Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix.

The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without a low probability of subsequent emergence of stable endolysin-resistant phenotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins' effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a twofold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3.

Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae.

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.

An amber-encoding helper phage for more efficient phage display of noncanonical amino acids.

Using an amber suppression-based noncanonical amino acid (ncAA) mutagenesis approach, the chemical space in phage display can be significantly expanded for drug discovery. In this work, we demonstrate the development of a novel helper phage, CMa13ile40, for continuous enrichment of amber obligate phage clones and efficient production of ncAA-containing phages. CMa13ile40 was constructed by insertion of a Candidatus Methanomethylophilus alvus pyrrolysyl-tRNA synthetase/PylT gene cassette into a helper phage genome. The novel helper phage allowed for a continuous amber codon enrichment strategy for two different libraries and demonstrated a 100-fold increase in packaging selectivity. CMa13ile40 was then used to create two peptide libraries containing separate ncAAs, Nϵ-tert-butoxycarbonyl-lysine and Nϵ-allyloxycarbonyl-lysine, respectively. These libraries were used to identify peptide ligands that bind to the extracellular domain of ZNRF3. Each selection showed differential enrichment of unique sequences dependent upon the ncAA used. Peptides from both selections were confirmed to have low micromolar affinity for ZNRF3 that was dependent upon the presence of the ncAA used for selection. Our results demonstrate that ncAAs in phages provide unique interactions for identification of unique peptides. As an effective tool for phage display, we believe that CMa13ile40 can be broadly applied to a wide variety of applications.

A Photomodulable Bacteriophage-Spike Nanozyme Enables Dually Enhanced Biofilm Penetration and Bacterial Capture for Photothermal-Boosted Catalytic Therapy of MRSA Infections.

Nanozymes, featuring intrinsic biocatalytic effects and broad-spectrum antimicrobial properties, are emerging as a novel antibiotic class. However, prevailing bactericidal nanozymes face a challenging dilemma between biofilm penetration and bacterial capture capacity, significantly impeding their antibacterial efficacy. Here, this work introduces a photomodulable bactericidal nanozyme (ICG@hMnOx ), composed of a hollow virus-spiky MnOx nanozyme integrated with indocyanine green, for dually enhanced biofilm penetration and bacterial capture for photothermal-boosted catalytic therapy of bacterial infections. ICG@hMnOx demonstrates an exceptional capability to deeply penetrate biofilms, owing to its pronounced photothermal effect that disrupts the compact structure of biofilms. Simultaneously, the virus-spiky surface significantly enhances the bacterial capture capacity of ICG@hMnOx . This surface acts as a membrane-anchored generator of reactive oxygen species and a glutathione scavenger, facilitating localized photothermal-boosted catalytic bacterial disinfection. Effective treatment of methicillin-resistant Staphylococcus aureus-associated biofilm infections is achieved using ICG@hMnOx , offering an appealing strategy to overcome the longstanding trade-off between biofilm penetration and bacterial capture capacity in antibacterial nanozymes. This work presents a significant advancement in the development of nanozyme-based therapies for combating biofilm-related bacterial infections.

Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

PhREEPred: Phage Resistance Emergence Prediction Web Tool to Foresee Encapsulated Bacterial Escape from Phage Cocktail Treatment.

Phages, as well as phage-derived proteins, especially lysins and depolymerases, are intensively studied to become prospective alternatives or supportive antibacterials used alone or in combination. In the common phage therapy approach, the unwanted emergence of phage-resistant variants from the treated bacterial population can be postponed or reduced by the utilization of an effective phage cocktail. In this work, we present a publicly available web tool PhREEPred (Phage Resistance Emergence Prediction) (https://phartner.shinyapps.io/PhREEPred/), which will allow an informed choice of the composition of phage cocktails by predicting the outcome of phage cocktail or phage/depolymerase combination treatments against encapsulated bacterial pathogens given a mutating population that escapes single phage treatment. PhREEPred simulates solutions of our mathematical model calibrated and tested on the experimental Klebsiella pneumoniae setup and Klebsiella-specific lytic phages: K63 type-specific phage KP34 equipped with a capsule-degrading enzyme (KP34p57), capsule-independent myoviruses KP15 and KP27, and recombinant capsule depolymerase KP34p57. The model can calculate the phage-resistance emergence depending on the bacterial growth rate and initial density, the multiplicity of infection, phage latent period, its infectiveness and the cocktail composition, as well as initial depolymerase concentration and activity rate. This model reproduced the experimental results and showed that (i) the phage cocktail of parallelly infecting phages is less effective than the one composed of sequentially infecting phages; (ii) depolymerase can delay or prevent bacterial resistance by unveiling an alternative receptor for initially inactive phages. In our opinion, this customer-friendly web tool will allow for the primary design of the phage cocktail and phage-depolymerase combination effectiveness against encapsulated pathogens.

