Crystal structure and location of gp131 in the bacteriophage phiKZ virion

Pseudomonas phage phi KZ and its two close relatives phi PA3 and 201 phi 2-1 are very large bacteriophages that form a separate branch in phage classification because their genomes are very different from the rest of GenBank sequence data. The contractile tail of phi KZ is built from at least 32 different proteins, but a definitive structural function is assigned to only one of them-the tail sheath protein. Here, we report the crystal structure of the C-terminal domain of another phiKZ tail protein, gene product 131 (gp131C). We show that gp131 is located at the periphery of the baseplate and possibly associates with fibers that emanate from the baseplate. Gp131C is a seven-bladed beta-propeller that has a shape of a skewed toroid. A small but highly conserved and negatively charged patch on the surface of gp131C might be important for substrate binding or for interaction with a different tail protein. (C) 2012 Elsevier Inc. All rights reserved

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

Production of the K16 capsular polysaccharide by Acinetobacter baumannii ST25 isolate D4 involves a novel glycosyltransferase encoded in the KL16 gene cluster

A new capsular polysaccharide (CPS) biosynthesis gene cluster, KL16, was found in the genome sequence of a clinical Acinetobacter baumannii ST25 isolate, D4. The variable part of KL16 contains a module of genes for synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-non-2-ulosonic acid (5,7-di-N-acetylpseudaminic acid, Pse5Ac7Ac), a gene encoding ItrA3 that initiates the CPS synthesis with d-GlcpNAc, and two glycosyltransferase (Gtr) genes. The K16 CPS was studied by sugar analysis and Smith degradation along with 1D and 2D H and C NMR spectroscopy, and shown to be built up of linear trisaccharide repeats containing d-galactose (d-Gal), N-acetyl-d-glucosamine (d-GlcNAc), and Pse5Ac7Ac. The d-Galp residue is linked to the d-GlcpNAc initiating sugar via a β-(1 → 3) linkage evidently formed by a Gtr5 variant, Gtr5, encoded in KL16. This reveals an altered or relaxed substrate specificity of this variant as the majority of Gtr5-type glycosyltransferases have previously been shown to form a β-d-Galp-(1 → 3)-d-GalpNAc linkage. The β-Psep5Ac7Ac-(2 → 4)-d-Galp linkage is predicted to be formed by the other glycosyltransferase, Gtr37, which does not match members of any known glycosyltransferase family

Function of bacteriophage G7C esterase tailspike in host cell adsorption.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.The work of the laboratory in the Winogradsky Institute was partially supported by Russian Science Foundation (RSF) grant #15–15‐0013

Inhibition of Influenza M2-Induced Cell Death Alleviates Its Negative Contribution to Vaccination Efficiency

The effectiveness of recombinant vaccines encoding full-length M2 protein of influenza virus or its ectodomain (M2e) have previously been tested in a number of models with varying degrees of success. Recently, we reported a strong cytotoxic effect exhibited by M2 on mammalian cells in vitro. Here we demonstrated a decrease in protection when M2 was added to a DNA vaccination regimen that included influenza NP. Furthermore, we have constructed several fusion proteins of conserved genes of influenza virus and tested their expression in vitro and protective potential in vivo. The four-partite NP-M1-M2-NS1 fusion antigen that has M2 sequence engineered in the middle part of the composite protein was shown to not be cytotoxic in vitro. A three-partite fusion protein (consisting of NP, M1 and NS1) was expressed much more efficiently than the four-partite protein. Both of these constructs provided statistically significant protection upon DNA vaccination, with construct NP-M1-M2-NS1 being the most effective. We conclude that incorporation of M2 into a vaccination regimen may be beneficial only when its apparent cytotoxicity-linked negative effects are neutralized. The possible significance of this data for influenza vaccination regimens and preparations is discussed

PAAR-repeat proteins sharpen and diversify the Type VI secretion system spike

The bacterial type VI secretion system (T6SS) is a large multi-component, dynamic macromolecular machine that plays an important role in the ecology of many Gram negative bacteria. T6SS is responsible for translocation of a wide range of toxic effector molecules allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells1-5. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike6,7. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (Proline-Alanine-Alanine-aRginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins function to sharpen the tip of the VgrG spike. We demonstrate that PAAR proteins are essential for T6SS- mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results suggest a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event

Contractile Tail Machines of Bacteriophages

Bacteriophages with contractile tails epitomize the concepts of "virus" and "phage" for many because the tails of these phages undergo a large conformational change - resembling the action of a syringe - upon the attachment to the host cell. The contractile tails belong to the recently recognized class of "contractile systems," which includes phage tails, their close relatives R-type pyocins, the bacterial type VI secretion system, and the virulence cassette of Photorhabdus. Their function is to deliver large proteins and/or DNA into the cytoplasm of a bacterial or eukaryotic cell. The structure of the core components of all contractile tail-like systems is conserved, but the corresponding genes have diverged to such a degree that the common ancestry can no longer be easily detected at the level of amino acid sequence. At present, it is unclear, whether the contractile systems originated in bacteria or in phages. This chapter describes the structure and function of phage contractile tails and compares them with other phage tails and with other known contractile systems

Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to “simple” contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains—two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction