As biology becomes increasingly data-driven, so does the field of phage lysins, enzymes that degrade bacterial cell walls and offer promising alternatives to traditional antibiotics. Five years ago, we introduced PhaLP, a centralized resource for Phage Lytic Protein sequences and associated metadata to support global research efforts. Here, we present PhaLP 2.0, an enhanced database designed to address key challenges in computational lysin research by integrating newly identified lysins from thousands of metagenomes. To expand the known diversity of lysins beyond that of cultured phages, we developed SUBLYME, a protein-embedding-based machine-learning Software designed to Uncover and classify Bacteriophage Lysins from Metagenomic datasets. Using embeddings derived from the well-curated sequences of the original PhaLP database, we trained support vector machines to distinguish lysins from non-lysins in viromes and classify them as endolysins or virion-associated lysins. The models achieved an average F1 score of 98% on held-out clusters. SUBLYME enabled the discovery of 743 000 new lysin sequences from EnVhogDB, a virome-derived protein database, increasing the number of known lysin clusters 40-fold, from 1000 to 40 000. SUBLYME and PhaLP 2.0 are accessible online at https://github.com/Rousseau-Team/sublyme and https://phalp.ugent.be, respectively. Together, these advances establish PhaLP 2.0 as a comprehensive and scalable portal for lysin discovery, classification, and sequence analysis, paving the way for future antibacterial applications and evolutionary insights.

Antimicrobial resistance in pathogenic mycobacteria remains a critical challenge due to poor drug penetration through their complex cell wall, which necessitates prolonged multidrug regimens. Mycobacteriophages encode a lytic machinery that can disrupt this barrier. In this research article, we describe a modular mycolysin platform combining phage enzymes Lysin A and Lysin B with outer membrane-permeabilizing peptides and protein transduction domains using VersaTile shuffling technology. Screening the chimeric libraries against Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG), followed by the evaluation of selected mycolysin hits, identified potent candidates with minimum inhibitory concentration values as low as 1.28 μg/ml against M. bovis BCG and up to 75 μg/ml against pathogenic nontuberculous mycobacterium Mycobacterium avium. The three most potent mycolysins showed intracellular efficacy, serum stability, noncytotoxicity, in vivo proof-of-concept efficacy in rat wound and pulmonary infection models, and synergy with rifampicin treatment. This biotechnology framework illustrates the promise of translating phage enzymes into next-generation antimycobacterial therapies.

INTRODUCTION:Antimicrobial resistance is escalating globally, while the development of new antibiotic classes has stagnated. This trend is reminiscent of the late-stage dynamics of S-curve innovation, where incremental advances no longer meet clinical needs. To overcome this impasse, disruptive innovation is required. As protein-based antibacterials, lysins represent a fundamentally different modality from traditional small-molecule antibiotics and offer new opportunities for disruptive innovation in terms of resistance development and microbiome preservation. AREAS COVERED:Sourced from a vast natural reservoir, lysins exhibit rapid, targeted bactericidal activity with low resistance potential and high specificity. Their narrow-spectrum nature supports a potential for microbiome preservation and the conceptual development of theranostic platforms for precision infectious disease management, combining ultra-fast pathogen detection with targeted therapeutic activity. EXPERT OPINION:To initiate new innovation cycles, disruptive modalities such as protein-based lysins will be needed, offering a paradigm shift in antimicrobial therapy. Their modular architecture and amenability to protein engineering enable a hit-to-lead development strategy akin to small-molecule pipelines. Their synergistic interactions with standard-of-care antibiotics and booster-like activity could facilitate incremental clinical integration into existing treatment protocols. This positioning supports regulatory acceptance and paves the way for lysins to become transformational components of precision antimicrobial therapy.

