@article{KunstmannEngstroemWehleetal.2020, author = {Kunstmann, Ruth Sonja and Engstr{\"o}m, Olof and Wehle, Marko and Widmalm, G{\"o}ran and Santer, Mark and Barbirz, Stefanie}, title = {Increasing the affinity of an O-Antigen polysaccharide binding site in Shigella flexneri bacteriophage Sf6 tailspike protein}, series = {Chemistry - A European Journal}, volume = {26}, journal = {Chemistry - A European Journal}, number = {32}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {0947-6539}, doi = {10.1002/chem.202000495}, pages = {7263 -- 7273}, year = {2020}, abstract = {Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen-binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O-polysaccharide of dysentery-causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D H-1,H-1-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O-polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design.}, language = {en} } @article{StephanBroekerSaragliadisetal.2020, author = {Stephan, Mareike Sophia and Br{\"o}ker, Nina K. and Saragliadis, Athanasios and Roos, Norbert and Linke, Dirk and Barbirz, Stefanie}, title = {In vitro analysis of O-antigen-specific bacteriophage P22 inactivation by Salmonella outer membrane vesicles}, series = {Frontiers in microbiology}, volume = {11}, journal = {Frontiers in microbiology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {1664-302X}, doi = {10.3389/fmicb.2020.510638}, pages = {12}, year = {2020}, abstract = {Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions ofSalmonella(S.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host.In vitro, these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.}, language = {en} } @article{GeorgievGrafmuellerBlegeretal.2018, author = {Georgiev, Vasil N. and Grafm{\"u}ller, Andrea and Bl{\´e}ger, David and Hecht, Stefan and Kunstmann, Sonja and Barbirz, Stefanie and Lipowsky, Reinhard and Dimova, Rumiana}, title = {Area increase and budding in giant vesicles triggered by light}, series = {Advanced science}, volume = {5}, journal = {Advanced science}, number = {8}, publisher = {Wiley}, address = {Hoboken}, issn = {2198-3844}, doi = {10.1002/advs.201800432}, pages = {9}, year = {2018}, abstract = {Biomembranes are constantly remodeled and in cells, these processes are controlled and modulated by an assortment of membrane proteins. Here, it is shown that such remodeling can also be induced by photoresponsive molecules. The morphological control of giant vesicles in the presence of a water-soluble ortho-tetrafluoroazobenzene photoswitch (F-azo) is demonstrated and it is shown that the shape transformations are based on an increase in membrane area and generation of spontaneous curvature. The vesicles exhibit budding and the buds can be retracted by using light of a different wavelength. In the presence of F-azo, the membrane area can increase by more than 5\% as assessed from vesicle electrodeformation. To elucidate the underlying molecular mechanism and the partitioning of F-azo in the membrane, molecular dynamics simulations are employed. Comparison with theoretically calculated shapes reveals that the budded shapes are governed by curvature elasticity, that the spontaneous curvature can be decomposed into a local and a nonlocal contribution, and that the local spontaneous curvature is about 1/(2.5 mu m). The results show that exo- and endocytotic events can be controlled by light and that these photoinduced processes provide an attractive method to change membrane area and morphology.}, language = {en} } @article{KunstmannGohlkeBroekeretal.2018, author = {Kunstmann, Ruth Sonja and Gohlke, Ulrich and Br{\"o}ker, Nina Kristin and Roske, Yvette and Heinemann, Udo and Santer, Mark and Barbirz, Stefanie}, title = {Solvent networks tune thermodynamics of oligosaccharide complex formation in an extended protein binding site}, series = {Journal of the American Chemical Society}, volume = {140}, journal = {Journal of the American Chemical Society}, number = {33}, publisher = {American Chemical Society}, address = {Washington}, issn = {0002-7863}, doi = {10.1021/jacs.8b03719}, pages = {10447 -- 10455}, year = {2018}, abstract = {The principles of protein-glycan binding are still not well understood on a molecular level. Attempts to link affinity and