The flagellotropic bacteriophage YSD1 targets Salmonella Typhi with a Chi‐like protein tail fibre
TL;DR: The results from genome sequencing of this phage, YSD1, are presented, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure and treatment of phage with the antibodies that bind Y SD1_29 inhibits phage infection of Salmonella.
Abstract: The discovery of a Salmonella-targeting phage from the waterways of the United Kingdom provided an opportunity to address the mechanism by which Chi-like bacteriophage (phage) engages with bacterial flagellae. The long tail fibre seen on Chi-like phages has been proposed to assist the phage particle in docking to a host cell flagellum, but the identity of the protein that generates this fibre was unknown. We present the results from genome sequencing of this phage, YSD1, confirming its close relationship to the original Chi phage and suggesting candidate proteins to form the tail structure. Immunogold labelling in electron micrographs revealed that YSD1_22 forms the main shaft of the tail tube, while YSD1_25 forms the distal part contributing to the tail spike complex. The long curling tail fibre is formed by the protein YSD1_29, and treatment of phage with the antibodies that bind YSD1_29 inhibits phage infection of Salmonella. The host range for YSD1 across Salmonella serovars is broad, but not comprehensive, being limited by antigenic features of the flagellin subunits that make up the Salmonella flagellum, with which YSD1_29 engages to initiate infection.
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TL;DR: The mechanisms behind phage resistance in bacterial pathogens and the physiological consequences of acquiringphage resistance phenotypes are explored and it may be possible to use phages to alter bacterial populations, making them more tractable to current therapeutic strategies.
Abstract: Bacteria that cause life-threatening infections in humans are becoming increasingly difficult to treat. In some instances, this is due to intrinsic and acquired antibiotic resistance, indicating that new therapeutic approaches are needed to combat bacterial pathogens. There is renewed interest in utilizing viruses of bacteria known as bacteriophages (phages) as potential antibacterial therapeutics. However, critics suggest that similar to antibiotics, the development of phage-resistant bacteria will halt clinical phage therapy. Although the emergence of phage-resistant bacteria is likely inevitable, there is a growing body of literature showing that phage selective pressure promotes mutations in bacteria that allow them to subvert phage infection, but with a cost to their fitness. Such fitness trade-offs include reduced virulence, resensitization to antibiotics, and colonization defects. Resistance to phage nucleic acid entry, primarily via cell surface modifications, compromises bacterial fitness during antibiotic and host immune system pressure. In this minireview, we explore the mechanisms behind phage resistance in bacterial pathogens and the physiological consequences of acquiring phage resistance phenotypes. With this knowledge, it may be possible to use phages to alter bacterial populations, making them more tractable to current therapeutic strategies.
96 citations
Cites background from "The flagellotropic bacteriophage YS..."
...enterica serovar Typhi uses flagella as the primary receptor for infection (99); however, ΔfliC and ΔfljB mutants that lack the flagellar filament can still be infected with reduced efficiency....
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TL;DR: How two nested sets of chainmail stabilise the viral head and a beta-hairpin regulates the formation of the robust yet pliable tail, characteristic of siphoviruses is shown.
Abstract: Flagellotropic bacteriophages engage flagella to reach the bacterial surface as an effective means to increase the capture radius for predation. Structural details of these viruses are of great interest given the substantial drag forces and torques they face when moving down the spinning flagellum. We show that the main capsid and auxiliary proteins form two nested chainmails that ensure the integrity of the bacteriophage head. Core stabilising structures are conserved in herpesviruses suggesting their ancestral origin. The structure of the tail also reveals a robust yet pliable assembly. Hexameric rings of the tail-tube protein are braced by the N-terminus and a β-hairpin loop, and interconnected along the tail by the splayed β-hairpins. By contrast, we show that the β-hairpin has an inhibitory role in the tail-tube precursor, preventing uncontrolled self-assembly. Dyads of acidic residues inside the tail-tube present regularly-spaced motifs well suited to DNA translocation into bacteria through the tail.
