus pyogenes were decolonized with a single dose of PlyC lysin delivered orally.[17]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Lysin

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from Grokipedia
Lysin, also known as endolysin or , is a highly evolved hydrolytic produced by bacteriophages that digests the layer of bacterial cell walls, enabling the release of progeny phages from infected host cells. These enzymes are encoded in the phage and are typically activated late in the , where they rapidly lyse the bacterial cell from within by targeting specific bonds in the structure, such as glycosidic linkages or cross-bridges. Lysins exhibit high specificity for their bacterial hosts, distinguishing between Gram-positive and Gram-negative , with many featuring a modular consisting of a catalytic domain for and a cell wall-binding domain for targeting. Beyond their natural role in phage replication, lysins have emerged as promising agents, often termed enzybiotics, due to their potent bactericidal activity against antibiotic-resistant pathogens without harming eukaryotic cells or the . As of 2025, engineered variants are in clinical development for treating infections caused by such as (e.g., Exebacase in Phase 3 trials) and Clostridium difficile, with preclinical and early-stage progress for , leveraging rapid killing kinetics—often within seconds to minutes—and low resistance potential compared to traditional antibiotics. Historically referred to by names such as phage-lysozyme, muralysin, or virolysin, lysins represent a diverse family with thousands of sequences identified in databases, and ongoing research as of 2025 focuses on therapeutic optimization, including fusions with or delivery systems to target Gram-negative outer membranes. Their potential extends to applications in (e.g., against in ), , and disruption, positioning lysins as a key tool in combating .

Overview

Definition and Types

Lysin, also known as phage lysin or endolysin, is a hydrolytic produced by bacteriophages that specifically degrades the layer of the bacterial , facilitating the release of phage progeny during the of infection. These enzymes, often referred to as murein hydrolases, target specific bonds in the structure, such as those in N-acetylmuramic acid or peptide cross-links, to cause rapid cell from within the host bacterium. The primary types of lysins are endolysins, which act internally after phage replication. Endolysins are synthesized within the infected bacterial cell and degrade from the side, triggered by a holin protein that forms pores in the inner membrane to allow access. Lysins must be distinguished from bacterial autolysins, which are endogenous enzymes produced by bacteria themselves for remodeling during growth and division, rather than for targeted phage-mediated destruction. Most lysins are compact proteins, typically ranging from 25 to 40 kDa for those targeting Gram-positive bacteria, though exceptions exist such as the multimeric PlyC lysin from a streptococcal phage, which assembles into a 114 kDa complex for enhanced activity. Lysins from phages infecting Gram-negative bacteria are generally smaller, around 15 to 20 kDa, reflecting their simpler domain architecture adapted to the outer membrane barrier. In biological context, lysins play a crucial role in the lytic lifecycle of bacteriophages infecting both Gram-positive and Gram-negative hosts: in Gram-positive bacteria, endolysins can access peptidoglycan directly upon exogenous application due to the absence of an outer membrane, while in Gram-negative infections, they primarily function internally.

Historical Discovery

The discovery of phage lysins emerged from early studies on bacteriophage-induced bacterial in the mid-20th century. In the , researchers observed "nascent " phenomena during phage infections of streptococci, where bacterial cultures cleared rapidly due to enzymatic activity separate from the phage itself. Key early work included W.R. Maxted's 1957 identification of a lytic factor in lysates of group C streptococci infected with phage B563, which he characterized as an capable of lysing , C, and E streptococci. Concurrently, Richard M. Krause at partially purified this lysin, renamed C1 phage lysin, from the same system, establishing it as a phage-encoded responsible for degradation during the phage . These findings laid the groundwork for understanding lysins as distinct from phages, highlighting their role in facilitating progeny release without requiring active . In the 1970s, advancements in purification techniques enabled deeper structural and biochemical characterization of lysins. Vincent A. Fischetti, working at , achieved the first homogeneous purification of C1 lysin in 1971 by stabilizing its sulfhydryl groups with , allowing detailed analysis of its molecular weight (approximately 80 kDa) and enzymatic properties. This milestone not only resolved the enzyme's instability issues but also positioned lysins as valuable tools for studying bacterial surface antigens, such as streptococcal M proteins, which were extracted using the purified lysin. Fischetti's contributions during this decade emphasized lysins' specificity and potency, setting the stage for broader applications beyond basic phage research. By the 1990s, rising antibiotic resistance prompted recognition of lysins as potential antimicrobial agents. Fischetti's laboratory began exploring their therapeutic promise, leveraging decades of characterization to propose lysins as targeted alternatives to broad-spectrum antibiotics, particularly against Gram-positive pathogens like streptococci and staphylococci. This shift culminated in the first in vivo demonstration of efficacy in 2001, when Fischetti and colleagues showed that purified C1 lysin (PlyC) eliminated group A Streptococcus colonization in the upper respiratory tract of mice, reducing bacterial loads by over 99% without toxicity. Early research focused predominantly on Gram-positive bacteria due to their accessible peptidoglycan layers, but by the 2010s, challenges with Gram-negative pathogens—stemming from their outer membrane barrier—drove innovations in lysin engineering to extend activity across bacterial types.