BACKGROUND:Branched receptor-binding protein (RBP) systems enable bacteriophages to broaden their host range through the incorporation of two or more RBPs. Using the Klebsiella podophages KP32, K11, and KP34 as model systems, we experimentally validated the interaction between the branching domain of the primary RBP (RBP1) and the conserved docking peptide of the secondary RBP (RBP2) as an essential architectural pair enabling dual-RBP incorporation into the virion. RESULTS:Systematic engineering revealed that loss of either of these domains, the branching domain or the conserved peptide, abolishes RBP2 assembly, underscoring their structural role in organizing the branched configuration and demonstrating that the anchor domain is the sole element directly attaching the RBP complex to the virion. Exploiting this interaction, we engineered a chimeric phage based on the Klebsiella phage KP32 scaffold that was capable of cross-genus infection and productive propagation on both Klebsiella and Escherichia hosts. In contrast to previous approaches that required replacement of the entire RBP, the adaptor and nozzle proteins, this strategy achieved host-range reprogramming through modular domain swapping and positional relocation of RBPs (i.e., exchanging RBP1 and RBP2 positions). Conversely, an Escherichia phage K1F scaffold was also successfully engineered to infect Klebsiella. CONCLUSIONS:Our study confirms that the RBP branching domain and the conserved peptide function as specific interacting partners. Our findings establish the conserved peptide as a docking element and highlight the structural flexibility of podoviruses to accommodate RBPs from different positional and taxonomic contexts. Collectively, this work provides a mechanistic framework for rational phage engineering and defines a general design principle for generating customized therapeutic phages with an expanded host spectrum, including cross-genus infectivity.

Our understanding of how depolymerase sequence and structure determine substrate specificity is fragmentary due to the limited number of experimentally characterized enzymes. Here we show DepoCatalog - an experimentally validated collection of 129 recombinantly prepared Klebsiella phage depolymerases (90 enzymes produced in this study and 39 homologs from the literature), with specificity spanning 75 KL-types. Enzymes originated from podo-, sipho-, myo-, jumbo phages, and prophages. Using activity profiling, structural modeling, and domain dissection, we propose a five‑class framework that captures the architectural and functional diversity of these enzymes. DepoCatalog uncovers cross-reactivity and taxa‑specific enzymes. Structural comparisons indicate that specificity switching or extension is associated with modifications to the C‑terminal domain. We further hypothesize that podoviruses encoding up to two RBPs show greater receptor adaptability than jumbo phages with multiple specialized RBPs. Finally, we develop a publicly accessible, DepoCat dataset ( https://depocat.uwr.edu.pl ) for specificity, structural classification and comparison of newly identified depolymerases.

Phage endolysins are increasingly investigated as novel protein-based antibiotics, offering solutions to the antibiotic resistance crisis. Endolysins targeting Gram-positive bacteria come in a variety of modular architectures, combining domains that bind the bacterial cell wall or enzymatically degrade it. While much research has focused on either understanding this multidomain architecture or leveraging it to create custom engineered lysins, far less is known about the oligopeptide linkers connecting their domains. Nevertheless, several engineering studies have observed a remarkable influence of the linker on lysin activity. In this work, we computationally investigated a broad set of Gram-positive endolysin linkers to bridge this knowledge gap. Relying on AlphaFold2-generated protein structural models, we collected 1072 linker sequences by finely delineating the domain limits using the SPAED tool, and described these through sixteen physicochemical and structural properties. Initial data exploration showed that endolysin linkers are highly diverse and feature similar amino acid compositions as previously described, general protein linkers. Subsequently, data mining and interpretable machine learning approaches were adopted to uncover the relationships between linkers and their endolysin domain architectures, as well as the associated phage host genus. These analyses revealed that such relationships do exist and are multidimensional in nature. Therefore, our findings provide evidence that the evolutionary pressure put on phages to adapt their lysis system to ever-changing environments and host requirements is not limited to the endolysin domains, but extends to the linkers connecting them. For instance, certain domain architectures were consistently associated with longer linkers, while others were highly stable. In summary, this work presents the first in-depth exploration of phage endolysin linkers, shedding light on their phage host- or domain architecture-specific design rules, and offering new perspectives for engineering endolysins as novel antimicrobials.