Identification and Characterization of a New Type of Holin-Endolysin Lysis Cassette in Acidovorax oryzae Phage AP1.

Phages utilize lysis systems to allow the release of newly assembled viral particles that kill the bacterial host. This is also the case for phage AP1, which infects the rice pathogen Acidovorax oryzae. However, how lysis occurs on a molecular level is currently unknown. We performed in silico bioinformatics analyses, which indicated that the lysis cassette contains a holin (HolAP) and endolysin (LysAP), which are encoded by two adjacent genes. Recombinant expression of LysAP caused Escherichia coli lysis, while HolAP arrested growth. Co-expression of both proteins resulted in enhanced lysis activity compared to the individual proteins alone. Interestingly, LysAP contains a C-terminal region transmembrane domain, which is different from most known endolysins where a N-terminal hydrophobic region is found, with the potential to insert into the membrane. We show that the C-terminal transmembrane domain is crucial for protein localization and bacterial lysis in phage AP1. Our study characterizes the new phage lysis cassette and the mechanism to induce cell disruption, giving new insight in the understanding of phage life cycles.

Bacteriophage-Encoded DNA Polymerases-Beyond the Traditional View of Polymerase Activities.

DNA polymerases are enzymes capable of synthesizing DNA. They are involved in replication of genomes of all cellular organisms as well as in processes of DNA repair and genetic recombination. However, DNA polymerases can also be encoded by viruses, including bacteriophages, and such enzymes are involved in viral DNA replication. DNA synthesizing enzymes are grouped in several families according to their structures and functions. Nevertheless, there are examples of bacteriophage-encoded DNA polymerases which are significantly different from other known enzymes capable of catalyzing synthesis of DNA. These differences are both structural and functional, indicating a huge biodiversity of bacteriophages and specific properties of their enzymes which had to evolve under certain conditions, selecting unusual properties of the enzymes which are nonetheless crucial for survival of these viruses, propagating as special kinds of obligatory parasites. In this review, we present a brief overview on DNA polymerases, and then we discuss unusual properties of different bacteriophage-encoded enzymes, such as those able to initiate DNA synthesis using the protein-priming mechanisms or even start this process without any primer, as well as able to incorporate untypical nucleotides. Apart from being extremely interesting examples of biochemical biodiversity, bacteriophage-encoded DNA polymerases can also be useful tools in genetic engineering and biotechnology.

A phage parasite deploys a nicking nuclease effector to inhibit viral host replication.

PLEs (phage-inducible chromosomal island-like elements) are phage parasites integrated into the chromosome of epidemic Vibrio cholerae. In response to infection by its viral host ICP1, PLE excises, replicates and hijacks ICP1 structural components for transduction. Through an unknown mechanism, PLE prevents ICP1 from transitioning to rolling circle replication (RCR), a prerequisite for efficient packaging of the viral genome. Here, we characterize a PLE-encoded nuclease, NixI, that blocks phage development likely by nicking ICP1's genome as it transitions to RCR. NixI-dependent cleavage sites appear in ICP1's genome during infection of PLE(+) V. cholerae. Purified NixI demonstrates in vitro nuclease activity specifically for sites in ICP1's genome and we identify a motif that is necessary for NixI-mediated cleavage. Importantly, NixI is sufficient to limit ICP1 genome replication and eliminate progeny production, representing the most inhibitory PLE-encoded mechanism revealed to date. We identify distant NixI homologs in an expanded family of putative phage parasites in vibrios that lack nucleotide homology to PLEs but nonetheless share genomic synteny with PLEs. More generally, our results reveal a previously unknown mechanism deployed by phage parasites to limit packaging of their viral hosts' genome and highlight the prominent role of nuclease effectors as weapons in the arms race between antagonizing genomes.

Parasites of viruses, often referred to as satellites, are found in all domains of life and have been co-opted for host defense across diverse virus-host systems multiple independent times. This study describes the mechanism by which such an element prevents a bacterial virus (a 'phage') from otherwise infecting Vibrio cholera and related bacteria. The study is of broad interest to investigators with interests in phage-host interactions and microbial genetics.

Petabase-scale sequence alignment catalyses viral discovery.

Public databases contain a planetary collection of nucleic acid sequences, but their systematic exploration has been inhibited by a lack of efficient methods for searching this corpus, which (at the time of writing) exceeds 20 petabases and is growing exponentially1. Here we developed a cloud computing infrastructure, Serratus, to enable ultra-high-throughput sequence alignment at the petabase scale. We searched 5.7 million biologically diverse samples (10.2 petabases) for the hallmark gene RNA-dependent RNA polymerase and identified well over 105 novel RNA viruses, thereby expanding the number of known species by roughly an order of magnitude. We characterized novel viruses related to coronaviruses, hepatitis delta virus and huge phages, respectively, and analysed their environmental reservoirs. To catalyse the ongoing revolution of viral discovery, we established a free and comprehensive database of these data and tools. Expanding the known sequence diversity of viruses can reveal the evolutionary origins of emerging pathogens and improve pathogen surveillance for the anticipation and mitigation of future pandemics.

The structure and function of modular Escherichia coli O157:H7 bacteriophage FTBEc1 endolysin, LysT84: defining a new endolysin catalytic subfamily.

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the ß(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Structural and Mechanistic Insight into CRISPR-Cas9 Inhibition by Anti-CRISPR Protein AcrIIC4Hpa.

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked.

Past and Future of Phage Therapy and Phage-Derived Proteins in Patients with Bone and Joint Infection.

Phage-derived therapies comprise phage therapy and the use of phage-derived proteins as anti-bacterial therapy. Bacteriophages are natural viruses that target specific bacteria. They were proposed to be used to treat bacterial infections in the 1920s, before the discovery and widespread over-commercialized use of antibiotics. Phage therapy was totally abandoned in Western countries, whereas it is still used in Poland, Georgia and Russia. We review here the history of phage therapy by focusing on bone and joint infection, and on the development of phage therapy in France in this indication. We discuss the rationale of its use in bacterial infection and show the feasibility of phage therapy in the 2020s, based on several patients with complex bone and joint infection who recently received phages as compassionate therapy. Although the status of phage therapy remains to be clarified by health care authorities, obtaining pharmaceutical-grade therapeutic phages (i.e., following good manufacturing practice guidelines or being "GMP-like") targeting bacterial species of concern is essential. Moreover, multidisciplinary clinical expertise has to determine what could be the relevant indications to perform clinical trials. Finally "phage therapy 2.0" has to integrate the following steps: (i) follow the status of phage therapy, that is not settled and defined; (ii) develop in each country a close relationship with the national health care authority; (iii) develop industrial-academic partnerships; (iv) create academic reference centers; (v) identify relevant clinical indications; (vi) use GMP/GMP-like phages with guaranteed quality bioproduction; (vii) start as salvage therapy; (vii) combine with antibiotics and adequate surgery; and (viii) perform clinical trials, to finally (ix) demonstrate in which clinical settings phage therapy provides benefit. Phage-derived proteins such as peptidoglycan hydrolases, polysaccharide depolymerases or lysins are enzymes that also have anti-biofilm activity. In contrast to phages, their development has to follow the classical process of medicinal products. Phage therapy and phage-derived products also have a huge potential to treat biofilm-associated bacterial diseases, and this is of crucial importance in the worldwide spread of antimicrobial resistance.

Genomic Analysis of the First European Bacteriophages with Depolymerase Activity and Biocontrol Efficacy against the Phytopathogen Ralstonia solanacearum.

Ralstonia solanacearum is the causative agent of bacterial wilt, one of the most destructive plant diseases. While chemical control has an environmental impact, biological control strategies can allow sustainable agrosystems. Three lytic bacteriophages (phages) of R. solanacearum with biocontrol capacity in environmental water and plants were isolated from river water in Europe but not fully analysed, their genomic characterization being fundamental to understand their biology. In this work, the phage genomes were sequenced and subjected to bioinformatic analysis. The morphology was also observed by electron microscopy. Phylogenetic analyses were performed with a selection of phages able to infect R. solanacearum and the closely related phytopathogenic species R. pseudosolanacearum. The results indicated that the genomes of vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 range from 40,688 to 41,158 bp with almost 59% GC-contents, 52 ORFs in vRsoP-WF2 and vRsoP-WM2, and 53 in vRsoP-WR2 but, with only 22 or 23 predicted proteins with functional homologs in databases. Among them, two lysins and one exopolysaccharide (EPS) depolymerase, this type of depolymerase being identified in R. solanacearum phages for the first time. These three European phages belong to the same novel species within the Gyeongsanvirus, Autographiviridae family (formerly Podoviridae). These genomic data will contribute to a better understanding of the abilities of these phages to damage host cells and, consequently, to an improvement in the biological control of R. solanacearum.

Non-canonical LexA proteins regulate the SOS response in the Bacteroidetes.

Lesions to DNA compromise chromosome integrity, posing a direct threat to cell survival. The bacterial SOS response is a widespread transcriptional regulatory mechanism to address DNA damage. This response is coordinated by the LexA transcriptional repressor, which controls genes involved in DNA repair, mutagenesis and cell-cycle control. To date, the SOS response has been characterized in most major bacterial groups, with the notable exception of the Bacteroidetes. No LexA homologs had been identified in this large, diverse and ecologically important phylum, suggesting that it lacked an inducible mechanism to address DNA damage. Here, we report the identification of a novel family of transcriptional repressors in the Bacteroidetes that orchestrate a canonical response to DNA damage in this phylum. These proteins belong to the S24 peptidase family, but are structurally different from LexA. Their N-terminal domain is most closely related to CI-type bacteriophage repressors, suggesting that they may have originated from phage lytic phase repressors. Given their role as SOS regulators, however, we propose to designate them as non-canonical LexA proteins. The identification of a new class of repressors orchestrating the SOS response illuminates long-standing questions regarding the origin and plasticity of this transcriptional network.

The Holin-Endolysin Lysis System of the OP2-Like Phage X2 Infecting Xanthomonas oryzae pv. oryzae.

Most endolysins of dsDNA phages are exported by a holin-dependent mechanism, while in some cases endolysins are exported via a holin-independent mechanism. However, it is still unclear whether the same endolysins can be exported by both holin-dependent and holin-independent mechanisms. This study investigated the lysis system of OP2-like phage X2 infecting Xanthomonas oryzae pv. oryzae, causing devastating bacterial leaf blight disease in rice. Based on bioinformatics and protein biochemistry methods, we show that phage X2 employs the classic "holin-endolysin" lysis system. The endolysin acts on the cell envelope and exhibits antibacterial effects in vitro, while the holin facilitates the release of the protein into the periplasm. We also characterized the role of the transmembrane domain (TMD) in the translocation of the endolysin across the inner membrane. We found that the TMD facilitated the translocation of the endolysin via the Sec secretion system. The holin increases the efficiency of protein release, leading to faster and more efficient lysis. Interestingly, in E. coli, the expression of either holin or endolysin with TMDs resulted in the formation of long rod shaped cells. We conclude that the TMD of X2-Lys plays a dual role: One is the transmembrane transport while the other is the inhibition of cell division, resulting in larger cells and thus in a higher number of released viruses per cell.

Bacteriophage origin of some minimal ATP-dependent DNA ligases: a new structure from Burkholderia pseudomallei with striking similarity to Chlorella virus ligase.

DNA ligases, the enzymes responsible for joining breaks in the phosphodiester backbone of DNA during replication and repair, vary considerably in size and structure. The smallest members of this enzyme class carry out their functions with pared-down protein scaffolds comprising only the core catalytic domains. Here we use sequence similarity network analysis of minimal DNA ligases from all biological super kingdoms, to investigate their evolutionary origins, with a particular focus on bacterial variants. This revealed that bacterial Lig C sequences cluster more closely with Eukaryote and Archaeal ligases, while bacterial Lig E sequences cluster most closely with viral sequences. Further refinement of the latter group delineates a cohesive cluster of canonical Lig E sequences that possess a leader peptide, an exclusively bacteriophage group of T7 DNA ligase homologs and a group with high similarity to the Chlorella virus DNA ligase which includes both bacterial and viral enzymes. The structure and function of the bacterially-encoded Chlorella virus homologs were further investigated by recombinantly producing and characterizing, the ATP-dependent DNA ligase from Burkholderia pseudomallei as well as determining its crystal structure in complex with DNA. This revealed that the enzyme has similar activity characteristics to other ATP-dependent DNA ligases, and significant structural similarity to the eukaryotic virus Chlorella virus including the positioning and DNA contacts of the binding latch region. Analysis of the genomic context of the B. pseudomallei ATP-dependent DNA ligase indicates it is part of a lysogenic bacteriophage present in the B. pseudomallei chromosome representing one likely entry point for the horizontal acquisition of ATP-dependent DNA ligases by bacteria.

The K26 capsular polysaccharide from Acinetobacter baumannii KZ-1098: Structure and cleavage by a specific phage depolymerase.

The KL26 gene cluster responsible for the synthesis of the K26 capsular polysaccharide (CPS) of Acinetobacter baumannii includes rmlBDAC genes for l-rhamnose (l-Rhap) synthesis, tle to generate 6-deoxy-l-talose (l-6dTalp) from l-Rhap, and a manC gene for D-mannose (D-Manp) that is rare in Acinetobacter CPS. K26 CPS material was isolated from A. baumannii isolate KZ-1098, and studied by sugar analysis, Smith degradation, and one and two-dimensional 1H and 13C NMR spectroscopy before and after O-deacetylation with aqueous ammonia. The following structure of the branched hexasaccharide repeating unit of the CPS was established: →2)-ß-D-Manp-1→4-ß-D-Glcp-1→3-α-L-6dTalp-1→3-ß-D-GlcpNAc-(1→3↑14│Acα-L-Rhap-2←1-α-D-Glcp The structural depolymerase of phage vB_AbaP_APK26 cleaved selectively the ß-GlcpNAc-(1 → 2)-α-Manp linkage in the K26 CPS formed by WzyK26 to give monomer, dimer, and trimer of the CPS repeating unit, which were characterized by high-resolution electrospray ionization mass spectrometry as well as 1H and 13C NMR spectroscopy. The wzyK26 gene responsible for this linkage and the manC gene were only found in six A. baumannii genomes carrying KL26 and one carrying the novel KL148 gene cluster, indicating the rare occurrence of ß-GlcpNAc-(1 → 2)-α-Manp in A. baumannii CPS structures. However, K26 shares a ß-d-Glcp-(1 → 3)-α-l-6dTalp-(1 → 3)-ß-d-GlcpNAc trisaccharide fragment with a group of related A. baumannii CPSs that have varying patterns of acetylation of l-6dTalp.

Novel Acinetobacter baumannii Bacteriophage Aristophanes Encoding Structural Polysaccharide Deacetylase.

Acinetobacter baumannii appears to be one of the most crucial nosocomial pathogens. A possible component of antimicrobial therapy for infections caused by extremely drug-resistant A. baumannii strains may be specific lytic bacteriophages or phage-derived enzymes. In the present study, we observe the biological features, genomic organization, and phage-host interaction strategy of novel virulent bacteriophage Aristophanes isolated on A. baumannii strain having K26 capsular polysaccharide structure. According to phylogenetic analysis phage Aristophanes can be classified as a representative of a new distinct genus of the subfamily Beijerinckvirinae of the family Autographiviridae. This is the first reported A. baumannii phage carrying tailspike deacetylase, which caused O-acetylation of one of the K26 sugar residues.

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.