Abstract P-46: Structure of A. Baumannii Phage Tapaz, Revealed with Cryo-Electron Microscopy

Background: Acinetobacter baumannii is an opportunistic pathogen and one of the six most important multidrug resistant microorganisms in hospitals worldwide. Some of its strains are resistant to most of the antibiotics, A. baumannii is included into the Priority 1 part of Global Priority List of Antibiotic-resistant Bacteria. Phage therapy is considered to be an alternative strategy to antibiotic treatments. Methods: A. baumannii strain NIPH601 cells were grown till OD6000.4 and infected with the phage at MOI 10:1. After complete lysis took place cell debris was spined down and phage particles were precipitated with the PEG6000 (final concentration 10% PEG 6000, 0.5 NaCl). Virus particles were collected by centrifugation, resuspended at SM buffer and applied on CsCl step gradient. Gradient was spinned down for 2 hours at 40000g and the fraction containing phage particles was collected and dialyzed against SM buffer. Purified phage particles were applied to Quantifoil 1.2/1.3 grids and plunge-froze in Vitrobot Mark IV (TFS) Micrographs were collected in HKU, Shenzhen campus with Titan Krios cryoelectron microscope (TFS), equipped with Gatan K3 direct electron detector. The micrographs were acquired with 1.06 Å pixel size and 1.5 um average defocus value in counting mode with 50 frames and 1.2 e/Å2/frame dose rate. All image processing was performed with Relion3.0 software, except for the particle picking step performed with cryolo. Results: Lytic A. baumannii phage TaPaz belongs to the family Myoviridae. BLAST search over NCBI “nr” (non-redundant) database revealed close homology with previously published sequences of Acinetobacter phage vB_AbaM_B9 and Acinetobacter phage BS46. However, no structural information about any homologous proteins was found among the PDB structures. The cryo-EM map was reconstructed with single particle analysis independently for the capsid, tail and baseplate regions. The capsid was reconstructed at 3.9 Å resolution with I3 symmetry applied (Fig. 1A). The baseplate region of the phage was reconstructed at 3.5 Å resolution with C3 symmetry (Fig. 1B). The tail region was reconstructed at 2.6 Å resolution with helical symmetry (Rise 36.4 Å, Twist 25.7 deg). Initial atomic model for the tail region was built from sequence with Deeptracer and was further refined in coot (Fig. 1C). Conclusion: We successfully obtained the near-atomic resolution structural map of phage TaPaz. The data obtained contribute to enhancing knowledge of structural diversity of bacterial viruses infecting A. baumannii

New <i>Obolenskvirus</i> Phages Brutus and Scipio: Biology, Evolution, and Phage-Host Interaction

Two novel virulent phages of the genus Obolenskvirus infecting Acinetobacter baumannii, a significant nosocomial pathogen, have been isolated and studied. Phages Brutus and Scipio were able to infect A. baumannii strains belonging to the K116 and K82 capsular types, respectively. The biological properties and genomic organization of the phages were characterized. Comparative genomic, phylogenetic, and pangenomic analyses were performed to investigate the relationship of Brutus and Scipio to other bacterial viruses and to trace the possible origin and evolutionary history of these phages and other representatives of the genus Obolenskvirus. The investigation of enzymatic activity of the tailspike depolymerase encoded in the genome of phage Scipio, the first reported virus infecting A. baumannii of the K82 capsular type, was performed. The study of new representatives of the genus Obolenskvirus and mechanisms of action of depolymerases encoded in their genomes expands knowledge about the diversity of viruses within this taxonomic group and strategies of Obolenskvirus–host bacteria interaction

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