What Is an Endolysin and How Does It Work?

An endolysin is an enzyme made by a bacteriophage (a virus that infects bacteria) to blow open its bacterial host from the inside at the end of the virus’s replication cycle. The enzyme chews through peptidoglycan, the mesh-like structural layer that holds a bacterial cell together, causing the cell to burst and release a fresh crop of phage particles.¹ That same cell-wall-destroying ability has attracted serious interest from researchers looking for alternatives to conventional antibiotics, because endolysins can kill bacteria rapidly and in ways that bacteria have a hard time evolving resistance to.

How Endolysins Work Inside the Phage Life Cycle

When a phage infects a bacterium, it hijacks the cell’s machinery to copy its own DNA and build new phage particles. At the very end of that process, the phage needs to destroy the host cell so the new particles can escape. This is where the endolysin comes in. The phage genome encodes the endolysin, which accumulates in the cell’s interior during infection. But the enzyme cannot reach the peptidoglycan on its own because the bacterial inner membrane stands in the way.

A second phage protein called a holin solves this problem. Holins form holes in the inner membrane at a precisely timed moment, allowing the endolysin to flood through and reach the peptidoglycan layer. In the well-studied phage lambda system, for example, three types of lysis proteins work in sequence: the S105 holin punches through the inner membrane, the R endolysin degrades the peptidoglycan, and a spanin complex disrupts the outer membrane.² The timing is critical. If the cell bursts too early, the new phage particles are not yet assembled. If it bursts too late, the phage misses out on infecting nearby hosts while conditions are favorable. Holins essentially act as a molecular clock, and the endolysin is the demolition charge that fires when the clock runs out.

The Modular Design of Endolysins

One reason endolysins have been so appealing to researchers is their modular architecture. Endolysins that target Gram-positive bacteria typically have at least two distinct functional regions: a catalytic domain that actually cuts bonds in the peptidoglycan, and a cell-wall binding domain (CBD) that anchors the enzyme to the bacterial surface.³ The binding domain grabs hold of the cell wall so the catalytic domain can work efficiently in close contact with its target.

Some endolysins go further and carry multiple catalytic domains with different cutting activities. PlySK1249, for instance, contains an amidase domain that lyses cells, an endopeptidase domain that breaks apart chains of bacteria, and a central binding domain holding the whole structure together.⁴ This multi-tool design means a single endolysin can attack the cell wall at different chemical bonds simultaneously, making it harder for the bacterium to survive.

How much each domain matters was demonstrated neatly in work on the endolysin LysPBC5, which kills Bacillus cereus. The full-length enzyme, with both its catalytic and binding domains intact, completed cell lysis within 30 minutes at low concentrations. When researchers stripped away the binding domain and used just the catalytic piece, they needed a 100-fold higher concentration and twice as much time to achieve comparable killing.⁵ The catalytic part could still do the job alone, but the binding domain made the whole process dramatically faster and more efficient.

Why Gram-Negative Bacteria Are Harder to Kill

Most early endolysin research focused on Gram-positive bacteria for a straightforward reason: these bacteria have their peptidoglycan layer on the outside, relatively exposed. When you apply an endolysin externally to Gram-positive bacteria, it can reach and degrade the cell wall directly, killing the cell from the outside just as effectively as it would from within during a phage infection.⁶

Gram-negative bacteria present a different challenge. They have an additional outer membrane surrounding the peptidoglycan, and that membrane acts as a shield that blocks externally applied endolysins from reaching their target.⁷ This is a significant problem because many of the most dangerous drug-resistant pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, are Gram-negative. Researchers have pursued several strategies to get around this barrier.