24 citations
TL;DR: In this article, a lytic coliphage PHH01 can hitchhike on carrier bacteria Bacillus cereus to facilitate its infection of host bacteria, Escherichia coli, in biofilms.
Abstract: Interactions between bacteriophages (phages) and biofilms remain poorly understood despite the broad implications for microbial ecology, water quality, and microbiome engineering. Here, we demonstrate that lytic coliphage PHH01 can hitchhike on carrier bacteria Bacillus cereus to facilitate its infection of host bacteria, Escherichia coli, in biofilms. Specifically, PHH01 could adsorb onto the flagella of B. cereus, and thus phage motility was increased, resulting in 4.36-fold more effective infection of E. coli in biofilm relative to free PHH01 alone. Moreover, phage infection mitigated interspecies competition and enhanced B. cereus colonization; the fraction of B. cereus in the final biofilm increased from 9% without phages to 43% with phages. The mutualistic relationship between the coliphage and carrier bacteria was substantiated by migration tests on an E. coli lawn: the conjugation of PHH01 and B. cereus enhanced B. cereus colonization by 6.54-fold compared to B. cereus alone (6.15 vs 0.94 cm2 in 24 h) and PHH01 migration by 5.15-fold compared to PHH01 alone (10.3 vs 2.0 mm in 24 h). Metagenomic and electron microscopic analysis revealed that the phages of diverse taxonomies and different morphologies could be adsorbed by the flagella of B. cereus, suggesting hitchhiking on flagellated bacteria might be a widespread strategy in aquatic phage populations. Overall, our study highlights that hitchhiking behavior in phages can facilitate phage infection of biofilm bacteria, promote carrier bacteria colonization, and thus significantly influence biofilm composition, which holds promise for mediating biofilm functions and moderating associated risks.
17 citations
TL;DR: In this article, the first structure of a depolymerase that targets the clinically relevant K2 capsule was determined and its putative active site was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides.
Abstract: 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-A 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.
14 citations
TL;DR: It is shown that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, and filaments containing the FljK flageLLin are the preferred substrate for bacteriophage ϕ CbK.
Abstract: Caulobacter crescentus is a Gram-negative alphaproteobacterium that commonly lives in oligotrophic fresh- and saltwater environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which require interaction with the bacterial flagellum and pilus complexes during adsorption. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, the bacteriophage ϕCbK likely recognizes a common motif among the six known flagellins in C. crescentus with various degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe posttranslational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 A. Altogether, this work builds a platform for future investigations of how phage ϕCbK infects C. crescentus at the molecular level. IMPORTANCE Bacterial flagellar filaments serve as an initial attachment point for many bacteriophages to bacteria. Some bacteria harbor numerous flagellin genes and are therefore able to generate flagellar filaments with complex compositions, which is thought to be important for evasion from bacteriophages. This study characterizes the importance of the six flagellin genes in C. crescentus for infection by bacteriophage ϕCbK. We find that filaments containing the FljK flagellin are the preferred substrate for bacteriophage ϕCbK. We also present a high-resolution structure of a flagellar filament containing only the FljK flagellin, which provides a platform for future studies on determining how bacteriophage ϕCbK attaches to flagellar filaments at the molecular level.
13 citations
References
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TL;DR: MUSCLE is a new computer program for creating multiple alignments of protein sequences that includes fast distance estimation using kmer counting, progressive alignment using a new profile function the authors call the log-expectation score, and refinement using tree-dependent restricted partitioning.
Abstract: We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the logexpectation score, and refinement using treedependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.
37,524 citations
Additional excerpts
...8.31 (Edgar, 2004) using the default settings....
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TL;DR: Two unusual extensions are presented: Multiscale, which adds the ability to visualize large‐scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales.
Abstract: The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
35,698 citations
"The flagellotropic bacteriophage YS..." refers methods in this paper
...…Typhimurium flagellar filament (PDB: 3A5X) was deposited as a single subunit, the rest of the filament was modelled based on the helical symmetry of the B. subtillis flagellar filament [PDB:5WJT, (Wang et al., 2017)] in Chimera 1.13.1 [(Pettersen et al., 2004); http://www.rbvi.ucsf. edu/chime ra/]....