Molecular Structure

Catalytic Domain

The catalytic domain of a lysin is located at the and typically comprises approximately 200–250 , forming the enzymatically active region responsible for hydrolyzing specific bonds in the bacterial layer. This domain's structure varies by class but generally features a compact fold that positions catalytic residues for efficient substrate access. Lysin catalytic domains are classified into five major enzymatic classes based on their peptidoglycan cleavage specificity: glycoside hydrolases, which include muramidases that cleave the β-1,4 between N-acetylmuramic acid (MurNAc) and (GlcNAc), and glucosaminidases that target the reducing end of GlcNAc; amidases, such as N-acetylmuramoyl-L-alanine amidases that hydrolyze the between MurNAc and L-alanine; endopeptidases that sever within the peptidoglycan stem peptides; transglycosylases that rearrange glycosidic linkages; and lytic transglycosylases that cleave β-1,4 glycosidic bonds while forming 1,6-anhydro-MurNAc products. Each class exhibits distinct folds, such as the α/β barrel in muramidases or the papain-like fold in endopeptidases, enabling targeted bond hydrolysis. The of these domains contains conserved residues critical for , with specificity tuned to bonds like β-1,4 glycosidic or linkages. For instance, amidases often feature a of , , and (Cys-His-Asn), where the acts as a to initiate bond cleavage. In endopeptidases like the CHAP domain, a similar Cys-His dyad or triad facilitates . A representative example is the catalytic domain of PlyC, a streptococcal phage lysin featuring a CHAP module that specifically cleaves amide linkages in the of s. PlyC's binding is mediated by its PlyCB subunit, which targets the group A carbohydrate side chains on the cell wall surface. This domain's activity underscores the structural adaptations in lysins for pathogen-specific .

Cell Wall Binding Domain

The binding domain (CBD) of lysins is predominantly situated at the C-terminal region of the protein in those targeting , serving as the key element for targeted attachment to the host cell surface. This domain typically spans 50–150 , exhibiting remarkable sequence and structural variability that enables specificity toward diverse bacterial hosts. Such diversity is reflected in the identification of several distinct CBD families (e.g., at least seven identified types), allowing lysins to adapt to various cell wall architectures across bacterial species. The binding motifs within the CBD include well-characterized domains such as SH3b and LysM, alongside unique repeat structures like choline-binding modules or PG_binding domains. These motifs facilitate non-covalent interactions with specific ligands; for example, in , they commonly target teichoic acids or peptidoglycan-associated components, while in , analogous regions may engage (LPS) outer membrane structures. This specificity ensures that the lysin is anchored proximal to its substrate, enhancing efficiency without affecting eukaryotic cells. Connecting the CBD to the N-terminal catalytic domain is a flexible linker region, often comprising 10–20 residues and featuring proline-rich or alpha-helical elements that provide structural mobility. This linker permits independent functioning of the domains, positioning the catalytic site optimally against the while maintaining overall protein stability. A representative example is the CBD of Pal, an endolysin from the phage Dp-1, which incorporates a modular choline-binding with beta-solenoid folds and multiple binding loci for teichoic acid-associated choline residues. This configuration not only confers high-affinity binding but also stabilizes the full lysin architecture upon cell wall engagement.

