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.

Exposure to redox‑active metals and sub‑inhibitory antibiotics represents a significant selective pressure shaping bacterial physiology and antimicrobial susceptibility. Here, we investigated how four genetically and phenotypically distinct Pseudomonas aeruginosa strains respond to sub‑MIC copper (Cu) and gentamicin (GE) stress, individually and in combination (Cu + GE). Across all strains, Cu acted as the dominant envelope‑active stressor, increasing biofilm biomass and extracellular DNA (eDNA) release, suppressing twitching and swarming motility, reducing quorum‑sensing‑linked protease activity, and enhancing pyomelanin production. GE alone produced limited physiological changes but modulated Cu‑driven outputs during co‑exposure, including attenuation of Cu‑induced eDNA release and strain‑specific shifts in motility and pigmentation. Early transcriptional profiling revealed consistent Cu‑dependent repression of lasI and mvfR, induction of the metal‑responsive regulator czcR, and downregulation of oprD under Cu or Cu + GE, corresponding to reduced imipenem inhibition zones when Cu was present during susceptibility testing. Combined Cu + GE exposure produced non‑additive, emergent effects that diverged from single‑stressor responses and varied across strains. These findings demonstrate that sub‑inhibitory Cu creates an envelope‑centered regulatory landscape into which gentamicin‑derived signals are integrated, generating heterogeneous and context‑dependent phenotypes. This work underscores the importance of metal-antibiotic interactions in shaping bacterial adaptation and highlights limitations of single‑stressor models for predicting antimicrobial behavior in combination.

The global rise of multidrug-resistant bacteria is a major health threat, with Klebsiella pneumoniae identified by the WHO as a critical priority pathogen. Phage-derived depolymerases have emerged as promising countermeasures because they can degrade the capsular polysaccharide (CPS) that shields bacteria from the immune system, thereby increasing their susceptibility to antibiotics and host defences. However, the limited understanding of how they recognise specific CPS structures remains a major obstacle to developing effective depolymerase-based therapeutics. In this study, we provide a comprehensive NMR and mutational analysis to characterise the mechanism of action of a miniaturised depolymerase, here mKP34gp57, targeting the clinically relevant K63 CPS. We show by NMR that mKP34gp57 hydrolyses CPS with high efficiency through an endoglycosidase-retaining mechanism. During substrate recognition, our data demonstrate that the enzyme interacts predominantly with the galactose and fucose moieties, which serve as the critical recognition features and thus the principal determinants of CPS specificity. Computational studies provide structural clues for the roles of the catalytic residues E266/E300 and D151. Finally, we prove that the hexasaccharide produced upon CPS hydrolysis stimulates dendritic cell maturation and T-helper-driven lymphocyte proliferation. Identifying the binding determinants that govern CPS recognition by mKP34gp57, and using this information to generate immunogenic fragments, deepens our understanding of how minidepolymerases can be rationally engineered to achieve tailored serotype specificity, and improved therapeutic and diagnostic potential.

Abstract Bacteriophage receptor-binding proteins (RBPs) determine bacterial host recognition and are central to phage host-range evolution, yet the structural principles governing how RBPs diversify and expand host range remain not fully understood. Although phage RBPs are thought to evolve through modular exchange of receptor-binding domains, it is unclear how this modularity is organised across diverse RBP architectures, at what structural scales recombination operates, and how it shapes host-range breadth. Here we combined large-scale AlphaFold3 structural modelling, domain-level annotation, de novo pseudo-domain segmentation, sequence modularity analysis and experimentally determined host-range phenotypes across 192 Klebsiella pneumoniae phages and 382 high-confidence RBPs spanning up to 96 K-types. We generated the first system-wide structural atlas of K. pneumoniae phage RBPs, resolving 39 structurally distinct RBP-classes. Although capsule-degrading beta-helix depolymerases dominated numerically, 161 RBPs across 37 RBP-classes employed non-depolymerase architectures, including 18 novel RBP-classes. Structural and sequence analyses show that this diversity arose through modular reuse of structural domains rather than independent invention. Conserved N-terminal scaffolds linked depolymerase and non-depolymerase RBPs across morphotypes, while receptor-binding regions diversified through recombination operating at and beyond domain boundaries. We found recent modular exchange both within genera, where capsule-specific and capsule-independent RBPs can be swapped to alter host-range strategy, and across morphotypes, where depolymerase modules moved between distant lineages altering capsule specificity. Together, these architectures resolve into six receptor-recognition strategies, establishing multi-scale modularity as the primary organising principle of RBP diversification and a structure-informed framework for guiding phage isolation and engineering against K. pneumoniae . The complete atlas is freely accessible as an interactive community resource at Klebsiella -Phage-RBP-Atlas.