Food contamination remains a major challenge for the food industry, including the dairy sector. Rich and complex composition of milk provides an excellent environment for the growth of pathogenic microorganisms, among which the most common bacterial contaminants include enterotoxin-producing Staphylococcus aureus, Listeria monocytogenes, Shiga toxin-producing Escherichia coli (STEC), and Salmonella spp. In this study we characterized the modular lytic enzyme MLE-19 obtained through modular engineering and its potential as an antibacterial agent to protect milk and dairy products against Salmonella enterica ssp. enterica PCM 2266. We demonstrated that MLE-19 with a catalytic domain of the thermostable endolysin Ph2119, exhibited high thermal stability with a Tm of 101.66°C. Furthermore, pre-treatment of MLE-19 under pasteurization conditions (80°C, 10 min) enhanced its anti-Salmonella activity compared with the unheated enzyme (reductions of 1.14 and 0.45 log units compared with the control), highlighting its applicability in the dairy industry. The minimum inhibitory concentration (MIC) of MLE-19 against Salmonella cells was 100 µg/mL, and the presence of 0.5 mM EDTA reduced this value 4-fold (to 25 µg/mL). MLE-19 caused significant decreases in bacterial load at concentrations of 12.5 and 200 µg/mL for planktonic and biofilm cells, respectively. Food matrix represented by milk, yogurt and cottage cheese and external conditions such as temperature and duration had a variable impact on the protective activity of MLE-19. Significant reductions were observed in all food matrices with the most pronounced antibacterial effects in milk, where up to 3 log reduction when storing at 4°C and 20°C were achieved. We propose that the enzyme exhibited greater mobility in milk, enabling more efficient access to and degradation of Salmonella cells. Our findings identify the thermostable MLE-19 as a new promising agent for controlling Salmonella spp. in the dairy industry.

Antibiotic-resistant Vibrio species pose a threat in aquaculture. This study evaluates the potential of phage vB_VibS_KarBoss under climate-influenced water conditions such as temperature, pH, and salinity. vB_VibS_KarBoss, isolated from fish market wastes, inhibited 48 of 89 nonredundant autochthonous Vibrio isolates. With a 10-min latent period and a burst size of 3 phages per cell, vB_VibS_KarBoss inhibited the growth of pathogenic species including Vibrio campbellii and Vibrio parahaemolyticus. With only 16.4% intergenomic similarity to related phages, we propose a new phage genus, KarBossvirus, for consideration by the International Committee on Taxonomy of Viruses. Considering the robustness of Artemia and their sensitivity to changes in environmental conditions, the virulence of the Vibrio isolates resulting in Artemia mortality and the efficacy of vB_VibS_KarBoss in these altered water conditions were studied. Results support the potential importance of vB_VibS_KarBoss in biocontrol in its ability to rescue >50% Artemia (p < 0.01) from mortality due to vibriosis.

Designer cellulosomes (DCs) are engineered multienzyme complexes inspired by natural cellulosomes, designed to improve lignocellulose breakdown. Their modular architecture enables the spatial colocalization of diverse catalytic activities, potentially enhancing depolymerization efficiency compared to free enzymes. Although conceptually promising, little is known about how they perform on complex lignocellulosic substrates. In this study, we developed a tetravalent DC using a modular VersaTile assembly approach, incorporating endoglucanase, cellobiohydrolase, β-glucosidase, and endoxylanase activities. The process involved (i) delineating catalytic modules from Cellvibrio japonicus enzymes, (ii) generating docking enzyme variants via combinatorial cloning, and (iii) selecting optimal candidates based on expression, activity, and cohesin-dockerin binding before assembling them onto a scaffoldin with four cohesins and a cellulose-binding module. The resulting DC was tested on two industrially relevant substrates: agro-industrial wheat fibers and genome-edited low-lignin poplar biomass under controlled laboratory conditions. It achieved cellulose-to-glucose conversion yields of 24.98% (150 pmol DC/ml) and 0.82% (200 pmol DC/ml), respectively, under the test conditions. By comparing the saccharification efficiencies of the enzymes in their free and complexed forms, we found that colocalization on a common scaffoldin significantly enhanced synergistic activity. This effect was most pronounced under low enzyme concentrations and when acting on complex lignocellulosic substrates, increasing glucose release compared to free enzymes. These observations highlight that the benefits of colocalization are substrate-dependent and occur under conditions that mimic the natural environment of biomass degradation, conditions that differ from typical industrial settings. This work advances our understanding of DC behavior on real-world substrates, providing essential insights for evaluating their economic viability in industrial applications. One-sentence summary Natural-like conditions helped customized DC release more sugars from biomass than standard industrial setups.