Engineering Endolysins to Penetrate the Outer Membrane

The modularity of endolysins turns out to be a gift for protein engineers, because you can swap, add, or rearrange the functional domains relatively easily. A key feature of endolysins is the opportunity this modularity creates: researchers can customize properties like specificity, activity, and stability through domain shuffling and fusion strategies.⁸

One of the most successful approaches has been the creation of “Artilysins,” engineered endolysins fused with short peptides that can penetrate or destabilize the outer membrane. In a landmark study, researchers attached a polycationic nonapeptide to a modular endolysin and found that the resulting Artilysin killed multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii with a four-to-five log reduction (meaning it eliminated roughly 99.99% of the bacteria) within 30 minutes.⁹

More recent work has pushed the concept further. An engineered endolysin called LNT103, created by substituting 15 amino acids and fusing an antimicrobial peptide called cecropin A to the enzyme, showed strong killing activity against a broad panel of Gram-negative pathogens including E. coli, Klebsiella pneumoniae, and Enterobacter cloacae, with minimum inhibitory concentrations as low as 4 micrograms per milliliter.¹⁰ Another strategy involves encapsulating endolysins inside liposomes, tiny fat-based vesicles that can fuse with the outer membrane and deliver the enzyme directly to the peptidoglycan. One liposome system reduced Salmonella cell counts by over two log units without needing any separate membrane-disrupting agent.¹¹

Why Resistance to Endolysins Is Rare

One of the most compelling arguments for endolysins as antimicrobials is the difficulty bacteria face in developing resistance to them. Conventional antibiotics typically target a single metabolic process or protein, and bacteria can evolve resistance through a single mutation that alters the target, pumps out the drug, or breaks it down. Endolysins, by contrast, target the peptidoglycan itself, a structural component so fundamental to bacterial survival that changing it in ways that would block the endolysin tends to compromise the cell’s integrity. Resistance to endolysins is considered rare, and the selection pressure they place on bacterial populations is thought to be lower than that of traditional antibiotics.¹²

Endolysins also tend to be highly specific, often targeting just one or a few related bacterial species. This specificity means they can kill the pathogen you are after without wiping out the broader microbial community in, say, the gut. Broad-spectrum antibiotics, by contrast, are notorious for collateral damage to the microbiome, which itself can create opportunities for secondary infections like Clostridioides difficile.

Breaking Down Biofilms

Bacteria do not always exist as free-floating individual cells. Many form biofilms, dense communities encased in a sticky matrix of sugars and proteins that antibiotics struggle to penetrate. Biofilms are responsible for a large share of chronic and device-related infections, and treating them is one of the biggest headaches in clinical medicine.

Endolysins have shown genuine promise against biofilms. The endolysin LysH5, for example, reduced attached populations of both Staphylococcus aureus and Staphylococcus epidermidis biofilms by one to three log units in a single treatment. A second application drove the remaining bacteria to undetectable levels, and the surviving cells after the first treatment were not resistant “persisters” — they remained fully susceptible to the endolysin.¹³ Work on a chimeric endolysin called CHAPk-SH3bk showed it could reduce MRSA (methicillin-resistant Staphylococcus aureus) biofilms on steel, glass, and even ex-vivo skin tissue, with reductions of roughly 50% or more on 24- and 48-hour biofilms.¹⁴

Pairing Endolysins with Antibiotics

Rather than replacing antibiotics entirely, endolysins may work best alongside them. Combination approaches have attracted growing attention, particularly for biofilm-associated infections where neither antibiotics nor endolysins alone are always sufficient.¹⁵

The endolysin SAL200, which targets Staphylococcus aureus, illustrates the idea well. In lab tests, SAL200 reduced the minimum inhibitory concentrations of standard-of-care antibiotics and achieved more than a three-log reduction in S. aureus counts within 30 minutes on its own. When combined with antibiotics, the effect was synergistic: the combined treatment produced a sustained drop of more than two log units, outperforming either treatment alone.¹⁶ The logic is intuitive: the endolysin weakens or breaches the cell wall, making it easier for antibiotics to reach their intracellular targets.

How Clinical Trials Have Gone So Far

The most advanced endolysin candidate in human medicine has been exebacase (also known as CF-301 or lysin CF-301), developed to treat Staphylococcus aureus bloodstream infections and endocarditis. Its journey through clinical trials illustrates both the promise and the difficulty of translating lab results into clinical drugs.

In a Phase 2 trial, patients who received exebacase plus standard antibiotics had a clinical response rate at day 14 of about 70%, compared with 60% for antibiotics alone. The difference was not statistically significant in the overall group, but in a prespecified subgroup of patients with MRSA infections, the response rate was dramatically higher in the exebacase group: roughly 74% versus 31%, a gap of over 40 percentage points.¹⁷ That MRSA finding, though exploratory, generated enormous excitement and propelled the drug into a larger Phase 3 trial.

