Autographivirinae Bacteriophage Arno 160 Infects Pectobacterium carotovorum via Depolymerization of the Bacterial O-Polysaccharide.

Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application.

Biological properties and genomics analysis of vB_KpnS_GH-K3, a Klebsiella phage with a putative depolymerase-like protein.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.

Functional domains of Acinetobacter bacteriophage tail fibers.

A rapid increase in antimicrobial resistant bacterial infections around the world is causing a global health crisis. The Gram-negative bacterium Acinetobacter baumannii is categorized as a Priority 1 pathogen for research and development of new antimicrobials by the World Health Organization due to its numerous intrinsic antibiotic resistance mechanisms and ability to quickly acquire new resistance determinants. Specialized phage enzymes, called depolymerases, degrade the bacterial capsule polysaccharide layer and show therapeutic potential by sensitizing the bacterium to phages, select antibiotics, and serum killing. The functional domains responsible for the capsule degradation activity are often found in the tail fibers of select A. baumannii phages. To further explore the functional domains associated with depolymerase activity, tail-associated proteins of 71 sequenced and fully characterized phages were identified from published literature and analyzed for functional domains using InterProScan. Multisequence alignments and phylogenetic analyses were conducted on the domain groups and assessed in the context of noted halo formation or depolymerase characterization. Proteins derived from phages noted to have halo formation or a functional depolymerase, but no functional domain hits, were modeled with AlphaFold2 Multimer, and compared to other protein models using the DALI server. The domains associated with depolymerase function were pectin lyase-like (SSF51126), tailspike binding (cd20481), (Trans)glycosidases (SSF51445), and potentially SGNH hydrolases. These findings expand our knowledge on phage depolymerases, enabling researchers to better exploit these enzymes for therapeutic use in combating the antimicrobial resistance crisis.

Genomic and Proteomic Analysis of Six Vi01-like Phages Reveals Wide Host Range and Multiple Tail Spike Proteins.

Enterobacteriaceae is a large family of Gram-negative bacteria composed of many pathogens, including Salmonella and Shigella. Here, we characterize six bacteriophages that infect Enterobacteriaceae, which were isolated from wastewater plants in the Wasatch front (Utah, United States). These phages are highly similar to the Kuttervirus vB_SenM_Vi01 (Vi01), which was isolated using wastewater from Kiel, Germany. The phages vary little in genome size and are between 157 kb and 164 kb, which is consistent with the sizes of other phages in the Vi01-like phage family. These six phages were characterized through genomic and proteomic comparison, mass spectrometry, and both laboratory and clinical host range studies. While their proteomes are largely unstudied, mass spectrometry analysis confirmed the production of five hypothetical proteins, several of which unveiled a potential operon that suggests a ferritin-mediated entry system on the Vi01-like phage family tail. However, no dependence on this pathway was observed for the single host tested herein. While unable to infect every genus of Enterobacteriaceae tested, these phages are extraordinarily broad ranged, with several demonstrating the ability to infect Salmonella enterica and Citrobacter freundii strains with generally high efficiency, as well as several clinical Salmonella enterica isolates, most likely due to their multiple tail fibers.

Agtrevirus phage AV101 recognizes four different O-antigens infecting diverse E. coli.

Bacteriophages in the Agtrevirus genus are known for expressing multiple tail spike proteins (TSPs), but little is known about their genetic diversity and host recognition apart from their ability to infect diverse Enterobacteriaceae species. Here, we aim to determine the genetic differences that may account for the diverse host ranges of Agrevirus phages. We performed comparative genomics of 14 Agtrevirus and identified only a few genetic differences including genes involved in nucleotide metabolism. Most notably was the diversity of the tsp gene cluster, specifically in the receptor-binding domains that were unique among most of the phages. We further characterized agtrevirus AV101 infecting nine diverse Extended Spectrum ß-lactamase (ESBL) Escherichia coli and demonstrated that this phage encoded four unique TSPs among Agtrevirus. Purified TSPs formed translucent zones and inhibited AV101 infection of specific hosts, demonstrating that TSP1, TSP2, TSP3, and TSP4 recognize O8, O82, O153, and O159 O-antigens of E. coli, respectively. BLASTp analysis showed that the receptor-binding domain of TSP1, TSP2, TSP3, and TSP4 are similar to TSPs encoded by E. coli prophages and distant related virulent phages. Thus, Agtrevirus may have gained their receptor-binding domains by recombining with prophages or virulent phages. Overall, combining bioinformatic and biological data expands the understanding of TSP host recognition of Agtrevirus and give new insight into the origin and acquisition of receptor-binding domains of Ackermannviridae phages.