The high capsular diversity restricting the host range of many Klebsiella phages has driven the evolution of branched receptor-binding protein (RBP) systems as a strategy for host range expansion. These dual RBP systems offer a unique opportunity for modular engineering. However, most previous approaches lacked a standardized and systematic framework to exploit an engineering platform that enables efficient and modular reprogramming of Klebsiella podophages with dual RBP systems. The workflow integrates (I) the VersaTile technique for rapid assembly of chimeric RBP gene clusters, (II) Gibson assembly for in vitro genome construction, and (III) electroporation-based rebooting. By retaining one native RBP, the system ensures that a suitable host is always available for rebooting, reducing technical failure, and allowing a clear distinction between biological incompatibility and assembly issues. Using this approach, we systematically swapped full-length RBPs and receptor-binding domains (RBDs) at both positions of the dual RBP system in podophages K11 and KP32, confirming the modularity and interchangeability of structural and enzymatic domains. Advanced designs, including cross-swapping and position swapping, were successfully implemented, while attempts to graft phylogenetically distant RBPs revealed structural constraints that inform future design strategies. This work introduces a standardized, scalable, and plug-and-play framework for phage engineering that leverages the modularity of dual RBP systems. By ensuring rebooting through an unmodified RBP, the platform provides a robust foundation for systematic host range reprogramming and functional studies of RBP architecture, paving the way for rational design of therapeutic phages.

ABSTRACT Insufficient product yield remains a major bottleneck in the development of microbial cell factories for fine chemical production. In vivo enzyme colocalisation on synthetic scaffolds has emerged as a promising strategy to enhance metabolic efficiency, yet current approaches are often labour‐intensive and inefficient. Here, we present the development of a ‘scaffoldomics’ platform, a generic synthetic framework designed to enable combinatorial scaffolding of biosynthetic pathways for the generation of diverse multi‐enzyme complexes. This system enables the assembly of up to four pathway enzymes onto a protein scaffold. As a proof of concept, the platform was applied to the biosynthesis of naringenin, a key flavonoid intermediate. Integration of a chromosomally encoded naringenin biosensor in Escherichia coli allowed for real‐time detection and pathway evaluation. The biosensor response curve was established and confirmed functional naringenin production. Moreover, comparative experiments demonstrated that the addition of a scaffold to docking enzymes significantly enhanced yield up to a ~19‐fold, indicating a positive colocalisation effect. These results highlight the utility of the scaffoldomics platform as a powerful tool for further efficient combinatorial design, construction, and optimisation of biosynthetic pathways in synthetic biology.

