Antimicrobial resistance (AMR) has emerged as one of the most pressing threats to global health, undermining the ability to effectively prevent and treat an ever-increasing range of infections caused by bacteria, viruses, parasites and fungi (Morrison and Zembower, 2020; Sati et al., 2025). Among all AMR pathogens, antibiotic-resistant bacteria pose the greatest burden and health-care costs, driven by the overuse and misuse of antibiotics over the past decades (Sati et al., 2025). Current projections estimate that AMR will directly cause nearly 40 million deaths between 2025 and 2050 with a predicted 1.91 million deaths attributable to and more than 8.2 million deaths associated with AMR annually by 2050 (Naghavi et al., 2024). Other studies predict even higher numbers up to 10 million directly attributable annual deaths in 2050, bypassing even current rates of cancer (Figure 1) (O’neill, 2014). With more and more bacterial strains gaining resistance even to last-resort antibiotics, coupled with a still slow-moving development of new antibiotics following the innovation gap over past decades, the global health community faces a severe crisis (European Commission OECD, 2024).

Figure 1: Projected rise in global annual deaths attributable to AMR, 2020–2050 (O’neill, 2014).

However, the impact of AMR extends far beyond its direct health consequences. It threatens to severely restrict the use of antibiotics across various fields, from prophylactic applications in surgery to food preservation and animal agriculture, thereby underscoring the interconnected One Health perspective, which links human, animal, and environmental well-being. If antibiotic use must be curtailed to preserve their effectiveness for the treatment of critical human infections, this will result in substantial collateral damage, including increased healthcare burdens, significant economic losses worldwide as well as disruptions in animal agriculture and food security (National Academies of Sciences et al., 2021). Estimates by the World Bank project that health costs could rise by up to $3.4 trillion by 2050 due to AMR, with effects on trade, livestock production, and millions pushed into poverty (O’neill, 2014; Jonas et al., 2017).

Since their discovery in the early 20th century, antibiotics have revolutionized the treatment of infectious diseases, dramatically reducing mortality from once-lethal bacterial infections. Yet, the efficacy of antibiotics is continually undermined by the relentless and near-inevitable emergence of resistances across bacterial populations. Virtually every new antibiotic class introduced is quickly met with adaptive bacterial strategies - including target site modification, enzymatic drug degradation, efflux pump overexpression or metabolic pathway alterations - often rendering drugs ineffective within just a decade of introduction (Figure 2). Resistance genes emerge through mutation and are quickly disseminated throughout bacterial populations via horizontal gene transfer and selective pressure in clinical and environmental settings (Fair and Tor, 2014; Kapoor, Saigal and Elongavan, 2017; Naghavi et al., 2024).

Figure 2: Timeline of antibiotic deployment and the evolution of antibiotic resistance from 1930 to 2005. Created with BioRender.com adapted from (Clatworthy, Pierson and Hung, 2007).

Beyond these established resistance mechanisms, a particular obstacle for antibiotics is the prevalence of bacterial biofilms. These biofilms are highly organized communities of bacteria encased in self-produced extracellular polymeric matrices that restrict drug penetration, facilitate genetic exchange, and enable the survival of “persister” cells that can withstand even high concentrations of antibiotics. These properties make biofilm-associated infections, such as those found in chronic wounds, medical devices, and certain lung or urinary tract infections, especially difficult to eradicate with standard drug therapies. Even new-generation antibiotics often struggle to clear these persistent biofilms (Stewart and Costerton, 2001; Høiby et al., 2010).

While new antibiotics will remain irreplaceable for the foreseeable future, reliance on them alone is neither sustainable nor sufficient. The rapid pace of resistance emergence, the protection afforded by biofilms, and the continued global burden of hard-to-treat pathogens underscore the urgent need for innovative solutions and alternative therapeutic strategies alongside robust antibiotic stewardship.

The so-called ESKAPE pathogens (Figure 3) – comprising the Gram-positive Enterococcus faecium and Staphylococcus aureus as well as the Gram-negative Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species – represent the most dangerous pathogens within the AMR crisis (De Oliveira et al., 2020).