The Phase 3 DISRUPT trial, however, was stopped early for futility. Among 97 patients with MRSA infections, the response rate was 50% in the exebacase group versus about 61% in the antibiotics-alone group, a reversal of the earlier trend. Adverse event rates were similar between groups, and no hypersensitivity reactions related to exebacase were reported, so safety was not the issue.¹⁸ The failure was a sobering reminder that promising early signals do not always survive larger, more rigorous testing. It did not invalidate the endolysin concept, but it made clear that the field has work to do before any endolysin reaches routine clinical use.

Safety and the Immune Response Question

Because endolysins are proteins, the body’s immune system will eventually recognize and respond to them, just as it does to any foreign protein. In mouse studies, the endolysin PlyC triggered a typical antibody response, with specific IgG levels rising about two weeks after injection and peaking around one month later.¹⁹ The practical concern is whether these antibodies would neutralize the endolysin in a repeat treatment, making it less effective the second time around.

In human safety data so far, the picture has been more reassuring than feared. In a Phase 1 dose-escalation study in healthy volunteers, SAL200 was well tolerated across doses ranging from 0.1 to 10 milligrams per kilogram, with no serious adverse events observed.²⁰ The exebacase Phase 3 trial similarly reported no hypersensitivity events.¹⁸ Still, the immunogenicity question has not been fully resolved for repeated or long-term dosing. Most infections treated with endolysins would likely involve short courses, which reduces the concern somewhat, but it remains an active area of investigation.

Endolysins in Food Safety

The medical applications get the most attention, but endolysins are arguably closer to real-world use in the food industry. Food safety is an appealing arena because the regulatory bar is different from pharmaceutical approval, and the need is urgent: foodborne pathogens like Listeria monocytogenes remain a persistent problem despite existing control measures.

Endolysins have shown promise for controlling Listeria during food production and storage, especially when combined with existing preservation methods like nisin (a naturally occurring antimicrobial peptide) or high hydrostatic pressure. The combination approach produces synergistic effects, with endolysins enhancing the killing achieved by conventional methods.²¹ Broader reviews of the field have cataloged successful endolysin treatments across various food types and processing environments.²² Because endolysins are proteins that break down naturally and target specific bacteria, they fit well into “clean label” food preservation, where consumers and regulators prefer agents that do not leave chemical residues.

Manufacturing Challenges

Producing endolysins at scale is trickier than it might seem. Most endolysins destined for research or commercial use are made as recombinant proteins in E. coli, a standard workhorse for protein production. But endolysins are often prone to forming insoluble clumps called inclusion bodies when expressed at high levels or at standard growth temperatures. Researchers have found that lowering the induction temperature significantly increases the fraction of endolysin that stays soluble and functional. Adding certain chemicals to the extraction process also helps: the surfactant N-lauroylsarcosine, for example, strongly boosted the recovery of soluble endolysin in a concentration-dependent manner.²³

These production challenges are not unique to endolysins — they are common across recombinant protein manufacturing — but they do add cost and complexity that factor into whether endolysins can become practical products at scale. For food safety applications where the endolysin is applied to surfaces or mixed into products, purity requirements may be somewhat less demanding than for injectable pharmaceutical formulations, which is another reason the food sector may see widespread endolysin adoption sooner than hospitals do.

The Diversity Hiding in Phage Genomes

Researchers have only scratched the surface of the endolysins that exist in nature. A comprehensive survey of endolysin genes across fully sequenced phage genomes found enormous diversity in catalytic domains, binding domains, and the ways these modules are arranged.²⁴ Each phage species has evolved endolysins fine-tuned to the specific cell-wall chemistry of its host bacterium, and because bacteria are endlessly diverse, so are the enzymes designed to destroy them. This natural library represents a vast resource for both discovering new antimicrobial candidates and finding novel building blocks that protein engineers can recombine into chimeric endolysins with customized properties. As genome sequencing becomes cheaper and metagenomic approaches allow researchers to mine viral DNA from environmental samples, the catalog of available endolysins will continue to grow, potentially yielding enzymes with activities no one has seen yet.