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.

The global rise of multidrug-resistant Klebsiella pneumoniae underscores the urgent need for alternative therapeutic strategies. Bacteriophage-derived depolymerases have emerged as promising antimicrobial factors, selectively degrading bacterial capsules and impairing key pathogenic traits. We characterize a novel depolymerase, PRA33gp45, associated with the structural protein of bacteriophage vB_KpnP_PRA33. Bioinformatic structural analyses predicted endo-N-acetyl neuraminidase-like activity and canonical depolymerase domain architecture. The recombinant PRA33gp45 specifically hydrolysed capsular polysaccharides (CPS) of K27 serotype K. pneumoniae and produced characteristic halo zones on bacterial lawns, confirming its enzymatic activity. Capsule staining demonstrated rapid and progressive capsule degradation within 120 min of treatment. PRA33gp45 significantly inhibited biofilm formation, disrupted mature biofilms, and altered biofilm architecture as visualized by confocal microscopy. Depolymerase pre-treatment markedly reduced K. pneumoniae survival within A549 human lung epithelial cells, without exhibiting any cytotoxic effect and sensitized bacteria to complement-mediated killing in human serum. Finally, PRA33gp45 treatment of K. pneumoniae lowers morbidity and mortality in the Galleria mellonella larvae model. Collectively, these findings identify PRA33gp45 as a novel and highly specific depolymerase that diminishes K. pneumoniae virulence by targeting its protective capsule, impairing persistence as biofilm, and enhancing innate immune clearance. Its safety and efficacy suggest potential as an antimicrobial or adjuvant therapeutic agent against K27-type K. pneumoniae infections, particularly in the context of multidrug resistance and emerging pathogens.

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.

Virulent bacteriophages infecting Klebsiella pneumoniae often show capsule-driven host tropism due to the presence of capsule-specific depolymerases. Yet for temperate phages the genetic and functional basis of such capsular specificity remains less well understood. Depolymerases appear unexpectedly rare in prophage genomes, raising unresolved questions about which prophage genes mediate capsular specificity, whether this apparent scarcity reflects biological or ecological differences versus annotation limitation, and whether prophage-encoded receptor-binding proteins (RBPs) are functionally active. To address these questions, we analysed 3,900 Klebsiella genomes from diverse ecological niches to identify prophage-encoded proteins mediating capsular specificity. We conducted a genome-wide association study (GWAS) correlating prophage protein clusters (from 8,105 prophages) with confidently assigned bacterial K-loci. GWAS revealed statistically supported predictors of capsular specificity for 16 of the 35 most diverse K-loci analysed. These predictors were dominated by diverse RBPs, including classical [Formula: see text]-helix depolymerases (6 predictors), SGNH-domain hydrolases predicted to deacetylate polysaccharides (6 predictors), and structurally novel RBPs lacking known depolymerase folds (2 predictors). Nearly one-third of K-loci yielded no statistically significant predictors. A targeted experimental screen of 50 candidate prophage depolymerases showed that 34 failed to yield detectable recombinant expression, and neither sequence similarity, structural prediction, nor prophage genomic context reliably predicted activity. Of the 14 active enzymes, 5 targeted a K-type different from that predicted of their bacterial host, and enzyme specificity was not consistently explained by sequence or structural homology. Comparison with GWAS predictions revealed that 10 of the 12 strongest GWAS predictors were experimentally validated, while 2 remained inconclusive. Together, these results highlight the intrinsic difficulty of predicting activity and capsular specificity of prophage-encoded RBPs from genomic information alone. Finally, analysis of 4,598 high-completeness prophages revealed that SGNH-domain hydrolases are among the most prevalent enzymatic domains in prophage RBPs. Two SGNH-domain RBPs identified by GWAS were experimentally confirmed as active esterases, supporting capsule deacetylation as a widespread alternative to polysaccharide depolymerisation in temperate phages. Our findings reveal that Klebsiella prophages encode structurally diverse RBPs, suggesting temperate phages may rely not only on depolymerisation but also on capsule modification-such as deacetylation-for infection. This also suggests that capsule modification may contribute to phage-host interactions in ways not fully captured by current K-locus assignments, with potential implications for phage specificity, competition and vaccine design.