Despite significant progress in understanding phage biology and their clinical applications, the specificity of phages remains only poorly understood and a matter of empirical testing. Phage receptor-binding proteins (RBPs), which mediate the initial contact with bacterial cells and govern host recognition, possess a modular architecture. The N-terminal domains primarily serve a structural role, facilitating the attachment of the RBP (or RBP complex) to the virion. In contrast, the function of the C-terminal modules, and their interplay with the central enzymatic domain in impacting RBP and phage specificity, remains underexplored. This study investigates the receptor-binding protein KP32gp38 of Klebsiella phage KP32, which contains an unusual C-terminal combination of a carbohydrate-binding module (CBM) and a lectin-like (LEC) domain, two elements that are typically found separately rather than in tandem. We dissected the roles of these modules in trimerization, substrate binding, and specificity at both the protein and phage level. By deletions, fusions, and exchanges of the modules through both protein and phage engineering, we examined the impact of the domains on the specificity of the RBP and the host range of the phage. Protein fusions with GFP were tested for their ability to bind the bacterial capsule. To verify the influence of the domains on RBP trimerization, different variants were analysed with SEC - MALLS. The results revealed that the LEC domain is essential for trimerization, whereas the CBM domain is crucial for enzymatic activity and capsule binding. Engineered phages lacking these domains confirmed the necessity of both CBM and LEC for full functionality. This work underscores the versatility and evolutionary adaptation of CBM and LEC folds in phage RBPs, providing valuable insights into phage specificity mechanisms. Our findings offer a blueprint for understanding the molecular determinants of phage-host interactions, crucial for advancing phage-based antibacterial therapies.

Phage lysins, enzymes encoded by bacteriophages that degrade bacterial cell walls, are emerging as a promising class of antimicrobial agents. This study aimed to discover novel lysins with activity against Enterococcus species using a sequence-based metagenomic discovery pipeline. Viral metagenomic DNA was extracted and sequenced from five environmental samples originating from pig feces or sewage. Putative lysins were first predicted with SUBLYME, a protein embedding-based classifier. Subsequently, a specific protein embedding-based classifier was developed to predict lysins with potential activity against Enterococcus . A total of 8 825 candidate lysins were predicted, including 129 with potential anti-enterococcal activity. Comparative analysis revealed differences in domain architectures and physicochemical properties between lysins derived from fecal and sewage samples, suggesting distinct phage host origins. A subset of the predicted lysins was expressed in Escherichia coli , partially purified and tested for muralytic activity against three enterococcal species ( Enterococcus faecium , Enterococcus faecalis , and Enterococcus hirae ). Among the 21 expressed lysins with variable expression yields, four exhibited lytic activity against all three Enterococcus species, two were active against Ent. faecalis and Ent. hirae , and seven showed activity exclusively against Ent. hirae . Six of these active proteins contained previously unreported domain architectures, indicating that this approach can uncover structurally novel functional lysins. While this pipeline was applied to Enterococcus , it is broadly adaptable for the discovery of lysins targeting other bacterial pathogens, offering a scalable approach to expand the antimicrobial arsenal.

Global food shortages and rising antimicrobial resistance require alternatives to antibiotics and agrichemicals for the management of agricultural bacterial pathogens. The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of kiwifruit canker and is responsible for major agricultural losses. Bacteriophage enzymes present an emerging antimicrobial option. Endolysins possess the ability to cleave peptidoglycan and are effective antimicrobials against gram-positive bacteria. Delivery of endolysins to the peptidoglycan of gram-negatives is impeded by the additional outer membrane. To overcome this barrier, we used VersaTile molecular shuffling to produce Psa-targeting chimeric proteins which were then tested for antimicrobial activity. These chimeras consist of endolysins linked by polypeptides to diverse phage proteins mined from Psa phage genomes. A preferential configuration for antibacterial activity was observed for enzymatic domains at the N terminus and alternative phage proteins at the C terminus. The lead variant possessed an N-terminal modular endolysin and a C-terminal lipase. Antibacterial activity was enhanced with the addition of the chemical permeabilizers such as citric acid or EDTA. Mutagenesis of the lipase active site eliminated exogenous antibacterial activity toward Psa. The endolysin-lipase chimera demonstrated specificity toward Psa, illustrating potential as a targeted biocontrol agent. Overall, we generated a chimeric endolysin with exogenous and specific activity toward Psa, the causative agent of kiwifruit canker.

ABSTRACT In systems biocatalysis, combining pathway enzymes in vitro allows for the conversion of basic substrates into more complex, valuable chemicals. However, in vitro enzyme cascades are not yet economically viable for large‐scale bio‐based chemical production. Enhancing pathway efficiency through enzyme colocalization on synthetic protein scaffolds is a proposed solution, though still debated. We constructed a synthetic protein scaffold that colocalises the first three glycolytic enzymes using cohesin–dockerin interactions. Initially, we converted wild‐type enzymes to the docking enzyme mode and evaluated their activity. Next, we demonstrate how the colocalisation of the three docking enzymes on distinct scaffolds enhances the enzyme cascade's production. Starting from glucose, the multi‐enzyme complexes produced fructose‐1,6‐bisphosphate, confirming the activity of each enzyme. PfkA, which converts fructose‐6‐phosphate and ATP to fructose‐1,6‐bisphosphate and ADP, was identified as the rate‐limiting enzyme. We demonstrated that scaffolding proximity effects lead to higher product output than free docking enzymes, particularly at lower enzyme densities. Further research is needed to determine the relevance of enzyme colocalisation under industrial production settings. In addition, optimising an enzyme cascade demands a thorough understanding of reaction mechanisms and kinetics. The VersaTile method streamlines optimisation studies of modular proteins and complexes, enabling analysis of a broader design space by bypassing technical preparatory hurdles.

Background: Phage tail-like bacteriocins, or tailocins, provide a competitive advantage to producer cells by killing closely related bacteria. Morphologically similar to headless phages, their narrow target specificity is determined by receptor-binding proteins (RBPs). While RBP engineering has been used to alter the target range of a selected R2 tailocin from Pseudomonas aeruginosa, the process is labor-intensive, limiting broader application. Methods: We introduce a VersaTile-driven R2 tailocin engineering and screening platform to scale up RBP grafting. Results: This platform achieved three key milestones: (I) engineering R2 tailocins specific to Escherichia coli serogroups O26, O103, O104, O111, O145, O146, and O157; (II) reprogramming R2 tailocins to target, for the first time, the capsule and a new species, specifically the capsular serotype K1 of E. coli and K11 and K63 of Klebsiella pneumoniae; (III) creating the first bivalent tailocin with a branched RBP and cross-species activity, effective against both E. coli K1 and K. pneumoniae K11. Over 90% of engineered tailocins were effective, with clear pathways for further optimization identified. Conclusions: This work lays the groundwork for a scalable platform for the development of engineered tailocins, marking an important step towards making R2 tailocins a practical therapeutic tool for targeted bacterial infections.

Designer cellulosomes (DCs) are precisely engineered multi-enzyme complexes aimed at lignocellulose saccharification. Achieving spontaneous designer cellulosome display on yeast cell surfaces has been a long-term objective to enhance consolidated bioprocesses with concurrent ethanol production. A "self-assembly" approach involves simultaneous scaffoldin display and docking enzyme secretion. However, challenges arise from the size and complexity of designer cellulosomes, coupled with the yeast cells' limited capacity for heterologous protein expression. A comprehensive examination of Saccharomyces cerevisiae as a host for DC expression remains unaddressed. We meticulously examined the capability of S. cerevisiae to produce and display a fluorescent protein complex, which mimics the designer cellulosome architecture and allows for convenient detection of all individual components on the cell surface, using flow cytometry and confocal microscopy. Population-wide analysis revealed a fluorescent protein complex production efficiency of approximately 10%. Single-cell analysis highlighted a clear mutual influence between the expression of scaffoldin and docking proteins, impacting cellular fitness. Newly emerging buds were identified as hotspots for scaffoldin display. The finite capacity of yeast cells to produce heterologous proteins was identified as a major bottleneck. While distributing the cellular load among multiple hosts within a synthetic yeast consortium can alleviate this burden, the use of fluorescent protein complex surface display has visualized the heterogeneity and constraints of S. cerevisiae as a host for designer cellulosome expression at the population and single cell level. This study provides a realistic assessment of the challenges in achieving efficient S. cerevisiae-based DC display for consolidated bioprocessing.IMPORTANCEEfficient and economically viable biomass conversion into fermentable sugars is a pivotal challenge in transitioning from a petroleum-based economy to a bio-economy. Drawing inspiration from nature, cellulosomes represent an exemplary solution for the effective digestion of lignocellulose. These multi-enzyme complexes can be precisely engineered to tailor their properties and transferred to the surface of yeast cells, which can subsequently ferment the sugars into bulk or fine chemicals. Achieving this transfer successfully necessitates a comprehensive understanding of how yeast cells can recombinantly produce and attach such multi-component complexes to their surface. This study employs a fluorescent surrogate to provide novel insights into the capabilities of yeast cells at both the single-cell and population levels.