specificity of glycan recognition to structure suffer from the general lack of model systems for experimental studies and the difficulty to describe the influence of solvent. We have experimentally and computationally addressed energetic contributions of solvent in protein-glycan complex formation in the tailspike protein (TSP) of E. coli bacteriophage HK620. HK620TSP is a 230 kDa native trimer of right-handed, parallel beta-helices that provide extended, rigid binding sites for bacterial cell surface O-antigen polysaccharides. A set of high affinity mutants bound hexa- or pentasaccharide O-antigen fragments with very similar affinities even though hexasaccharides introduce an additional glucose branch into an occluded protein surface cavity. Remarkably different thermodynamic binding signatures were found for different mutants; however, crystal structure analyses indicated that no major oligosaccharide or protein topology changes had occurred upon complex formation. This pointed to a solvent effect. Molecular dynamics simulations using a mobility-based approach revealed an extended network of solvent positions distributed over the entire oligosaccharide binding site. However, free energy calculations showed that a small water network inside the glucose-binding cavity had the most notable influence on the thermodynamic signature. The energy needed to displace water from the glucose binding pocket depended on the amino acid at the entrance, in agreement with the different amounts of enthalpy-entropy compensation found for introducing glucose into the pocket in the different mutants. Studies with small molecule drugs have shown before that a few active water molecules can control protein complex formation. HK620TSP oligosaccharide binding shows that similar fundamental principles also apply for glycans, where a small number of water molecules can dominate the thermodynamic signature in an extended binding site.}, language = {en} } @article{BroekerRoskeVallerianietal.2019, author = {Broeker, Nina K. and Roske, Yvette and Valleriani, Angelo and Stephan, Mareike Sophia and Andres, Dorothee and Koetz, Joachim and Heinemann, Udo and Barbirz, Stefanie}, title = {Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail}, series = {The journal of biological chemistry}, volume = {294}, journal = {The journal of biological chemistry}, number = {31}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {1083-351X}, doi = {10.1074/jbc.RA119.008133}, pages = {11751 -- 11761}, year = {2019}, abstract = {Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 angstrom resolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an E15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.}, language = {en} } @article{DunsingIrmscherBarbirzetal.2019, author = {Dunsing, Valentin and Irmscher, Tobias and Barbirz, Stefanie and Chiantia, Salvatore}, title = {Purely Polysaccharide-Based Biofilm Matrix Provides Size-Selective Diffusion Barriers for Nanoparticles and Bacteriophages}, series = {Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences}, volume = {20}, journal = {Biomacromolecules : an interdisciplinary journal focused at the interface of polymer science and the biological sciences}, number = {10}, publisher = {American Chemical Society}, address = {Washington}, issn = {1525-7797}, doi = {10.1021/acs.biomac.9b00938}, pages = {3842 -- 3854}, year = {2019}, abstract = {Biofilms are complex mixtures of proteins, DNA, and polysaccharides surrounding bacterial communities as protective barriers that can be biochemically modified during the bacterial life cycle. However, their compositional heterogeneity impedes a precise analysis of the contributions of individual matrix components to the biofilm structural organization. To investigate the structural properties of glycan-based biofilms, we analyzed the diffusion dynamics of nanometer-sized objects in matrices of the megadalton-sized anionic polysaccharide, stewartan, the major biofilm component of the plant pathogen, Pantoea stewartii. Fluorescence correlation spectroscopy and single-particle tracking of nanobeads and bacteriophages indicated notable subdiffusive dynamics dependent on probe size and stewartan concentration, in contrast to free diffusion of small molecules. Stewartan enzymatic depolymerization by bacteriophage tailspike proteins rapidly restored unhindered diffusion. We, thus, hypothesize that the glycan polymer stewartan determines the major physicochemical properties of the biofilm, which acts as a selective diffusion barrier for nanometer-sized objects and can be controlled by enzymes.