This study introduces the Annotated Germs for Automated Recognition (AGAR) dataset, an image database designed to advance the automation of microbial colony detection and classification. It encompasses an extensive collection of 18,000 high-resolution images depicting five distinct microorganisms, meticulously curated as single or mixed cultures, taken under diverse lighting conditions using two different camera setups. The dataset categorizes images into countable, uncountable, and empty, with the countable images further labeled by expert microbiologists to mark colony locations and species identification, totaling 336,442 colonies. We detail the comprehensive methodology employed in the creation of the AGAR database, emphasizing the rigorous annotation process by a team of professional microbiologists who have worked with microbial collection and identification for over 20 years. This process ensures the high-quality data necessary for training machine learning models. Our investigation further explores the performance of deep neural network architectures for object detection, namely Faster R-CNN and Cascade R-CNN with four different backbones, in leveraging the AGAR dataset for the tasks of microbial colony localization and species classification. The results confirmed the great potential of deep learning methods to automate the process of microbe localization and classification based on Petri dish photos. AGAR stands as the first dataset of this kind, both in scale and in public availability, laying the groundwork for future advancements in the field of machine learning applied to microbiology (https://agar.neur osys.com/).

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.

Bacteria and phages have coexisted for billions of years engaging in continuous evolutionary arms races that drive reciprocal adaptations and resistance mechanisms. Among the diverse antiviral strategies developed by bacteria, modification or masking phage receptors as well as their physical removal via extracellular vesicles are the first line of defense. These vesicles play a pivotal role in bacterial survival by mitigating the effects of various environmental threats, including predation by bacteriophages. The secretion of extracellular vesicles represents a highly conserved evolutionary trait observed across all domains of life. Bacterial extracellular vesicles (BEVs) are generated by a wide variety of Gram (+), Gram (−), and atypical bacteria, occurring under both natural and stress conditions, including phage infection. This review addresses the multifaceted role of BEVs in modulating bacteria–phage interactions, considering the interplay from both bacterial and phage perspectives. We focus on the dual function of BEVs as both defensive agents that inhibit phage infection and as potential facilitators that may inadvertently enhance bacterial susceptibility to phages. Furthermore, we discuss how bacteriophages can influence BEV production, affecting both the quantity and molecular composition of vesicles. Finally, we provide an overview of the ecological relevance and efficacy of BEV–phage interplay across diverse environments and microbial ecosystems.

Gram-negative pathogens are surrounded by lipopolysaccharide-containing outer membrane that is crucial in protecting them against antimicrobials and host defence proteins. Some phage endolysins, which are peptidoglycan-degrading enzymes, can overcome this barrier and kill bacteria. However studies on their structure-function relationships remain scarce. The present study verified that the muralytic activity of Klebsiella myovirus endopeptidase EndoKP27 is not required for Pseudomonas aeruginosa killing. EndoKP27 is a 14 kDa protein with moderate thermostability and a cationic N-terminus. We show that the antibacterial activity of EndoKP27 is boosted in the presence of membrane-targeting agents such as polymyxin B, cathelicidin LL-37, or human serum complement, as well as innate antimicrobials from Galleria mellonella in response to P. aeruginosa and A.baumannii infection. Using potential zeta measurements and dynamic light scattering, we documented that EndoKP27 binds effectively to the pseudomonal surface, causing disorganization of the LPS layer. We established the structure-function relationship by demonstrating the permeabilizing activity of native and heat-inactivated EndoKP27 against liposomal vesicles resembling bacterial inner and outer membranes, and against P. aeruginosa strains with altered LPS structures and varying susceptibility to polymyxins. Computational molecular dynamics simulations revealed the interaction between EndoKP27 and its N-terminal part with bacterial membranes leading to channel formation. Finally, we showed that synthesized 30-amino-acid N-terminal peptide of EndoKP27 exhibits antibacterial activity against a range of P. aeruginosa strains. By integrating experimental findings with computational simulation, we propose the possible LPS-destabilizing and membranolytic mechanism underlying P. aeruginosa sensitization to exogenously applied endolysin without structural modifications. Our findings identify EndoKP27 as a promising antipseudomonal agent. (c) 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license (http://creati-vecommons.org/licenses/by-nc/4.0/).

Bacterial biofilms, characterized by complex structures, molecular communication, adaptability to environmental changes, insensitivity to chemicals, and immune response, pose a big problem both in clinics and in everyday life. The increasing bacterial resistance to antibiotics also led to the exploration of lytic bacteriophages as alternatives. Nevertheless, bacteria have co-evolved with phages, developing effective antiviral strategies, notably modification or masking phage receptors as the first line of defense mechanism. This study investigates viral-host interactions between non-host-specific phages and Pseudomonas aeruginosa, assessing whether bacteria can detect phage particles and initiate protective mechanisms. Using real-time biofilm monitoring via impedance and optical density techniques, we monitored the phage effects on biofilm and planktonic populations. Three Klebsiella phages, Slopekvirus KP15, Drulisvirus KP34, and Webervirus KP36, were tested against the P. aeruginosa PAO1 population, as well as Pseudomonas Pbunavirus KTN6. The results indicated that Klebsiella phages (non-specific to P. aeruginosa), particularly podovirus KP34, accelerated biofilm formation without affecting planktonic cultures. Our hypothesis suggests that bacteria sense phage virions, regardless of specificity, triggering biofilm matrix formation to block potential phage adsorption and infection. Nevertheless, further research is needed to understand the ecological and evolutionary dynamics between phages and bacteria, which is crucial for developing novel antibiofilm therapies.

Understanding host-range determinants in temperate bacteriophages is critical for elucidating phage-host co-evolution and advancing phage therapies.We analysed 3,900 Klebsiella genomes from diverse ecological niches to identify prophage-encoded proteins mediating capsule tropism. We applied a genome-wide association study (GWAS) correlating prophage protein clusters (from 8,105 prophages) with confidently assigned bacterial K-loci. GWAS identified high-confidence predictors for 16 out of 35 most diverse K-loci, of which 14 were receptor-binding proteins (RBPs) belonging to classical depolymerases ( n = 6), SGNH hydrolases which deacetylate polysaccharides ( n = 6), and structurally novel RBPs ( n = 2). When we relaxed the filtering thresholds, we identified 26 putative depolymerases, of which 12 were deemed as strong predictions against 10 K-types. In parallel, 50 depolymerases manually found in prophages from the representative subset of 99 bacterial isolates, together with an additional 10 depolymerases based on GWAS predictions, were prepared as recombinant proteins and tested on a Klebsiella reference panel of 119 K-types. Most predicted prophage depolymerases (34/60) failed to yield soluble products, 6 were not active on the K-types panel, and 5/14 targeted a different K-type than their bacterial host, highlighting the unpredictability of prophages as a source of functional enzymes. A comparison of GWAS-predicted enzymes, from the manual search and from virulent phages showed that depolymerase specificity was often difficult to infer from sequence or structure alone. Our findings reveal that Klebsiella prophages encode structurally diverse RBPs, suggesting temperate phages may rely not only on depolymerisation but also on capsule modification—such as deacetylation—for infection. This suggests capsule diversity in K. pneumoniae may be substantially underestimated, with implications for phage specificity, competition and vaccine design. ### Competing Interest Statement The authors have declared no competing interest.