Evolution and Diversity

Modular Evolution

Lysin proteins, also known as endolysins, exhibit a modular architecture where distinct functional domains—typically an N-terminal catalytic domain and a C-terminal binding domain—evolve independently within genomes. This modularity arises primarily through (HGT) and events that allow phages to exchange genetic modules, enabling adaptation to diverse bacterial hosts. Such independent evolution of domains facilitates the assembly of chimeric lysins tailored to specific targets, enhancing phage efficiency. Sequence analyses of lysin genes from diverse phages provide compelling evidence for this domain shuffling, revealing chimeric structures where the catalytic domain originates from one phage lineage and the binding domain from another unrelated phage or even bacterial sources. For instance, of 723 endolysins identified 89 unique architectural combinations, with many displaying patterns indicative of inter-phage module exchanges via HGT. These findings underscore how recombination-driven interchange of complete functional modules occurs naturally, contributing to the structural diversity observed in lysin repertoires. Phylogenetically, lysins trace their ancient origins to approximately 3 billion years ago, coinciding with the emergence of bacterial cell walls and the onset of phage-bacteria interactions. This timeline aligns with the co-evolution of lysins in the context of an ongoing between phages and their bacterial hosts, where selective pressures drive the refinement of lysis mechanisms to counter evolving bacterial defenses. The primary mechanisms underlying this modular evolution include , often mediated by phage-encoded integrases or self-splicing introns within lysin genes, which promote precise module exchanges without disrupting overall gene function. Transposition events, facilitated by in phage genomes, further contribute to the dissemination of lysin domains across phage populations, amplifying .

Diversity Across Phages

lysins exhibit significant sequence and functional diversity, reflecting adaptations to a wide array of bacterial hosts and phage lifestyles within the dominant order Caudovirales, which encompasses tailed phages responsible for the majority of characterized lysins. This diversity manifests in variations of enzymatic activities, domain architectures, and substrate specificities, enabling phages to efficiently degrade diverse structures during host . For instance, the T7 phage lysin functions primarily as a simple muramidase, hydrolyzing the β-1,4 between N-acetylmuramic acid and in the backbone. In contrast, the lysin operates as an amidase, targeting the amide bond between N-acetylmuramic acid and L-alanine, highlighting the enzymatic specialization across even closely related phages. Such variations underscore the broad evolutionary divergence within Caudovirales, where lysin genes often show low sequence similarity despite conserved overall functions. Lysins are finely tuned to the cell wall architecture of their bacterial hosts, with distinct adaptations for Gram-positive versus Gram-negative targets. In Gram-positive bacteria, which lack an outer membrane, lysins like PlySs2—derived from a Streptococcus suis phage—directly access and degrade the exposed peptidoglycan layer, exhibiting potent activity against Staphylococcus aureus and related streptococci. PlySs2's cell wall-binding domain confers specificity to staphylococcal peptidoglycan, enabling rapid lysis without additional facilitators. For Gram-negative hosts, however, the outer membrane poses a barrier, necessitating lysins that either incorporate signal peptides for translocation or rely on co-factors like outer membrane permeabilizers to access the peptidoglycan. This host-specific tailoring is evident in phages infecting Escherichia coli or Pseudomonas species, where lysins often feature modular elements adapted for periplasmic navigation. Comprehensive sequence databases reveal the scale of this diversity, with the PhaLP database cataloging over 16,000 characterized lysin entries, showcasing extensive variability in domain combinations such as catalytic domains paired with diverse binding motifs. These combinations allow for tailored specificity, with amidases, glucosaminidases, and endopeptidases comprising the most common catalytic classes, often rearranged across phage genomes. Earlier compilations, such as those analyzing over 2,000 lysin sequences, further illustrate how domain shuffling contributes to functional breadth without altering core hydrolytic mechanisms. Natural variants from extremophile phages expand the functional repertoire, including thermostable lysins like TSPphg from the Thermus phage TSP4, which retains activity at temperatures exceeding 70°C due to enhanced . Similarly, broad-spectrum lysins such as LysK from a phage demonstrate lytic activity across multiple Gram-positive genera, including staphylococci, streptococci, and enterococci, arising from versatile binding domains that recognize conserved motifs. These extremophile-derived and broad-host variants highlight how environmental pressures drive lysin evolution, providing templates for understanding phage-bacteria interactions in diverse ecosystems.

