Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Endolysin NC5 improves early cloxacillin treatment in a mouse model of Streptococcus uberis mastitis.

Streptococcus uberis frequently causes bovine mastitis, an infectious udder disease with significant economic implications for dairy cows. Conventional antibiotics, such as cloxacillin, sometimes have limited success in eliminating S. uberis as a stand-alone therapy. To address this challenge, the study objective was to investigate the VersaTile engineered endolysin NC5 as a supplemental therapy to cloxacillin in a mouse model of bovine S. uberis mastitis. NC5 was previously selected based on its intracellular killing and biofilm eradicating activity. To deliver preclinical proof-of-concept of this supplemental strategy, lactating mice were intramammarily infected with a bovine S. uberis field isolate and subsequently treated with cloxacillin (30.0 µg) combined with either a low (23.5 µg) or high (235.0 µg) dose of NC5. An antibiotic monotherapy group, as well as placebo treatment, was included as controls. Two types of responders were identified: fast (n = 17), showing response after 4-h treatment, and slow (n = 10), exhibiting no clear response at 4 h post-treatment across all groups. The high-dose combination therapy in comparison with placebo treatment impacted the hallmarks of mastitis in the fast responders by reducing (i) the bacterial load 13,000-fold (4.11 ± 0.78 Δlog10; p < 0.001), (ii) neutrophil infiltration 5.7-fold (p > 0.05), and (iii) the key pro-inflammatory chemokine IL-8 13-fold (p < 0.01). These mastitis hallmarks typically followed a dose response dependent on the amount of endolysin added. The current in vivo study complements our in vitro data and provides preclinical proof-of-concept of NC5 as an adjunct to intramammary cloxacillin treatment. KEY POINTS: • Engineered endolysin NC5 was preclinically evaluated as add-on to cloxacillin treatment. • Two types of mice (slow and fast responding) were observed. • The add-on treatment decreased bacterial load, neutrophil influx, and pro-inflammatory mediators.

Phage tailspike modularity and horizontal gene transfer reveals specificity towards E. coli O-antigen serogroups.

BACKGROUND: The interaction between bacteriophages and their hosts is intricate and highly specific. Receptor-binding proteins (RBPs) of phages such as tail fibers and tailspikes initiate the infection process. These RBPs bind to diverse outer membrane structures, including the O-antigen, which is a serogroup-specific sugar-based component of the outer lipopolysaccharide layer of Gram-negative bacteria. Among the most virulent Escherichia coli strains is the Shiga toxin-producing E. coli (STEC) pathotype dominated by a subset of O-antigen serogroups. METHODS: Extensive phylogenetic and structural analyses were used to identify and validate specificity correlations between phage RBP subtypes and STEC O-antigen serogroups, relying on the principle of horizontal gene transfer as main driver for RBP evolution. RESULTS: We identified O-antigen specific RBP subtypes for seven out of nine most prevalent STEC serogroups (O26, O45, O103, O104, O111, O145 and O157) and seven additional E. coli serogroups (O2, O8, O16, O18, 4s/O22, O77 and O78). Eight phage genera (Gamaleya-, Justusliebig-, Kaguna-, Kayfuna-, Kutter-, Lederberg-, Nouzilly- and Uetakeviruses) emerged for their high proportion of serogroup-specific RBPs. Additionally, we reveal sequence motifs in the RBP region, potentially serving as recombination hotspots between lytic phages. CONCLUSION: The results contribute to a better understanding of mosaicism of phage RBPs, but also demonstrate a method to identify and validate new RBP subtypes for current and future emerging serogroups.

Current challenges in designer cellulosome engineering.