Background: Klebsiella pneumoniae is one of the most critical Gram-negative bacteria according to the World Health Organization (WHO). Due to the ability of this bacterium to evade antibiotics, phage therapy is becoming a promising tool. However, the use of isolated proteins rather than entire phages could reduce several risks associated with phage replication. Thus, understanding the protein composition and structural organization of bacteriophages is crucial for unlocking their biology and holds great potential for medicine and biotechnology. Methods: In this study, artificial intelligence with AlphaFold 3.0 (AF3) and bioinformatic analysis were used to model the hitherto unknown structure of the Klebsiella phage KP32 (KP32), a complex and selective phage that targets K. pneumoniae strains with the K3 and K21/KL163 capsular serotypes. Results: By combining AF3 with sequence and structure analysis, we reconstructed the entire phage KP32. This complex phage is composed of over 500 protein chains, of which 415 compose its capsid and 104 its core-portal-tail complex, a platform that allows the phage to adhere to K. pneumoniae, hydrolyze its capsular sugars and finally inject its genetic code into the bacterium. Conclusions: Phage therapy is a potentially promising tool for controlling antimicrobial resistance (AMR). However, one limitation arises from the limited knowledge of their nature and mechanisms of action, as only a few phages have been structurally characterized. The reconstruction of entire phages is currently a viable strategy for elucidating their mechanistic properties, knowledge that will enhance their potential applications as therapeutic alternatives.

ABSTRACT Pyroptosis is an inflammatory immune response of eukaryotic cells to bacterial lipopolysaccharide (LPS) and other pathological stimuli, leading to the activation of the gasdermin D (GSDMD) and secretion of pore-forming domain GSDMD Nterm , facilitating the release of cytokines. Additionally, GSDMD Nterm exhibits antibacterial properties through interactions with bacterial outer membranes (OM). We explored alternative antimicrobial strategy to determine whether inducing natural pyroptosis via GSDMD activation by P. aeruginosa LPS could enhance the effectiveness of recombinant phage endopeptidase KP27 (peptidoglycan-degrading enzyme) against P. aeruginosa , enabling penetration through OM and bacterial killing synergistically. Our findings demonstrated that recombinant GSDMD alone exhibited antibacterial effects against wild-type P. aeruginosa with smooth LPS, while recombinant GSDMD Nterm efficiently permeabilized both smooth LPS-bearing and O-chain-deficient P. aeruginosa potentially synergizing with endolysin KP27. Transcriptomic analyses revealed the activation of the immune system pathways in response to LPS, mainly in monocytic cells, in contrast to epithelial A549 or HeLa cell lines. LPS-induced pyroptosis in monocytes led to GSDMD cleavage and the release of interleukins, regardless of the nature/origin of the LPS used. However, the pyroptosis stimulation by LPS in the antibacterial assay was not effective enough for bacterial OM permeabilization and enhancement of endolysin activity. We assume that leveraging pyroptosis induction in monocytic cells to augment the bactericidal activity of endolysins may be limited. IMPORTANCE Recombinant GSDMD Nterm protein was able to efficiently permeabilize P. aeruginosa outer membranes and increase endolysin activity against bacteria, producing either long LPS O-chains or lack them entirely. The obtained results suggest the limited possibility of using the natural process of pyroptosis occurring in monocytic cells to enhance the bactericidal effect of recombinant phage endolysins against Gram-negative bacteria infection.

OBJECTIVE:Klebsiella pneumoniae Przondovirus KP32 presents a complex capsular degradation machinery comprised of two serotype-specific depolymerases, KP32gp38 and KP32gp37. METHODS:In this work, we performed capsular polysaccharide (CPS) degradation assays combined with mass spectrometry approaches to identify the reaction product of K21 serotype CPS degradation by KP32gp38. We determined the crystal structure of the KP32gp38 depolymerase in complex with the identified degradation product, a pyruvated pentasaccharide, called K21-pyr5. RESULTS:The structure showed that K21-pyr5 binds to the inter-chain catalytic site, allowing the identification of important residues for CPS recognition. Importantly, we observed that the production of K21-pyr5 through CPS degradation by KP32gp38 is able to induce the maturation and differentiation of monocyte-derived dendritic cells, which, in turn, induce lymphocyte proliferation and Th polarization. By employing a T7 phage of Escherichia coli analogy, we were able to provide insights into the portal assembly of the Przondovirus K32. Our modeling studies suggest that the KP32 portal, attached to its icosahedral capsid shell, carries 12 depolymerase molecules on a single virion, arranged in 6 branches; in each branch, KP32gp38 depolymerase adheres to KP32gp37, which is directly connected to the phage portal. CONCLUSIONS:Overall, our results suggest that depolymerases act as anti-virulent agents, not only by depleting the bacteria of their CPS but also by producing immunostimulatory CPS degradation products. This indicates the use of CPS degradation products by depolymerases as potential antigens in K. pneumoniae vaccination strategies.