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TL;DR: An updated protocol for Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants for a user's protein sequence.
Abstract: Phyre2 is a web-based tool for predicting and analyzing protein structure and function. Phyre2 uses advanced remote homology detection methods to build 3D models, predict ligand binding sites, and analyze amino acid variants in a protein sequence. Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2
. A typical structure prediction will be returned between 30 min and 2 h after submission.
7,941 citations
"The flagellotropic bacteriophage YS..." refers methods in this paper
...0 (Kelley et al., 2015) using the S. Typhimurium FliC structure (PDB:3A5X) as a template....
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...…2018); https :// www.ebi.ac.uk/Tools/ hmmer/ ], HHpred [(Zimmermann et al., 2018); https ://toolk it.tuebi ngen.mpg.de/#/tools/ hhpred] and Phyre2 [(Kelley et al., 2015); http://www.sbg. bio.ic.ac.uk/phyre 2/html/page.cgi?id=index ] searching for domains generally characteristic of phage tail…...
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...de/#/tools/ hhpred] and Phyre2 [(Kelley et al., 2015); http://www....
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...0 [(Kelley et al., 2015); http://www.sbg.bio.ic.ac.uk/phyre 2/html/ page.cgi?id=index ]....
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TL;DR: SeaView version 4 combines all the functions of the widely used programs SeaView and Phylo_win, and expands them by adding network access to sequence databases, alignment with arbitrary algorithm, maximum-likelihood tree building with PhyML, and display, printing, and copy-to-clipboard of rooted or unrooted, binary or multifurcating phylogenetic trees.
Abstract: We present SeaView version 4, a multiplatform program designed to facilitate multiple alignment and phylogenetic tree building from molecular sequence data through the use of a graphical user interface. SeaView version 4 combines all the functions of the widely used programs SeaView (in its previous versions) and Phylo_win, and expands them by adding network access to sequence databases, alignment with arbitrary algorithm, maximum-likelihood tree building with PhyML, and display, printing, and copy-to-clipboard of rooted or unrooted, binary or multifurcating phylogenetic trees. In relation to the wide present offer of tools and algorithms for phylogenetic analyses, SeaView is especially useful for teaching and for occasional users of such software. SeaView is freely available at http://pbil.univ-lyon1.fr/software/seaview.
5,074 citations
TL;DR: This work has focused on minimizing search times and the ability to rapidly display tabular results, regardless of the number of matches found, developing graphical summaries of the search results to provide quick, intuitive appraisement of them.
Abstract: HMMER is a software suite for protein sequence similarity searches using probabilistic methods Previously, HMMER has mainly been available only as a computationally intensive UNIX command-line tool, restricting its use Recent advances in the software, HMMER3, have resulted in a 100-fold speed gain relative to previous versions It is now feasible to make efficient profile hidden Markov model (profile HMM) searches via the web A HMMER web server (http://hmmerjaneliaorg) has been designed and implemented such that most protein database searches return within a few seconds Methods are available for searching either a single protein sequence, multiple protein sequence alignment or profile HMM against a target sequence database, and for searching a protein sequence against Pfam The web server is designed to cater to a range of different user expertise and accepts batch uploading of multiple queries at once All search methods are also available as RESTful web services, thereby allowing them to be readily integrated as remotely executed tasks in locally scripted workflows We have focused on minimizing search times and the ability to rapidly display tabular results, regardless of the number of matches found, developing graphical summaries of the search results to provide quick, intuitive appraisement of them
4,159 citations
"The flagellotropic bacteriophage YS..." refers methods in this paper
...Table 1 documents outcomes from analyses with BLASTp (https ://blast.ncbi.nlm.nih.gov/Blast. cgi), HMMer [(Finn et al., 2011; Potter et al., 2018); https :// www.ebi.ac.uk/Tools/ hmmer/ ], HHpred [(Zimmermann et al., 2018); https ://toolk it.tuebi ngen.mpg.de/#/tools/ hhpred] and Phyre2 [(Kelley et…...
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