Discovery and characterisation of new phage targeting uropathogenic Escherichia coli.

Antimicrobial resistance is an escalating threat with few new therapeutic options in the pipeline. Urinary tract infections (UTIs) are one of the most prevalent bacterial infections globally and are prone to becoming recurrent and antibiotic resistant. We discovered and characterized six novel Autographiviridae and Guernseyvirinae bacterial viruses (phage) against uropathogenic Escherichia coli (UPEC), a leading cause of UTIs. The phage genomes were between 39,471 bp - 45,233 bp, with 45.0%-51.0% GC%, and 57-84 predicted coding sequences per genome. We show that tail fiber domain structure, predicted host capsule type, and host antiphage repertoire correlate with phage host range. In vitro characterisation of phage cocktails showed synergistic improvement against a mixed UPEC strain population and when sequentially dosed. Together, these phage are a new set extending available treatments for UTI from UPEC, and phage vM_EcoM_SHAK9454 represents a promising candidate for further improvement through engineering.

Host interactions of novel Crassvirales species belonging to multiple families infecting bacterial host, Bacteroides cellulosilyticus WH2.

Bacteroides, the prominent bacteria in the human gut, play a crucial role in degrading complex polysaccharides. Their abundance is influenced by phages belonging to the Crassvirales order. Despite identifying over 600 Crassvirales genomes computationally, only few have been successfully isolated. Continued efforts in isolation of more Crassvirales genomes can provide insights into phage-host-evolution and infection mechanisms. We focused on wastewater samples, as potential sources of phages infecting various Bacteroides hosts. Sequencing, assembly, and characterization of isolated phages revealed 14 complete genomes belonging to three novel Crassvirales species infecting Bacteroides cellulosilyticus WH2. These species, Kehishuvirus sp. 'tikkala' strain Bc01, Kolpuevirus sp. 'frurule' strain Bc03, and 'Rudgehvirus jaberico' strain Bc11, spanned two families, and three genera, displaying a broad range of virion productions. Upon testing all successfully cultured Crassvirales species and their respective bacterial hosts, we discovered that they do not exhibit co-evolutionary patterns with their bacterial hosts. Furthermore, we observed variations in gene similarity, with greater shared similarity observed within genera. However, despite belonging to different genera, the three novel species shared a unique structural gene that encodes the tail spike protein. When investigating the relationship between this gene and host interaction, we discovered evidence of purifying selection, indicating its functional importance. Moreover, our analysis demonstrated that this tail spike protein binds to the TonB-dependent receptors present on the bacterial host surface. Combining these observations, our findings provide insights into phage-host interactions and present three Crassvirales species as an ideal system for controlled infectivity experiments on one of the most dominant members of the human enteric virome.

StenM_174: A Novel Podophage That Infects a Wide Range of Stenotrophomonas spp. and Suggests a New Subfamily in the Family Autographiviridae.

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family.

Tailed Lytic Bacteriophages of Soft Rot Pectobacteriaceae.

The study of the ecological and evolutionary traits of Soft Rot Pectobacteriaceae (SRP) comprising genera Pectobacterium and Dickeya often involves bacterial viruses (bacteriophages). Bacteriophages are considered to be a prospective tool for the ecologically safe and highly specific protection of plants and harvests from bacterial diseases. Information concerning bacteriophages has been growing rapidly in recent years, and this has included new genomics-based principles of taxonomic distribution. In this review, we summarise the data on phages infecting Pectobacterium and Dickeya that are available in publications and genomic databases. The analysis highlights not only major genomic properties that assign phages to taxonomic families and genera, but also the features that make them potentially suitable for phage control applications. Specifically, there is a discussion of the molecular mechanisms of receptor recognition by the phages and problems concerning the evolution of phage-resistant mutants.

Treating Bacterial Infections with Bacteriophage-Based Enzybiotics: In Vitro, In Vivo and Clinical Application.

Over the past few decades, we have witnessed a surge around the world in the emergence of antibiotic-resistant bacteria. This global health threat arose mainly due to the overuse and misuse of antibiotics as well as a relative lack of new drug classes in development pipelines. Innovative antibacterial therapeutics and strategies are, therefore, in grave need. For the last twenty years, antimicrobial enzymes encoded by bacteriophages, viruses that can lyse and kill bacteria, have gained tremendous interest. There are two classes of these phage-derived enzymes, referred to also as enzybiotics: peptidoglycan hydrolases (lysins), which degrade the bacterial peptidoglycan layer, and polysaccharide depolymerases, which target extracellular or surface polysaccharides, i.e., bacterial capsules, slime layers, biofilm matrix, or lipopolysaccharides. Their features include distinctive modes of action, high efficiency, pathogen specificity, diversity in structure and activity, low possibility of bacterial resistance development, and no observed cross-resistance with currently used antibiotics. Additionally, and unlike antibiotics, enzybiotics can target metabolically inactive persister cells. These phage-derived enzymes have been tested in various animal models to combat both Gram-positive and Gram-negative bacteria, and in recent years peptidoglycan hydrolases have entered clinical trials. Here, we review the testing and clinical use of these enzymes.

Subtypes of tail spike proteins predicts the host range of Ackermannviridae phages.

Phages belonging to the Ackermannviridae family encode up to four tail spike proteins (TSPs), each recognizing a specific receptor of their bacterial hosts. Here, we determined the TSPs diversity of 99 Ackermannviridae phages by performing a comprehensive in silico analysis. Based on sequence diversity, we assigned all TSPs into distinctive subtypes of TSP1, TSP2, TSP3 and TSP4, and found each TSP subtype to be specifically associated with the genera (Kuttervirus, Agtrevirus, Limestonevirus, Taipeivirus) of the Ackermannviridae family. Further analysis showed that the N-terminal XD1 and XD2 domains in TSP2 and TSP4, hinging the four TSPs together, are preserved. In contrast, the C-terminal receptor binding modules were only conserved within TSP subtypes, except for some Kuttervirus TSP1s and TSP3s that were similar to specific TSP4s. A conserved motif in TSP1, TSP3 and TSP4 of Kuttervirus phages may allow recombination between receptor binding modules, thus altering host recognition. The receptors for numerous uncharacterized phages expressing TSPs in the same subtypes were predicted using previous host range data. To validate our predictions, we experimentally determined the host recognition of three of the four TSPs expressed by kuttervirus S117. We confirmed that S117 TSP1 and TSP2 bind to their predicted host receptors, and identified the receptor for TSP3, which is shared by 51 other Kuttervirus phages. Kuttervirus phages were thus shown encode a vast genetic diversity of potentially exchangeable TSPs influencing host recognition. Overall, our study demonstrates that comprehensive in silico and host range analysis of TSPs can predict host recognition of Ackermannviridae phages.

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.

Morphologically Different Pectobacterium brasiliense Bacteriophages PP99 and PP101: Deacetylation of O-Polysaccharide by the Tail Spike Protein of Phage PP99 Accompanies the Infection.

Soft rot caused by numerous species of Pectobacterium and Dickeya is a serious threat to the world production of potatoes. The application of bacteriophages to combat bacterial infections in medicine, agriculture, and the food industry requires the selection of comprehensively studied lytic phages and the knowledge of their infection mechanism for more rational composition of therapeutic cocktails. We present the study of two bacteriophages, infective for the Pectobacterium brasiliense strain F152. Podoviridae PP99 is a representative of the genus Zindervirus, and Myoviridae PP101 belongs to the still unclassified genomic group. The structure of O-polysaccharide of F152 was established by sugar analysis and 1D and 2D NMR spectroscopy: → 4)-α-D-Manp6Ac-(1→ 2)-α-D-Manp-(1→ 3)-ß-D-Galp-(1→ 3 ↑ 1 α -l- 6 dTal p Ac 0 - 2 The recombinant tail spike protein of phage PP99, gp55, was shown to deacetylate the side chain talose residue of bacterial O-polysaccharide, thus providing the selective attachment of the phage to the cell surface. Both phages demonstrate lytic behavior, thus being prospective for therapeutic purposes.

