Nearly complete structure of bacteriophage DT57C reveals architecture of head-to-tail interface and lateral tail fibers.

The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.

The metastable associations of bacteriophages and Erwinia amylovora.

Bacteriophages are often considered as possible agents of biological control of unwanted bacterial populations in medicine, agriculture and food industry. Although the virulent phages can efficiently kill the infected host cells but at the population level phage attack not always leads to the host population collapse but may result in establishment of a more or less stable co-existence. The mechanism of the long-term stabilization of the mixed phage-host cultures is poorly understood. Here we describe bacteriophages VyarbaL and Hena2, the members of the Molineuxvirinae and the Ounavirinae subfamilies, respectively, that are able to form the pseudolysogenic associations (PA) with their host Erwinia amylovora 1/79Sm on solid media. These PAs were stable through multiple passages. The phenomenon of the PA formation between a bacterial culture and bacteriophages decreases the effectiveness of bacteriophage-mediated biological control agents based on lytic bacteriophages.

Prophage-Derived Regions in Curtobacterium Genomes: Good Things, Small Packages.

Curtobacterium is a genus of Gram-positive bacteria within the order Actinomycetales. Some Curtobacterium species (C. flaccumfaciens, C. plantarum) are harmful pathogens of agricultural crops such as soybean, dry beans, peas, sugar beet and beetroot, which occur throughout the world. Bacteriophages (bacterial viruses) are considered to be potential curative agents to control the spread of harmful bacteria. Temperate bacteriophages integrate their genomes into bacterial chromosomes (prophages), sometimes substantially influencing bacterial lifestyle and pathogenicity. About 200 publicly available genomes of Curtobacterium species, including environmental metagenomic sequences, were inspected for the presence of sequences of possible prophage origin using bioinformatic methods. The comparison of the search results with several ubiquitous bacterial groups showed the relatively low level of the presence of prophage traces in Curtobacterium genomes. Genomic and phylogenetic analyses were undertaken for the evaluation of the evolutionary and taxonomic positioning of predicted prophages. The analyses indicated the relatedness of Curtobacterium prophage-derived sequences with temperate actinophages of siphoviral morphology. In most cases, the predicted prophages can represent novel phage taxa not described previously. One of the predicted temperate phages was induced from the Curtobacterium genome. Bioinformatic analysis of the modelled proteins encoded in prophage-derived regions led to the discovery of some 100 putative glycopolymer-degrading enzymes that contained enzymatic domains with predicted cell-wall- and cell-envelope-degrading activity; these included glycosidases and peptidases. These proteins can be considered for the experimental design of new antibacterials against Curtobacterium phytopathogens.

RB49-like Bacteriophages Recognize O Antigens as One of the Alternative Primary Receptors.

The power of most of the enterobacterial O antigen types to provide robust protection against direct recognition of the cell surface by bacteriophage receptor-recognition proteins (RBP) has been recently recognized. The bacteriophages infecting O antigen producing strains of E. coli employ various strategies to tackle this nonspecific protection. T-even related phages, including RB49-like viruses, often have wide host ranges, being considered good candidates for use in phage therapy. However, the mechanisms by which these phages overcome the O antigen barrier remain unknown. We demonstrate here that RB49 and related phages Cognac49 and Whisky49 directly use certain types of O antigen as their primary receptors recognized by the virus long tail fibers (LTF) RBP gp38, so the O antigen becomes an attractant instead of an obstacle. Simultaneously to recognize multiple O antigen types, LTFs of each of these phages can bind to additional receptors, such as OmpA protein, enabling them to infect some rough strains of E. coli. We speculate that the mechanical force of the deployment of the short tail fibers (STF) triggered by the LTF binding to the O antigen or underneath of it, allows the receptor binding domains of STF to break through the O polysaccharide layer.

Two novel Erwinia amylovora bacteriophages, Loshitsa2 and Micant, isolated in Belarus.

The complete genomes of the new Erwinia amylovora bacteriophages Loshitsa2 and Micant are 43,092 bp and 43,028 bp long, respectively, encode 51 putative proteins, and have two tRNA genes. Comparative analysis with representatives of the class Caudoviricetes suggests that bacteriophages Loshitsa2 and Micant are related to LIMElight bacteriophage belonging to the family Autographiviridae and could be proposed to be members of a novel subfamily.

Ayka, a Novel Curtobacterium Bacteriophage, Provides Protection against Soybean Bacterial Wilt and Tan Spot.

Diseases caused by the Gram-positive bacterium Curtobacteriumflaccumfaciens pv. flaccumfaciens (Cff) inflict substantial economic losses in soybean cultivation. Use of specific bacterial viruses (bacteriophages) for treatment of seeds and plants to prevent the development of bacterial infections is a promising approach for bioprotection in agriculture. Phage control has been successfully tested for a number of staple crops. However, this approach has never been applied to treat bacterial diseases of legumes caused by Cff, and no specific bacteriophages have been known to date. This paper presents detailed characteristics of the first lytic bacteriophage infecting this pathogen. Phage Ayka, related to φ29-like (Salasmaviridae) viruses, but representing a new subfamily, was shown to control the development of bacterial wilt and tan spot in vitro and in greenhouse plants.

Bacteriophage Control of Pseudomonas savastanoi pv. glycinea in Soybean.

Bacterial viruses (bacteriophages) have been considered as potential agents for the biological control of bacterial phytopathogens due to their safety and host specificity. Pseudomonas savastanoi pv. glycinea (Psg) is a causative agent of the bacterial spotting of soybean (Glycine max Willd). The harm caused by this bacterium to crop production and the development of antibiotic resistance in Psg and other pathogenic microorganisms has led to the pursuit of alternative management strategies. In this study, three Psg-specific lytic bacteriophages were isolated from soybean field soil in geographically distant regions of Russia, and their potential for protective action on plants was assessed. Sequencing of phage genomes has revealed their close relatedness and attribution to the genus Ghunavirus, subfamily Studiervirinae, family Autographiviridae. Extensive testing of the biological properties of P421, the representative of the isolated phage group, has demonstrated a relatively broad host range covering closely related Pseudomonas species and stability over wide temperature (4-40 °C) and pH (pH 4-7) ranges, as well as stability under ultraviolet irradiation for 30 min. Application of the phages to prevent, and treat, Psg infection of soybean plants confirms that they are promising as biocontrol agents.

Novel Klebsiella pneumoniae K23-Specific Bacteriophages From Different Families: Similarity of Depolymerases and Their Therapeutic Potential.

Antibiotic resistance is a major public health concern in many countries worldwide. The rapid spread of multidrug-resistant (MDR) bacteria is the main driving force for the development of novel non-antibiotic antimicrobials as a therapeutic alternative. Here, we isolated and characterized three virulent bacteriophages that specifically infect and lyse MDR Klebsiella pneumoniae with K23 capsule type. The phages belonged to the Autographiviridae (vB_KpnP_Dlv622) and Myoviridae (vB_KpnM_Seu621, KpS8) families and contained highly similar receptor-binding proteins (RBPs) with polysaccharide depolymerase enzymatic activity. Based on phylogenetic analysis, a similar pattern was also noted for five other groups of depolymerases, specific against capsule types K1, K30/K69, K57, K63, and KN2. The resulting recombinant depolymerases Dep622 (phage vB_KpnP_Dlv622) and DepS8 (phage KpS8) demonstrated narrow specificity against K. pneumoniae with capsule type K23 and were able to protect Galleria mellonella larvae in a model infection with a K. pneumoniae multidrug-resistant strain. These findings expand our knowledge of the diversity of phage depolymerases and provide further evidence that bacteriophages and phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Equine Intestinal O-Seroconverting Temperate Coliphage Hf4s: Genomic and Biological Characterization.

Tailed bacteriophages constitute the bulk of the intestinal viromes of vertebrate animals. However, the relationships between lytic and lysogenic lifestyles of phages in these ecosystems are not always clear and may vary between the species or even between the individuals. The human intestinal (fecal) viromes are dominated mostly by temperate phages, while in horse feces virulent phages are more prevalent. To our knowledge, all the previously reported isolates of horse fecal coliphages are virulent. Temperate coliphage Hf4s was isolated from horse feces, from the indigenous equine Escherichia coli 4s strain. It is a podovirus related to the Lederbergvirus genus (including the well-characterized Salmonella bacteriophage P22). Hf4s recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by the same bacteriophage and also abolishes the adsorption of some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. IMPORTANCE The relationships between virulent and temperate bacteriophages and their impact on high-density symbiotic microbial ecosystems of animals are not always clear and may vary between species or even between individuals. The horse intestinal virome is dominated by virulent phages, and Hf4s is the first temperate equine intestinal coliphage characterized. It recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. These findings raise questions on the significance of bacteriophage-bacteriophage interactions within the ecology of microbial viruses in mammal intestinal ecosystems.

Free SARS-CoV-2 Spike Protein S1 Particles May Play a Role in the Pathogenesis of COVID-19 Infection.

The imbalance of the renin-angiotensin system is currently considered as a potentially important factor of the pathogenesis of COVID-19 disease. It has been shown previously in the murine model, that the expression of angiotensin-converting enzyme 2 (ACE2) on the cell surface is downregulated in response to the infection by SARS-CoV virus or recombinant spike protein (S protein) alone. In the case of natural infection, circulation of the S protein in a soluble form is unlikely. However, in SARS-CoV-2, a large fraction of S protein trimers is pre-processed during virion morphogenesis due to the presence of furin protease cleavage site between the S1 and S2 subunits. Therefore, S protein transition into the fusion conformation may be accompanied by the separation of the S1 subunits carrying the receptor-binding domains from the membrane-bound S2 subunits. The fate of the S1 particles shed due to the spontaneous "firing" of some S protein trimers exposed on the virions and on the surface of infected cells has been never investigated. We hypothesize that the soluble S1 subunits of the SARS-CoV-2 S protein shed from the infected cells and from the virions in vivo may bind to the ACE2 and downregulate cell surface expression of this protein. The decrease in the ACE2 activity on the background of constant or increased ACE activity in the lungs may lead to the prevalence of angiotensin II effects over those of angiotensin (1-7), thus promoting thrombosis, inflammation, and pulmonary damage. This hypothesis also suggests the association between less pronounced shedding of the S1 particles reported for the S protein carrying the D614G mutation (vs. the wild type D614 protein), and lack of increased severity of the COVID-19 infection caused by the mutant (D614G) SARS-CoV-2 strain, despite its higher infectivity and higher in vivo viral load.

Origin and Evolution of Studiervirinae Bacteriophages Infecting Pectobacterium: Horizontal Transfer Assists Adaptation to New Niches.

Black leg and soft rot are devastating diseases causing up to 50% loss of potential potato yield. The search for, and characterization of, bacterial viruses (bacteriophages) suitable for the control of these diseases is currently a sought-after task for agricultural microbiology. Isolated lytic Pectobacterium bacteriophages Q19, PP47 and PP81 possess a similar broad host range but differ in their genomic properties. The genomic features of characterized phages have been described and compared to other Studiervirinae bacteriophages. Thorough phylogenetic analysis has clarified the taxonomy of the phages and their positioning relative to other genera of the Autographiviridae family. Pectobacterium phage Q19 seems to represent a new genus not described previously. The genomes of the phages are generally similar to the genome of phage T7 of the Teseptimavirus genus but possess a number of specific features. Examination of the structure of the genes and proteins of the phages, including the tail spike protein, underlines the important role of horizontal gene exchange in the evolution of these phages, assisting their adaptation to Pectobacterium hosts. The results provide the basis for the development of bacteriophage-based biocontrol of potato soft rot as an alternative to the use of antibiotics.

Transcriptional Landscape of Staphylococcus aureus Kayvirus Bacteriophage vB_SauM-515A1.

The Twort-like myoviruses (Kayvirus genus) of S. aureus are promising agents for bacteriophage therapy due to a broad host range and high killing activity against clinical isolates. This work improves the current understanding of the phage infection physiology by transcriptome analysis. The expression profiles of a typical member of the Kayvirus genus (vB_SauM-515A1) were obtained at three time-points post-infection using RNA sequencing. A total of 35 transcription units comprising 238 ORFs were established. The sequences for 58 early and 12 late promoters were identified in the phage genome. The early promoters represent the strong sigma-70 promoters consensus sequence and control the host-dependent expression of 26 transcription units (81% of genes). The late promoters exclusively controlled the expression of four transcription units, while the transcription of the other five units was directed by both types of promoters. The characteristic features of late promoters were long -10 box of TGTTATATTA consensus sequence and the absence of -35 boxes. The data obtained are also of general interest, demonstrating a strategy of the phage genome expression with a broad overlap of the early and late transcription phases without any middle transcription, which is unusual for the large phage genomes (>100 kbp).

Contribution of Podoviridae and Myoviridae bacteriophages to the effectiveness of anti-staphylococcal therapeutic cocktails.

Bacteriophage therapy is considered one of the most promising therapeutic approaches against multi-drug resistant bacterial infections. Infections caused by Staphylococcus aureus are very efficiently controlled with therapeutic bacteriophage cocktails, containing a number of individual phages infecting a majority of known pathogenic S. aureus strains. We assessed the contribution of individual bacteriophages comprising a therapeutic bacteriophage cocktail against S. aureus in order to optimize its composition. Two lytic bacteriophages vB_SauM-515A1 (Myoviridae) and vB_SauP-436A (Podoviridae) were isolated from the commercial therapeutic cocktail produced by Microgen (Russia). Host ranges of the phages were established on the panel of 75 S. aureus strains. Phage vB_SauM-515A1 lysed 85.3% and vB_SauP-436A lysed 68.0% of the strains, however, vB_SauP-436A was active against four strains resistant to vB_SauM-515A1, as well as to the therapeutic cocktail per se. Suboptimal results of the therapeutic cocktail application were due to extremely low vB_SauP-436A1 content in this composition. Optimization of the phage titers led to an increase in overall cocktail efficiency. Thus, one of the effective ways to optimize the phage cocktails design was demonstrated and realized by using bacteriophages of different families and lytic spectra.

Untangling the Metabolic Reprogramming in Brain Cancer: Discovering Key Molecular Players Using Mass Spectrometry.

Cells metabolism alteration is the new hallmark of cancer, as well as an important method for carcinogenesis investigation. It is well known that the malignant cells switch to aerobic glycolysis pathway occurring also in healthy proliferating cells. Recently, it was shown that in malignant cells de novo synthesis of the intracellular fatty acid replaces dietary fatty acids which change the lipid composition of cancer cells noticeably. These alterations in energy metabolism and structural lipid production explain the high proliferation rate of malignant tissues. However, metabolic reprogramming affects not only lipid metabolism but many of the metabolic pathways in the cell. 2-hydroxyglutarate was considered as cancer cell biomarker and its presence is associated with oxidative stress influencing the mitochondria functions. Among the variety of metabolite detection methods, mass spectrometry stands out as the most effective method for simultaneous identification and quantification of the metabolites. As the metabolic reprogramming is tightly connected with epigenetics and signaling modifications, the evaluation of metabolite alterations in cells is a promising approach to investigate the carcinogenesis which is necessary for improving current diagnostic capabilities and therapeutic capabilities. In this paper, we overview recent studies on metabolic alteration and oncometabolites, especially concerning brain cancer and mass spectrometry approaches which are now in use for the investigation of the metabolic pathway.

High-throughput LPS profiling as a tool for revealing of bacteriophage infection strategies.

O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.

Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup.

Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CF3CO2H along with one- and two-dimensional 1H and 13C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-glucosamine; about 2/3 units bear a side-chain glucose residue. To our knowledge, the F17 O-polysaccharide structure established is unique among known bacterial polysaccharide structures. The O-antigen gene cluster of E. coli F17 between the conserved genes galF and gnd was sequenced and found to be 99% identical to that of E. coli 102,755 assigned to a novel OgN8 genotype (A. Iguchi, S. Iyoda, K. Seto, H. Nishii, M. Ohnishi, H. Mekata, Y. Ogura, T. Hayashi, Front. Microbiol. 7 (2016) 765). Genes in the cluster were annotated taking into account the F17 O-polysaccharide structure. The data obtained confirm that E. coli F17 and E. coli strains belonging to the OgN8 genotype can be considered as a candidate to a new E. coli O-serogroup. The O antigen of this novel type was demonstrated to make for an effective shield protecting the intimate outer membrane surface of bacteria from direct interaction with bacteriophages.

Escherichia coli bacteriophage Gostya9, representing a new species within the genus T5virus.

Escherichia coli bacteriophage Gostya9 (genus T5virus) was isolated from horse feces collected in Moscow, Russia, in 2013. This phage was associated in a single plaque with the previously reported phage 9g and was subsequently purified. Analysis of the complete genomic sequence of Gostya9 revealed that it is closely related to the T5-like bacteriophage DT57C, which had been isolated at the same location in 2007. These two viruses share 79.5% nucleotide sequence identity, which is below the 95% threshold applied currently to demarcate bacteriophage species. The most significant features distinguishing Gostya9 from DT57C include 1) the presence of one long tail fiber protein gene, 122c (ltf), instead of the two genes, ltfA and ltfB, that are present in DT57C; 2) the absence of the gene for the receptor-blocking lytic conversion lipoprotein precursor llp; and 3) the divergence of the receptor-recognition protein, pb5, which is only distantly related at the amino acid sequence level. The observed features of the Gostya9 adsorption apparatus are suggestive of a possible novel specificity for the final receptor and make this phage interesting for possible direct application in phage therapy of E. coli infections or as a source of receptor-recognition protein for engineering new phage specificities.

Genomic characteristics of vB_PpaP_PP74, a T7-like Autographivirinae bacteriophage infecting a potato pathogen of the newly proposed species Pectobacterium parmentieri.

Bacteriophage vB_PpaP_PP74 (PP74) is a novel virulent phage that infects members of the species Pectobacterium parmentieri, a newly established species of soft-rot-causing bacteria in the family Pectobacteriaceae, derived from potato-specific Pectobacterium wasabiae. vB_PpaP_PP74 was identified as a member of the family Podoviridae by transmission electron microscopy. The phage has a 39,790-bp dsDNA genome containing 50 open reading frames (ORFs). Because of the absence of genes encoding toxins or lysogeny factors, PP74 may be considered a candidate phage for pathogen biocontrol applications. The genome layout is similar to genomes of T7-like phages within the subfamily Autographivirinae, and therefore, functions can be attributed to most of ORFs. However, the closest nucleotide sequence homologs of phage PP74 are unclassified Escherichia phages. Based on phylogenetic analysis, vB_PpaP_PP74 is a sensu lato T7-like phage, but it forms a distant subgenus group together with homologous enterobacterial phages.

Complete Genome Sequence of Escherichia coli Bacteriophage PGT2.

Bacteriophage PGT2 was isolated from horse feces by using an uncharacterized Escherichia coli strain, 7s, isolated from the same sample as the host. Bacteriophage PGT2 and a related phage, phiKT, which was previously isolated from the same source, are likely to represent a new genus within the Autographivirinae subfamily of the Podoviridae family of viruses.