Designer cellulosomes (DCs) are engineered multi-enzyme complexes, comprising carbohydrate-active enzymes attached to a common backbone, the scaffoldin, via high-affinity cohesin-dockerin interactions. The use of DCs in the degradation of renewable biomass polymers is a promising approach for biorefineries. Indeed, DCs have shown significant hydrolytic activities due to the enhanced enzyme-substrate proximity and inter-enzyme synergies, but technical hurdles in DC engineering have hindered further progress towards industrial application. The challenge in DC engineering lies in the large diversity of possible building blocks and architectures, resulting in a multivariate and immense design space. Simultaneously, the precise DC composition affects many relevant parameters such as activity, stability, and manufacturability. Since protein engineers face a lack of high-throughput approaches to explore this vast design space, DC engineering may result in an unsatisfying outcome. This review provides a roadmap to guide researchers through the process of DC engineering. Each step, starting from concept to evaluation, is described and provided with its challenges, along with possible solutions, both for DCs that are assembled in vitro or are displayed on the yeast cell surface. KEY POINTS: • Construction of designer cellulosomes is a multi-step process. • Designer cellulosome research deals with multivariate construction challenges. • Boosting designer cellulosome efficiency requires exploring a vast design space.

CkP1 bacteriophage, a S16-like myovirus that recognizes Citrobacter koseri lipopolysaccharide through its long tail fibers.

Citrobacter koseri is an emerging Gram-negative bacterial pathogen, which causes urinary tract infections. We isolated and characterized a novel S16-like myovirus CKP1 (vB_CkoM_CkP1), infecting C. koseri. CkP1 has a host range covering the whole C. koseri species, i.e., all strains that were tested, but does not infect other species. Its linear 168,463-bp genome contains 291 coding sequences, sharing sequence similarity with the Salmonella phage S16. Based on surface plasmon resonance and recombinant green florescence protein fusions, the tail fiber (gp267) was shown to decorate C. koseri cells, binding with a nanomolar affinity, without the need of accessory proteins. Both phage and the tail fiber specifically bind to bacterial cells by the lipopolysaccharide polymer. We further demonstrate that CkP1 is highly stable towards different environmental conditions of pH and temperatures and is able to control C. koseri cells in urine samples. Altogether, CkP1 features optimal in vitro characteristics to be used both as a control and detection agent towards drug-resistant C. koseri infections. KEY POINTS: • CkP1 infects all C. koseri strains tested • CkP1 recognizes C. koseri lipopolysaccharide through its long tail fiber • Both phage CkP1 and its tail fiber can be used to treat or detect C. koseri pathogens.

Phages and engineered lysins as an effective tool to combat Gram-negative foodborne pathogens.

One of the biggest challenges faced by food producers is ensuring microbiological safety. Despite strict criteria for food products, foodborne diseases are a global problem and pose a real risk to consumers. Therefore, it is necessary to identify new and more effective methods for eliminating pathogens from food and the food processing environment. According to the European Food Safety Authority (EFSA), the most common foodborne diseases are caused by Campylobacter, Salmonella, Yersinia, Escherichia coli, and Listeria. Out of the five listed, four are Gram-negative bacteria. Our review focuses on the use of bacteriophages, which are ubiquitous bacterial viruses, and bacteriophage endolysins to eliminate Gram-negative pathogens. Endolysins cleave specific bonds within the peptidoglycan (PG) of the bacterial cell, causing the cell to burst. Single phages or phage cocktails, which are, in some instances, commercially available products, eliminate pathogenic bacteria in livestock and various food matrices. Endolysins have matured as the most advanced class of antibacterial agents in the clinical sector, but their use in food protection is highly unexplored. Advanced molecular engineering techniques, different formulations, protein encapsulation, and the addition of outer membrane (OM) permeabilization agents enhance the activity of lysins against Gram-negative pathogens. This creates space for groundbreaking research on the use of lysins in the food sector.

Engineering a Lysin with Intrinsic Antibacterial Activity (LysMK34) by Cecropin A Fusion Enhances Its Antibacterial Properties against Acinetobacter baumannii.