Reprogramming Bacteriophage Host Range through Structure-Guided Design of Chimeric Receptor Binding Proteins.

Bacteriophages provide excellent tools for diagnostics, remediation, and targeted microbiome manipulation, yet isolating viruses with suitable host specificity remains challenging. Using Listeria phage PSA, we present a synthetic biology blueprint for host-range engineering through targeted modification of serovar-specific receptor binding proteins (RBPs). We identify Gp15 as the PSA RBP and construct a synthetic phage library featuring sequence-randomized RBPs, from which host range mutants are isolated and subsequently integrated into a synthetic, polyvalent phage with extended host range. To enable rational design of chimeric RBPs, we determine the crystal structure of the Gp15 receptor-binding carboxyl terminus at 1.7-Å resolution and employ bioinformatics to identify compatible, prophage-encoded RBPs targeting different Listeria serovars. Structure-guided design enables exchange of heterologous RBP head, neck, or shoulder domains to generate chimeric phages with predictable and extended host ranges. These strategies will facilitate the development of phage biologics based on standardized virus scaffolds with tunable host specificities.

Host Specificity of the Dickeya Bacteriophage PP35 Is Directed by a Tail Spike Interaction With Bacterial O-Antigen, Enabling the Infection of Alternative Non-pathogenic Bacterial Host.

Dickeya solani is a recently emerged virulent bacterial potato pathogen that poses a major threat to world agriculture. Because of increasing antibiotic resistance and growing limitations in antibiotic use, alternative antibacterials such as bacteriophages are being developed. Myoviridae bacteriophages recently re-ranked as a separate Ackermannviridae family, such as phage PP35 described in this work, are the attractive candidates for this bacterial biocontrol. PP35 has a very specific host range due to the presence of tail spike protein PP35 gp156, which can depolymerize the O-polysaccharide (OPS) of D. solani. The D. solani OPS structure, →2)-ß-D-6-deoxy-D-altrose-(1→, is so far unique among soft-rot Pectobacteriaceae, though it may exist in non-virulent environmental Enterobacteriaceae. The phage tail spike depolymerase degrades the shielding polysaccharide, and launches the cell infection process. We hypothesize that non-pathogenic commensal bacteria may maintain the population of the phage in soil environment.

Novel Fri1-like Viruses Infecting Acinetobacter baumannii-vB_AbaP_AS11 and vB_AbaP_AS12-Characterization, Comparative Genomic Analysis, and Host-Recognition Strategy.

Acinetobacter baumannii is a gram-negative, non-fermenting aerobic bacterium which is often associated with hospital-acquired infections and known for its ability to develop resistance to antibiotics, form biofilms, and survive for long periods in hospital environments. In this study, we present two novel viruses, vB_AbaP_AS11 and vB_AbaP_AS12, specifically infecting and lysing distinct multidrug-resistant clinical A. baumannii strains with K19 and K27 capsular polysaccharide structures, respectively. Both phages demonstrate rapid adsorption, short latent periods, and high burst sizes in one-step growth experiments. The AS11 and AS12 linear double-stranded DNA genomes of 41,642 base pairs (bp) and 41,402 bp share 86.3% nucleotide sequence identity with the most variable regions falling in host receptor-recognition genes. These genes encode tail spikes possessing depolymerizing activities towards corresponding capsular polysaccharides which are the primary bacterial receptors. We described AS11 and AS12 genome organization and discuss the possible regulation of transcription. The overall genomic architecture and gene homology analyses showed that the phages are new representatives of the recently designated Fri1virus genus of the Autographivirinae subfamily within the Podoviridae family.