Mechanism of Action

Enzymatic Hydrolysis

Lysin enzymes catalyze the hydrolysis of specific bonds within the layer of bacterial cell walls, enabling targeted degradation during the . , one major class of lysins, specifically cleave the bond linking N-acetylmuramic acid (MurNAc) to L-alanine in the stem . , another prevalent type, hydrolyze the β-1,4 between MurNAc and (GlcNAc), disrupting the glycan backbone of the structure. Other classes include endopeptidases, which cleave cross-bridges between stem peptides, and glucosaminidases, which target bonds involving GlcNAc. These cleavage reactions weaken the lattice, compromising bacterial integrity. Lysins exhibit high substrate specificity for bacterial peptidoglycan components, particularly targeting motifs involving N-acetylmuramic acid, which distinguishes prokaryotic cell walls from eukaryotic ones. This selectivity arises from the interaction between the enzyme's catalytic domain and the unique sugar-amino acid linkages in , ensuring precise enzymatic action without off-target effects on host cells. The kinetics of lysin-mediated are efficient, requiring only a small number of molecules, typically fewer than 1000 per bacterial cell, to achieve substantial peptidoglycan degradation once access is granted. Optimal activity often occurs around neutral , with some lysins enhanced by divalent cations. In the natural phage infection process, holin proteins synergize with lysins by forming pores in the cytoplasmic membrane, permitting the enzymes to reach and hydrolyze the peptidoglycan substrate from within the cell. The catalytic domains of lysins underpin these events, integrating structural features for bond recognition and cleavage.

Lysis Process

In the late of bacteriophages, holin proteins accumulate in the bacterial cytoplasmic membrane and trigger at a genetically determined time, forming discrete membrane holes that permit endolysins, or lysins, to access and degrade the layer of the from within. This timed deployment ensures synchronized release of progeny phages, with occurring approximately 45-60 minutes post-infection in model systems like phage λ. Following hydrolysis, the resulting becomes susceptible to osmotic in hypotonic environments, where influx of water due to the internal causes the cytoplasmic membrane to rupture and the cell to explode. When applied externally as purified enzymes, lysins induce rapid bacterial lysis in Gram-positive species by directly binding to and degrading the exposed peptidoglycan, often within seconds to minutes at concentrations as low as nanograms per milliliter. In contrast, exhibit slower lysis times, typically requiring minutes to hours, because the outer membrane serves as a permeability barrier that limits lysin access to the peptidoglycan until disrupted by additional agents or . Lysins hydrolyze specific peptidoglycan bonds, such as β-1,4 glycosidic linkages, to initiate this process. Effective lysis demands the cooperative action of multiple lysin molecules, which collectively hydrolyze numerous cross-links to generate sufficient lesions in the , ultimately leading to instability and explosive bursting. This multi-molecular requirement ensures that isolated enzymatic events are insufficient for complete disruption, with lysis efficiency scaling with lysin concentration and exposure duration. The efficacy of lysins is modulated by environmental factors, including buffer and , where physiological conditions (e.g., isotonic saline approximating 150 mM NaCl) often reduce lytic activity compared to hypotonic lab buffers due to minimized osmotic gradients and stabilized cell walls. In high-ionic-strength environments mimicking serum, lysin-induced killing can decrease by up to 10-fold relative to low-salt lab media, highlighting the need for condition-specific optimization.

Therapeutic Potential

Efficacy and Spectrum

Phage lysins display a spectrum of antibacterial activity that can be highly specific or relatively broad, depending on the and its target bacteria. For example, the lysin PlyG exhibits potent lytic activity exclusively against , including its vegetative cells and spores, making it suitable for targeted therapeutics. In contrast, CF-301 (exebacase), derived from a staphylococcal phage, shows broader efficacy against various species, encompassing methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), and coagulase-negative staphylococci. In vitro studies demonstrate the high efficacy of lysins, with minimum inhibitory concentrations (MICs) typically ranging from 0.1 to 10 µg/mL against susceptible Gram-positive pathogens. These enzymes exert rapid bactericidal effects, often achieving a 3–5 log₁₀ reduction in viable bacteria within 1–5 minutes by directly hydrolyzing the peptidoglycan layer. Resistance development is minimal, as lysins target conserved, essential components of the bacterial cell wall that are not subject to frequent mutation. In vivo efficacy has been validated in murine models of systemic infections, where lysins like LysGH15 achieve 90–100% survival rates in MRSA-induced sepsis when administered post-infection. CF-301 has also demonstrated similar protective effects in murine S. aureus bacteremia models. For instance, a single dose of LysGH15 (50 µg) injected 1 hour after challenge resulted in complete protection and undetectable bacterial loads in blood. However, efficacy against Gram-negative bacteria remains limited due to the outer membrane acting as a permeability barrier, necessitating engineering strategies for broader applicability. Key factors enhancing lysin performance include their stability in biological fluids and lack of to host cells. Lysins maintain activity in serum, with reported half-lives of 20–60 minutes in murine models, though optimizations can extend this for therapeutic use. They exhibit no to mammalian cells at concentrations effective against , supporting their safety profile.

Applications

Lysin-based therapies have emerged as promising alternatives to traditional antibiotics for treating infections caused by antibiotic-resistant pathogens, particularly Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and Clostridium difficile. These enzymes demonstrate rapid bactericidal activity against these targets, making them suitable for serious infections including endocarditis and pneumonia. For instance, the endolysin Cpl-1 has shown efficacy in animal models of pneumococcal pneumonia when delivered via inhalation, reducing bacterial burden in the lungs. Similarly, lysins like Ply113 exhibit potent lytic activity against VRE strains, offering potential for treating enterococcal endocarditis. For example, exebacase (CF-301) is currently in phase 3 clinical trials for Staphylococcus aureus bacteremia as of 2025. Delivery formulations for lysins include intravenous administration for systemic infections and topical applications for localized ones, such as and infections caused by MRSA. Intravenous has been tested in phase 1 clinical trials with endolysins like SAL200, confirming safety and tolerability in healthy volunteers. Topical formulations, including gels and creams, leverage lysins' specificity to treat superficial infections without disrupting the skin . Lysins also exhibit effects when combined with , often reducing the (MIC) by 4-fold or more, which allows for lower antibiotic doses and mitigates resistance development. This , observed in combinations like endolysin LysAB1245 with against Pseudomonas aeruginosa, enhances overall therapeutic efficacy while minimizing toxicity. Beyond human medicine, lysins find applications in food safety, where they effectively decontaminate dairy products from Listeria monocytogenes, a common contaminant in cheese and milk. Endolysins such as PlyP100 reduce L. monocytogenes counts in fresh cheese without altering product quality or sensory attributes. In veterinary settings, lysins target bovine mastitis caused by streptococci and staphylococci, with engineered variants showing intracellular activity against Streptococcus uberis in mammary epithelial cells. PlyC, for example, has been developed as a potential therapeutic for lactating dairy cows, demonstrating reduced bacterial loads in udder infections. Regulatory progress includes FDA orphan drug designation for certain lysins targeting rare infections; for instance, BAL200 received this status in 2018 for inhalational . While no lysin has full FDA approval specifically for C. difficile as of 2025, ongoing research supports their advancement toward clinical use in recurrent infections.

Immunological Aspects

Host Immune Interactions

Lysins, derived from bacteriophages, are recognized as foreign antigens by the mammalian , primarily eliciting a humoral response through the production of IgG . In models, administration of the endolysin PlyC induces robust IgG production, with levels peaking approximately one month post-injection following a single dose of 100 µg. Similarly, serological surveys in humans reveal that 10–12.5% of individuals exhibit elevated IgG reactivity to PlyC or its PlyCB binding subunit, often attributed to with environmental exposures. Studies with other lysins, such as Cpl-1, demonstrate that repeated intravenous dosing in generates IgG titers around 1:10, yet these exhibit limited neutralizing capacity . The primary mechanism driving this immunogenicity involves B-cell activation and antibody production targeting specific linear epitopes on the lysin structure. For PlyC, immunogenic epitopes are predominantly located in the PlyCA catalytic domain, with key regions spanning 1–9, 91–146, 171–226, and 351–406, as identified through and next-generation sequencing of B-cell repertoires. Due to their prokaryotic origin, lysins generally provoke minimal T-cell mediated responses, as they lack motifs optimized for effective presentation by mammalian molecules, focusing the immune interaction on humoral pathways. In models, hyperimmune sera against Cpl-1 partially slow lytic activity but do not fully inhibit it, indicating that epitope accessibility in binding domains may contribute to antibody binding without complete neutralization. The therapeutic implications of these interactions include the potential development of neutralizing antibodies following multiple doses, which could diminish lysin efficacy over time. However, in preclinical evaluations, such as with Cpl-1 in immunized versus naïve mice, bacteremia reduction remains comparable, suggesting antibodies do not substantially impair activity in practice; approximately 10–20% of exposed individuals may develop detectable neutralizing titers, but this varies by lysin and dosing regimen. No cases of have been reported, as IgE responses to lysins like PlyC are negligible in both sera (n=104) and challenged mice. Lysins also demonstrate evasion of certain host innate immune responses, particularly avoiding activation of the . In mouse studies with endolysins such as SAL-1, no significant complement activation or elevation in C3 levels was observed post-administration, allowing sustained enzymatic activity without rapid clearance via opsonization or . This property, combined with reduced proinflammatory induction (e.g., lower IL-1β and IL-6 levels in models treated with Cpl-1), underscores lysins' compatibility with host innate defenses during antibacterial therapy.

Mitigation Strategies

To address the immunogenicity challenges posed by bacteriophage lysins in therapeutic applications, deimmunization strategies involve targeted mutations to eliminate immunogenic epitopes, thereby reducing immune recognition while preserving enzymatic activity. For instance, structure-based computational design has been used to deplete T-cell epitopes in lysostaphin, a staphylolytic enzyme analogous to phage lysins, resulting in variants with 14 substitutions that eliminate T-cell activation in peripheral blood mononuclear cells, dropping responder rates from 47-53% to 0%. These modifications also substantially lower anti-drug (ADA) responses, with deimmunized variants showing 18- to 100-fold reductions in ADA titers compared to wild-type in HLA-transgenic models, effectively minimizing B-cell mediated neutralization. Similar epitope mutation approaches for phage lysins target both T- and B-cell sites to decrease binding affinity, enabling sustained efficacy without significant immune interference. For phage lysins, epitope scanning and design have been applied to Cpl-1 and Pal, identifying and substituting immunogenic epitopes to avoid cross-neutralization by IgG while preserving antibacterial efficacy. Fusion proteins represent another key tactic to enhance lysin tolerability by promoting immune evasion and extending . Linking lysin domains to the Fc region of IgG creates lysibodies that leverage FcRn-mediated recycling to prolong serum , reducing clearance rates and limiting exposure to immune mechanisms. This fusion imparts a "stealth" effect by mimicking host antibodies, which helps evade innate immune detection and . Complementarily, —covalent attachment of chains—shields lysins from proteolytic degradation and immune recognition, extending plasma from hours to days while decreasing in preclinical settings. For example, PEGylated peptidoglycan hydrolases (a class including lysins) demonstrate reduced ADA formation and improved biodistribution in murine models of . Optimized dosing regimens further mitigate immune responses by limiting exposure. Single-dose or intermittent administration schedules prevent chronic stimulation of adaptive immunity, as evidenced by lysin therapies where one-time intravenous dosing achieves bacterial clearance without eliciting detectable neutralizing antibodies in bacteremia models. Route-specific delivery, such as topical application, circumvents systemic immune activation by confining lysins to localized sites like or mucosa, resulting in negligible humoral responses. Preclinical studies validate these strategies, with engineered lysin variants exhibiting less than 10% neutralization by host antibodies in animal models of recurrent . In HLA-DR4 transgenic mice challenged with MRSA, deimmunized lysostaphin supported seven repeated doses with full efficacy and survival, contrasting with wild-type failure after four doses due to ADA accumulation. These findings underscore the potential for tailored mitigation to enable safe, repeated lysin use in clinical scenarios.