Bacteriophage-encoded lysins are increasingly reported as alternatives to combat Acinetobacter baumannii infections, for which limited therapeutic options are available. Some lysins, such as LysMK34, have a C-terminal amphipathic helix allowing them to penetrate the otherwise-impermeable outer membrane barrier. Another approach to kill Gram-negative pathogens with lysins relies on fusion of a peptide with outer membrane-permeabilizing properties to the lysin. In this work, we aimed to leverage the intrinsic antibacterial activity of LysMK34 by fusing the peptide cecropin A to its N terminus via a linker of three Ala-Gly repeats, resulting in engineered LysMK34 (eLysMK34). The engineered lysin has an improved antibacterial activity compared to that of the parental lysin, LysMK34, in terms of MICs (0.45 to 1.2 µM), killing rate, and killing extent. eLysMK34 has a ≥2-fold-increased activity against stationary-phase cells, and the bactericidal effect becomes less dependent on the intracellular osmotic pressure. In particular, colistin-resistant strains become highly susceptible to eLysMK34, and enhanced antibacterial activity is observed in complement-deactivated human serum. These observations demonstrate that fusion of a lysin with intrinsic antibacterial activity with a selected outer membrane-permeabilizing peptide is a useful strategy to further improve the in vitro antibacterial properties of such lysins. IMPORTANCE Phage lysins are a new class of enzyme-based antibiotics that increasingly gain interest. Lysins kill cells through rapid degradation of the peptidoglycan layer, resulting in sudden osmotic lysis. Whereas Gram-positive bacteria are readily susceptible to the actions of lysins, Gram-negative bacteria are naturally resistant, as the outer membrane protects their peptidoglycan layer. This work reveals that fusing an outer membrane-permeabilizing peptide to a lysin with intrinsic antibacterial activity results in a superior lysin that shows improved robustness in its antibacterial activity, including against the most worrisome colistin-resistant A. baumannii strains.

Conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode.

Cellulosomes are multi-enzyme complexes produced by specialised micro-organisms. The spatial proximity of synergistically acting enzymes incorporated in these naturally occurring complexes supports the efficient hydrolysis of lignocellulosic biomass. Several functional designer cellulosomes, incorporating naturally non-cellulosomal cellulases, have been constructed and can be used for cellulose saccharification. However, in lignocellulosic biomass, cellulose is tightly intertwined with several hemicelluloses and lignin. One of the most abundant hemicelluloses interacting with cellulose microfibrils is xyloglucan, and degradation of these polymers is crucial for complete saccharification. Yet, designer cellulosome studies focusing on the incorporation of hemicellulases have been limited. Here, we report the conversion of the free Cellvibrio japonicus xyloglucan degradation system to the cellulosomal mode. Therefore, we constructed multiple docking enzyme variants of C. japonicus endoxyloglucanase, ß-1,2-galactosidase, α-1,6 xylosidase and ß-1,4-glucosidase, using the combinatorial VersaTile technique dedicated to the design and optimisation of modular proteins. We individually optimised the docking enzymes to degrade the xyloglucan backbone and side chains. Finally, we show that a purified designer xyloglucanosome comprising these docking enzymes was able to release xyloglucan oligosaccharides, galactose, xylose and glucose from tamarind xyloglucan. KEY POINTS: • Construction of xyloglucan-degrading designer cellulosome. • Conversion of free Cellvibrio japonicus enzymes to cellulosomal mode. • Type of linker inserted between dockerin and enzyme module affects docking enzyme activity.

Transcriptional Organization of the Salmonella Typhimurium Phage P22 pid ORFan Locus.

Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5' untranslated region that is instructive for a secondary pid mRNA species, and has a 3' Rho-independent termination loop that is responsible for stability of the pid transcript.

Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages.

Pseudomonas virus vB_PaeM_PA5oct is proposed as a model jumbo bacteriophage to investigate phage-bacteria interactions and is a candidate for phage therapy applications. Combining hybrid sequencing, RNA-Seq and mass spectrometry allowed us to accurately annotate its 286,783 bp genome with 461 coding regions including four non-coding RNAs (ncRNAs) and 93 virion-associated proteins. PA5oct relies on the host RNA polymerase for the infection cycle and RNA-Seq revealed a gradual take-over of the total cell transcriptome from 21% in early infection to 93% in late infection. PA5oct is not organized into strictly contiguous regions of temporal transcription, but some genomic regions transcribed in early, middle and late phases of infection can be discriminated. Interestingly, we observe regions showing limited transcription activity throughout the infection cycle. We show that PA5oct upregulates specific bacterial operons during infection including operons pncA-pncB1-nadE involved in NAD biosynthesis, psl for exopolysaccharide biosynthesis and nap for periplasmic nitrate reductase production. We also observe a downregulation of T4P gene products suggesting mechanisms of superinfection exclusion. We used the proteome of PA5oct to position our isolate amongst other phages using a gene-sharing network. This integrative omics study illustrates the molecular diversity of jumbo viruses and raises new questions towards cellular regulation and phage-encoded hijacking mechanisms.