Figure 3: ESKAPE pathogens (Created with BioRender.com)

These bacteria exhibit remarkable versatility in acquiring and exchanging resistance genes and are frequently associated with biofilm-driven infections, enabling resistance to multiple antibiotic classes and rendering many infections extremely difficult to treat (De Oliveira et al., 2020; Sati et al., 2025). Examples of infections caused by these strains include ventilator-associated pneumonia as well as skin and soft tissue infections for A. baumannii; pneumonia and chronic respiratory infections for P. aeruginosa; as well as intra-abdominal infections and urinary tract infections for Enterobacter species (De Oliveira et al., 2020). As a more recent and local example, pathogenic enterohaemorrhagic Escherichia coli , i.e. responsible for the ongoing outbreak in Germany, can cause hemolytic uremic syndrome, leading to kidney failure and possibly death (Wadl et al., 2011).

In the context of bacterial infections, the therapeutic use of bacteriophages to treat bacterial diseases has long been regarded as one of the most promising approaches. First reported nearly a decade before the discovery of antibiotics, bacteriophages were quickly recognized for their potential to combat bacterial infections, showing promising results in early treatments of bacterial infections such as cholera. However, the subsequent introduction and mass production of antibiotics in the 1940s led to the decline of phage therapy and research in Western medicine, while it continued to be developed in countries such as Russia, Poland, and Georgia (Summers, 2012; Wittebole, De Roock and Opal, 2014).

With the emergence of antibiotic-resistant bacterial strains, the interest in phage related therapeutics has since been renewed worldwide. The ongoing clinical usage of phages and phage cocktails in multiple European countries has demonstrated their great potential to complement conventional antibiotics (Wittebole, De Roock and Opal, 2014). Most importantly, phages exhibit very high specificity in their targeting, typically able to infect only a few bacterial strains within a single species, which significantly minimizes off-target effects during treatment (Harper et al., 2021).

The secret behind this specificity lies in the phages’ structure and mode of action against the bacteria. Phages recognize and bind to distinct molecular patterns on bacterial cell surfaces, enabling the injection of their genetic material into the host. The bacterial translational machinery then expresses phage-encoded proteins, including structural components to form new phage particles and endolysins, potent enzymes responsible for bacterial cell wall degradation (Harper et al., 2021).

Endolysins act by cleaving specific bonds within the peptidoglycan layer of the bacterial cell wall, ultimately weakening its structure. There is a broad diversity of endolysins, each specialized to target distinct molecular bonds within the peptidoglycan, ultimately weakening the cell wall. Their expression marks the final stage of the phage’s lytic cycle, culminating in the rupture and release of new phage particles as the host cell bursts under osmotic pressure (Harper et al., 2021).

In recent decades, studies have demonstrated that externally applied endolysins can rapidly and specifically destroy Gram-positive bacteria by binding to and degrading peptidoglycan, making them highly promising candidates for next-generation antimicrobial agents. Their effectiveness and specificity are largely attributed to their molecular structure, which allows for precise targeting of bacterial pathogens (Zheng and Zhang, 2024).

Endolysins typically rely on either signal peptides or another phage-encoded protein family called holins, which form pores in the bacterial inner membrane to allow endolysins to access the peptidoglycan layer located in the extra- or periplasmic space. Once secreted, endolysins exhibit their catalytic activity through enzymatically active domains (EADs) that cleave specific bonds within the bacterial peptidoglycan layer (Figure 4) . These EADs belong to several protein families, each with distinct enzymatic functions such as glucosaminidase/muramidase (red/green), amidase, or endopeptidase (blue), targeting the glycosidic bonds, sugar-peptide amide linkages, or peptide crosslinks in the peptidoglycan, respectively (Figure 4) (Gontijo, Jorge and Brocchi, 2021; Zheng and Zhang, 2024).

Figure 4: Schematic of endolysin-mediated peptidoglycan degradation in Gram-positive and Gram-negative bacteria. Key differences in cell membrane and catalytic cleavage sites are highlighted. Endolysins are depicted as red, green, blue and purple “Pac-Man-like” circles, indicating their lysis activity (Zheng and Zhang, 2024) (Created with BioRender.com).

Structurally, endolysins differ depending on their bacterial targets, with Gram-positive endolysins containing a cell-wall binding domain (CBD) in addition to the EAD, while Gram-negative endolysins typically lack a CBD. This structural distinction arises because Gram-negative bacteria possess an additional outer membrane, locking the endolysins in the periplasm, while Gram-positive endolysins require the specific binding capability of the CBD to recognize their bacterial host (Zheng and Zhang, 2024).

The CBD-mediated specificity equips endolysins with remarkable selectivity, making them highly potent antibacterial agents against Gram-positive bacteria even when applied externally. However, for Gram-negative bacteria, endolysins face the challenge of penetrating the outer membrane to reach the peptidoglycan. To overcome this barrier, researchers have engineered hybrid or fusion endolysins by combining them with antimicrobial peptides (AMPs) capable of penetrating the outer membrane or by using membrane destabilizers such as EDTA together with endolysin (Sisson et al., 2024; Zheng and Zhang, 2024). As the outer membrane composition of Gram-negative bacteria varies substantially between species and even subspecies, the effectiveness of AMPs depends highly on the targeted strain. Selecting suitable AMPs is therefore complex, but when combined with the selectivity of the EAD, this approach could enable therapeutic agents as precisely targeted as Gram-positive endolysins (Sisson et al., 2024).

During the initial phase of our project, we explored bacteriophages as a potential alternative to antibiotics and engaged with key stakeholders from industry, healthcare, academia and regulatory authorities as part of our Integrated Human Practices. Although we learned that phage therapy is recognized as a versatile and established method for specifically targeting bacterial infections, our discussions revealed its regulatory complexities, medical limitations, and practical challenges in clinical application. Ultimately, these insights led us to pivot from whole phages to focusing on endolysins, leveraging their phage-derived specificity while avoiding many of the associated constraints. Overall, this direct exchange also strengthened our awareness of the responsibility scientists hold in engaging with society and highlighted the crucial role of Education and communication in ensuring the societal relevance and acceptance of such biotechnological innovations. We therefore made it our mission to communicate this topic with the public in visiting schools, senior citizen homes, organizing an art exhibition and publishing in a local newspaper reaching a broad and diverse range of target groups.

To advance our goal of harnessing the potential of endolysins as novel antimicrobials, we sought to understand the current primary limitations and challenges of endolysin research and application. Through consultations with experts, including leading endolysin researcher Prof. Vincent Fischetti as well as representatives from pioneering companies such as Micreos and Invitris, two main obstacles became evident: First, the limited applicability of endolysins to Gram-negative bacteria, primarily due to their protective outer membrane and second, the difficulties in recombinant production and expression, which continue to hinder their practical implementation. That’s where we come into play:

Antibiotic resistance is one of today’s largest threats to global health and we aim to redefine how we fight back against these superbugs. With Bactolyze, we are building a modular pipeline for endolysin discovery and optimization, combining cell-free in vitro production and functional screening with molecular dynamics-based modeling of antimicrobial peptides. These peptides are fused to endolysins to enable extended targeting of Gram-negative pathogens. Prospectively, our endolysins could then be applied topically or inside the body in combination with phages as shuttle vectors or inside of nanocarriers. This would open up new possibilities for more precise and effective applications against bacteria and biofilms, offering an alternative to antibiotic therapies.

Antimicrobial peptides represent a promising complement to endolysins as novel antibiotics. When fused with endolysins, AMPs can enhance the activity of these enzymes, notably against Gram-negative bacteria . The AMP component facilitates penetration through the outer membrane, thereby enabling the endolysin to more effectively traverse this barrier and reach the peptidoglycan cell wall, where it exerts its lytic function (Sisson et al., 2024).

The mechanism of AMP insertion into the outer membrane of bacteria is still not well understood and most mechanistic studies of AMP focus on the interaction between AMP and the inner membrane. Therefore, we provided AMP-MD , an Molecular Dynamics (MD) simulation platform that studies atomic-scale membrane insertion of peptides.

By combining Umbrella Sampling, unbiased simulations (Figure 5), and analysis tools we investigated peptide-membrane interactions and demonstrated that the destabilization of our model membrane through peptide insertion follows the “self-induced uptake” mechanism proposed by Hancock (Hancock, 1997), a mechanism that has not been analyzed by any molecular dynamics simulation study before.

Figure 5: Unbiased simulation of CM15 peptide insertion into the E. coli R3 O111 bacterial outer membrane. System trajectory is shown from 1 µs to 4 µs.

Through targeted case studies, we identified why some AMPs are more effective than others to enhance the membrane permeability when fused to endolysins.

In order to overcome the production issues of endolysins when using recombinant expression, such as low protein yields due to formation of inclusion bodies or cell lysis of host cells, we aimed to explore the possibility of using cell-free expression systems (Cremelie, Vázquez and Briers, 2024). In our project we used the ALiCE® system, based on crude lysate of Nicotiana tabacum Bright Yellow 2 cells (Buntru et al., 2014; LenioBio®, 2024), while also considering other, prokaryotic systems.

Building on our goal to extend the efficacy of endolysins targeting Gram-negative pathogens, we aimed to develop a modular toolbox approach, incorporating endolysins, linkers, and AMPs, each equipped with specific position tags. This design would enable the rapid and standardized Golden Gate-based cloning into our expression vector to generate and screen different variants for enhanced efficiency and targeting toward Gram-negative strains of interest. In respect to specific target strains, the selection of AMPs would herein be guided by computational modeling of AMP-membrane interactions, as described in our preceding modeling section, allowing for rational design of constructs tailored to the membrane composition of the respective pathogens.

With our approach for producing and optimizing endolysins in place, our next focus was on working on targeted delivery strategies to bring these engineered endolysins precisely to their site of action. In the course of this, we also focused on biofilms as a major obstacle to the effective application of antibacterial agents.

Biofilms pose a major challenge in combating bacteria, as they increase bacterial resistance and persistence, and cause problems with implants, food production, and water systems (Ban-Cucerzan et al., 2025). Biofilms consist of bacteria communities embedded in a self-produced extracellular protective polymeric slime layer, which provide protection against external influences such as antibiotics, disinfectants and the host’s immune system (Mayorga-Ramos et al., 2024; Ban-Cucerzan et al., 2025). To overcome this, we used M13 bacteriophages to infiltrate biofilms, which are ideal for phage display due to their large surface area. Additionally, unlike lytic phages, M13 does not lyse its bacterial host, which makes them an optimal delivery platform to test multiple endolysin variants in parallel. At the same time, it hinders the horizontal gene transfer via conjugation (Wan and Goddard, 2012). In addition, we are evaluating a range of antimicrobial peptides (AMPs) and signal peptides, designed to target both bacterial cells and the extracellular biofilm matrix simultaneously. This combinatorial approach aims to destabilize biofilm integrity and enhance bacterial susceptibility, thereby paving the way for more effective biofilm-targeted antimicrobial strategies.

Our journey with Bactolyze began with a simple yet ambitious vision: to take part in the global fight against antimicrobial resistance by contributing to developing a new generation of enzyme-based therapeutics. With our project, we explored endolysins as the innovative solution for complementing antibiotics and through extensive research and interdisciplinary collaboration, we gained a holistic understanding of both their potential and current challenges.

However, along the way, we not only explored the scientific challenges of utilizing endolysins but also the practical requirements for medical application, the legal and regulatory frameworks shaping its accessibility and the public perception of such novel therapeutics. In addition to our Integrated Human Practices, we thus also directly engaged with the public throughout educational endeavors.

Prospectively, our project opens up several paths for future development. In the short-term, the Bactolyze modular system could be expanded to different expression systems to assemble and screen AMP-endolysin combinations, targeting clinically relevant ESKAPE pathogens. Long-term applications may include integrating our engineered endolysins into various delivery systems such as phage vectors, enabling targeted application even through biofilms.

Ultimately, our vision is to establish Bactolyze not only as a proof of concept for an innovative antimicrobial approach but as a platform for continuous endolysin evolution - bridging computational modeling, synthetic biology, and in vitro expression for rapid design and functional validation. By sharing our model, protocols, and insights with the iGEM and wider scientific community, we aim to empower future teams to build upon our foundation, advancing the field of endolysin and AMP therapeutics and contributing long-term to global efforts against antimicrobial resistance.

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