Abstract In this study, we evaluated the combined effect between MLE-15, a modular lytic enzyme composed of four building blocks, and reline, a natural deep eutectic solvent. The bioinformatic analysis allowed us to determine the spatial architecture of MLE-15, whose components were bactericidal peptide cecropin A connected via a flexible linker to the cell wall binding domain (CBD) of mesophilic 201ϕ2 − 1 endolysin and catalytic domain (EAD) of highly thermostable Ph2119 endolysin. The modular enzyme showed high thermostability with the melting temperature of 93.97 ± 0.38 °C, significantly higher than their natural counterparts derived from mesophilic sources. The minimum inhibitory concentration (MIC) of MLE-15 was 100 µg/mL for a panel of Gram-positive and Gram-negative bacteria, while the MIC of reline ranged from 6.25 to 25% v/v for the same strains. The addition of reline effectively reduced the MIC of MLE-15 from 100 µg/mL to 3.15–50 µg/mL. This combination displayed additive effects for most strains and synergism for extensively antibiotic-resistant Acinetobacter baumannii and Bacillus subtilis. The subsequent evaluation revealed that MLE-15 eliminated planktonic cells of A. baumannii RUH134, but was ineffective against matured biofilms. However, combined with reline, MLE-15 reduced the bacterial load in the matured biofilm by 1.39 log units. Confocal fluorescence microscopy indicated that reline damaged the structure of the biofilm, allowing MLE-15 to penetrate it. Additionally, MLE-15 and its combination with reline eradicated meropenem-persistent cells of A. baumannii RUH134. Effectiveness in lowering the MIC value of MLE-15 as well as protection against antibiotic-tolerant persister cells, indicate that MLE-15 and reline combination is a promising candidate for effective therapies in bacterial infections, which is especially important in the light of the global crisis of antimicrobial resistance.

SPAED is an accessible tool for the accurate segmentation of protein domains that applies hierarchical clustering to the predicted aligned error (PAE) matrix obtained from AlphaFold predictions. It leverages information contained in the PAE matrix to better identify domain-linker boundaries and detect disordered regions. On a dataset of 376 bacteriophage endolysins (proteins that degrade the bacterial cell wall), SPAED achieves a mean intersect-over-union score of 96% and a domain-boundary-distance score of 89% compared to 94% and 70%, respectively, for the state-of-the-art tool Chainsaw. SPAED is available on the web at http://spaed.ca and available for download at https://github.com/Rousseau-Team/spaed . Elsa Rousseau - elsa.rousseau@ift.ulaval.ca , Roberto Vázquez - rvazqf@gmail.com

SUMMARY:SPAED is an accessible tool for the accurate segmentation of protein domains that leverages information contained in the predicted aligned error (PAE) matrix obtained from AlphaFold to better identify domain-linker boundaries and detect terminal disordered regions. On a dataset of 376 bacteriophage endolysins (proteins that degrade the bacterial cell wall), SPAED achieves a mean intersect-over-union score of 96% and a domain-boundary-distance score of 89% compared to 94% and 70%, respectively, for the state-of-the-art tool Chainsaw. AVAILABILITY AND IMPLEMENTATION:Implemented in Python, SPAED is accessible on the web (https://spaed.ca) and available for download from https://github.com/Rousseau-Team/spaed or https://pypi.org/project/spaed. The data used to test SPAED can be found at https://doi.org/10.5281/zenodo.15285860.