High-Throughput Generation of Product Profiles for Arabinoxylan-Active Enzymes from Metagenomes.

Metagenomics is an exciting alternative to seek carbohydrate-active enzymes from a range of sources. Typically, metagenomics reveals dozens of putative catalysts that require functional characterization for further application in industrial processes. High-throughput screening methods compatible with adequate natural substrates are crucial for an accurate functional elucidation of substrate preferences. Based on DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) analysis of enzymatic-reaction products, we generated product profiles to consequently infer substrate cleavage positions, resulting in the generation of enzymatic-degradation maps. Product profiles were produced in high throughput for arabinoxylan (AX)-active enzymes belonging to the glycoside hydrolase families GH43 (subfamilies 2 [MG432], 7 [MG437], and 28 [MG4328]) and GH8 (MG8) starting from 12 (arabino)xylo-oligosaccharides. These enzymes were discovered through functional metagenomic studies of feces from the North American beaver (Castor canadensis). This work shows how enzyme loading alters the product profiles of all enzymes studied and gives insight into AX degradation patterns, revealing sequential substrate preferences of AX-active enzymes.IMPORTANCE Arabinoxylan is mainly found in the hemicellulosic fractions of rice straw, corn cobs, and rice husk. Converting arabinoxylan into (arabino)xylo-oligosaccharides as added-value products that can be applied in food, feed, and cosmetics presents a sustainable and economic alternative for the biorefinery industries. Efficient and profitable AX degradation requires a set of enzymes with particular characteristics. Therefore, enzyme discovery and the study of substrate preferences are of utmost importance. Beavers, as consumers of woody biomass, are a promising source of a repertoire of enzymes able to deconstruct hemicelluloses into soluble oligosaccharides. High-throughput analysis of the oligosaccharide profiles produced by these enzymes will assist in the selection of the most appropriate enzymes for the biorefinery.

Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance.

The prevalence of extensively and pandrug-resistant strains of Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes; nonetheless, its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8-kb genome (50 open reading frames), encoding an endolysin (LysMK34) with potent muralytic activity (1,499.9 ± 131 U/µM), a typical mesophilic thermal stability up to 55°C, and a broad pH activity range (4 to 10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. The addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are resensitized in both Mueller-Hinton broth and 50% human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic.IMPORTANCEA. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last-resort antibiotic, and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure-dependent intrinsic antibacterial activity, providing new insights in the mode of action of lysins with intrinsic activity against Gram-negative bacteria.

Advanced engineering of third-generation lysins and formulation strategies for clinical applications.

One of the possible solutions for the current antibiotic resistance crisis may be found in (often bacteriophage-derived) peptidoglycan hydrolases. The first clinical trials of these natural enzymes, coined here as first-generation lysins, are currently ongoing. Moving beyond natural endolysins with protein engineering established the second generation of lysins. In second-generation lysins, the focus lies on improving antibacterial and biochemical properties such as antimicrobial activity and stability, as well as expanding their activities towards Gram-negative pathogens. However, solutions to particular key challenges regarding clinical applications are only beginning to emerge in the third generation of lysins, in which protein and biochemical engineering efforts focus on improving properties relevant under clinical conditions. In addition, increasingly advanced formulation strategies are developed to increase the bioavailability, antibacterial activity, and half-life, and to reduce pro-inflammatory responses. This review focuses on third-generation and advanced formulation strategies that are developed to treat infections, ranging from topical to systemic applications. Together, these efforts may fully unlock the potential of lysin therapy and will propel it as a true antibiotic alternative or supplement.

Engineering of receptor-binding proteins in bacteriophages and phage tail-like bacteriocins.