Research and Development

Engineering of Lysins

Engineering of lysins leverages their natural modular , consisting of catalytic and cell wall-binding domains, to enhance therapeutic properties such as , stability, and activity. Domain shuffling involves recombining catalytic domains from one lysin with binding domains from another to broaden the lytic or target specific pathogens. For instance, Artilysins are engineered by fusing lysin domains with outer permeabilizing peptides, enabling activity against by disrupting the outer barrier. Site-directed target key residues to improve lysin stability and enzymatic efficiency. Thermostable variants have been developed that retain activity at 60°C, suitable for applications requiring resistance, through stabilizing the protein fold. optimizations, such as altering catalytic residues, can increase lytic activity by up to 10-fold, enhancing bacterial killing rates without compromising specificity. Fusion constructs combine lysins with additional functional modules to augment their mechanism. Examples include fusions of endolysins with membrane-disrupting , which permeabilize bacterial membranes to facilitate peptidoglycan access and inhibit cell wall synthesis pathways. Recent advances from 2024–2025 utilize AI-driven design to create chimeric lysins, optimizing domain interfaces for superior stability and broad-spectrum activity against multidrug-resistant strains, including tools like DeepLysin for mining novel antibacterial proteins from uncharacterized phages. Recombinant expression systems enable scalable production of engineered lysins, primarily in Escherichia coli or yeast hosts. Yields in E. coli can reach up to 100 mg/L through optimized codon usage and fermentation conditions, while yeast systems offer advantages for post-translational modifications in complex variants.

Clinical Trials and Future Prospects

Clinical trials of lysins have advanced into human studies, primarily targeting multidrug-resistant bacterial infections, with exebacase (CF-301) representing a pivotal example against Staphylococcus aureus. In a Phase 2 randomized trial completed in 2020 but with follow-up analyses extending into 2023, exebacase combined with standard-of-care antibiotics achieved a clinical responder rate of 70.4% at day 14 in patients with S. aureus bacteremia and right-sided endocarditis, compared to 60.0% for antibiotics alone, indicating a 10.4% improvement (90% CI: -7.8 to 28.6). However, the subsequent Phase 3 superiority trial (NCT04160468), enrolling patients from 2019 to 2023 and reporting results in 2024, failed to demonstrate significant improvement in clinical response at day 14 for exebacase plus antibiotics versus antibiotics alone in methicillin-resistant S. aureus (MRSA) bacteremia and endocarditis, highlighting the challenges in replicating preclinical synergy in larger cohorts. Another notable advancement is the Phase 1 trial of HY-133, a recombinant chimeric endolysin developed by HYpharm, initiated in June 2024 at University Hospital Tübingen to evaluate safety, tolerability, and pharmacokinetics in healthy male volunteers for preventing S. aureus nasal colonization. As of the latest available information in 2024, the trial is ongoing in Phase 1, with no interim efficacy data released, underscoring the shift toward prophylactic applications of lysins in high-risk populations. For Clostridioides difficile infections, no dedicated lysin-specific Phase 1 trials were completed in 2024, though broader phage-derived therapies have shown safety in compassionate use cases, informing potential lysin adaptations. By 2025, research has emphasized synergy between lysins and antibiotics, with preclinical and early translational studies demonstrating enhanced bactericidal effects; for instance, exebacase combined with exhibited synergistic activity against staphylococcal bacteremia models, reducing bacterial loads more effectively than either agent alone. Ongoing trials are building on these findings for applications including and infections, though full human data remain pending. For Gram-negative pathogens like , no lysin approvals have been granted as of 2025, but engineered endolysins such as PlyKp104 are advancing through preclinical validation, with Phase 1 initiations anticipated by 2026. Key challenges in lysin development include regulatory hurdles as biologics, requiring extensive and stability assessments under FDA and EMA guidelines, which have delayed approvals beyond small-molecule antibiotics. issues, such as achieving consistent recombinant production without loss of enzymatic activity, further complicate large-scale manufacturing for intravenous formulations. Future prospects for lysins involve personalized therapies derived from patient-specific phages, enabling rapid sequencing and matching to isolate-derived lysins for targeted treatment of resistant infections. Integration with -phage hybrids offers potential for enhanced specificity and reduced off-target effects, as explored in recent engineering studies combining for bacterial targeting with lysin payloads. The global market, encompassing lysin applications, is projected to reach $1.51 billion by 2030, driven by rising and investments in biologics.

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