Bacteriophages and phage tail-like bacteriocins (PTLBs) rely on receptor-binding proteins (RBPs) located in tail fibers or spikes for an initial and specific interaction with susceptible bacteria. Bacteriophages kill bacteria through a lytic, replicative cycle, whereas PTLBs kill the target through membrane depolarization in a single hit mechanism. Extensive efforts in the engineering of RBPs of both phages and PTLBs have been undertaken to obtain a greater understanding of the structural organization of RBPs. In addition, a major goal of engineering RBPs of phages and PTLBs is the production of antibacterials with a customized spectrum. Swapping of the RBP of phages and PTLBs results in a shift in activity spectrum in accordance with the spectrum of the new RBP. The engineering of strictly virulent phages with new RBPs required significant technical advances in the past decades, whereas the engineering of RBPs of PTLBs relied on the traditional molecular techniques used for the manipulation of bacteria and was thus relatively straightforward. While phages and PTLBs share their potential for specificity tuning, specific features of phages such as their lytic killing mechanism, their self-replicative nature and thus different pharmacokinetics and their potential to co-evolve are clear differentiators compared with PTLBs in terms of their antibacterial use.

Enzybiotics: Enzyme-Based Antibacterials as Therapeutics.

Antibiotics have saved millions of lives. However, the overuse and misuse of antibiotics have contributed to a rapid emergence of antibiotic resistance worldwide. In addition, there is an unprecedented void in the development of new antibiotic classes by the pharmaceutical industry since the first introduction of antibiotics. This antibiotic crisis underscores the urgent and increasing necessity of new, innovative antibiotics. Enzybiotics are such a promising class of antibiotics. They are derived from endolysins, bacteriophage-encoded enzymes that degrade the bacterial cell wall of the infected cell at the end of the lytic replication cycle. Enzybiotics are featured by a rapid and unique mode-of-action, a high specificity to kill pathogens, a low probability for bacterial resistance development and a proteinaceous nature. (Engineered) endolysins have been demonstrated to be effective in a variety of animal models to combat both Gram-positive and Gram-negative bacteria and have entered different phases of preclinical and clinical trials. In addition, mycobacteriophage-encoded endolysins have been successfully used to inhibit mycobacteria in vitro. In this chapter we focus on the (pre)clinical progress of enzybiotics as potent therapeutic agent against human pathogenic bacteria.

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis.

Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted ß-D-xylosyl residues, whereas a GH43 α-L-arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process.

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes.

Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus "Kp15virus" within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between L-alanine and D-glutamic acid.

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage.

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7 %) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7-49.5 % identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin, and spanins) and shows 29-98 % homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60 °C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest that the AP3 phage is a promising potent agent against bacteria belonging to the most common B. cenocepacia IIIA lineage strains.

Efficacy of Artilysin Art-175 against Resistant and Persistent Acinetobacter baumannii.

Bacteriophage-encoded endolysins have shown promise as a novel class of antibacterials with a unique mode of action, i.e., peptidoglycan degradation. However, Gram-negative pathogens are generally not susceptible due to their protective outer membrane. Artilysins overcome this barrier. Artilysins are optimized, engineered fusions of selected endolysins with specific outer membrane-destabilizing peptides. Artilysin Art-175 comprises a modified variant of endolysin KZ144 with an N-terminal fusion to SMAP-29. Previously, we have shown the high susceptibility of Pseudomonas aeruginosa to Art-175. Here, we report that Art-175 is highly bactericidal against stationary-phase cells of multidrug-resistant Acinetobacter baumannii, even resulting in a complete elimination of large inocula (≥10(8) CFU/ml). Besides actively dividing cells, Art-175 also kills persisters. Instantaneous killing of A. baumannii upon contact with Art-175 could be visualized after immobilization of the bacteria in a microfluidic flow cell. Effective killing of a cell takes place through osmotic lysis after peptidoglycan degradation. The killing rate is enhanced by the addition of 0.5 mM EDTA. No development of resistance to Art-175 under selection pressure and no cross-resistance with existing resistance mechanisms could be observed. In conclusion, Art-175 represents a highly active Artilysin against both A. baumannii and P. aeruginosa, two of the most life-threatening pathogens of the order Pseudomonadales.