}, language = {en} } @article{KangGohlkeEngstroemetal.2016, author = {Kang, Yu and Gohlke, Ulrich and Engstr{\"o}m, Olof and Hamark, Christoffer and Scheidt, Tom and Kunstmann, Ruth Sonja and Heinemann, Udo and Widmalm, G{\"o}ran and Santer, Mark and Barbirz, Stefanie}, title = {Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions}, series = {Journal of the American Chemical Society}, volume = {138}, journal = {Journal of the American Chemical Society}, publisher = {American Chemical Society}, address = {Washington}, issn = {0002-7863}, doi = {10.1021/jacs.6b00240}, pages = {9109 -- 9118}, year = {2016}, abstract = {Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S. flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.}, language = {en} } @article{SchmidtRabschBroekeretal.2016, author = {Schmidt, Andreas and Rabsch, Wolfgang and Br{\"o}ker, Nina Kristin and Barbirz, Stefanie}, title = {Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens}, series = {BMC microbiology}, volume = {16}, journal = {BMC microbiology}, publisher = {BioMed Central}, address = {London}, issn = {1471-2180}, doi = {10.1186/s12866-016-0826-0}, pages = {2214 -- 2226}, year = {2016}, abstract = {Background: Non-typhoid Salmonella Typhimurium (S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable. Results: We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state. Conclusions: Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.}, language = {en} } @article{KunstmannScheidtBuchwaldetal.2018, author = {Kunstmann, Ruth Sonja and Scheidt, Tom and Buchwald, Saskia and Helm, Alexandra and Mulard, Laurence A. and Fruth, Angelika and Barbirz, Stefanie}, title = {Bacteriophage Sf6 Tailspike Protein for Detection of Shigella flexneri Pathogens}, series = {Viruses}, volume = {10}, journal = {Viruses}, number = {8}, publisher = {Molecular Diversity Preservation International (MDPI)}, address = {Basel}, issn = {1999-4915}, doi = {10.3390/v10080431}, pages = {1 -- 18}, year = {2018}, abstract = {Bacteriophage research is gaining more importance due to increasing antibiotic resistance. However, for treatment with bacteriophages, diagnostics have to be improved. Bacteriophages carry adhesion proteins, which bind to the bacterial cell surface, for example tailspike proteins (TSP) for specific recognition of bacterial O-antigen polysaccharide. TSP are highly stable proteins and thus might be suitable components for the integration into diagnostic tools. We used the TSP of bacteriophage Sf6 to establish two applications for detecting Shigella flexneri (S. flexneri), a highly contagious pathogen causing dysentery. We found that Sf6TSP not only bound O-antigen of S. flexneri serotype Y, but also the glucosylated O-antigen of serotype 2a. Moreover, mass spectrometry glycan analyses showed that Sf6TSP tolerated various O-acetyl modifications on these O-antigens. We established a microtiter plate-based ELISA like tailspike adsorption assay (ELITA) using a Strep-tag®II modified Sf6TSP. As sensitive screening alternative we produced a fluorescently labeled Sf6TSP via coupling to an environment sensitive dye. Binding of this probe to the S. flexneri O-antigen Y elicited a fluorescence intensity increase of 80\% with an emission maximum in the visible light range. The Sf6TSP probes thus offer a promising route to a highly specific and sensitive bacteriophage TSP-based Shigella detection system.}, language = {en} } @article{KangBarbirzLipowskyetal.2014, author = {Kang, Yu and Barbirz, Stefanie and Lipowsky, Reinhard and Santer, Mark}, title = {Conformational Diversity of O-Antigen Polysaccharides of the Gram-Negative Bacterium Shigella flexneri Serotype Y}, series = {The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces \& biophysical chemistry}, volume = {118}, journal = {The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces \& biophysical chemistry}, number = {9}, publisher = {American Chemical Society}, address = {Washington}, issn = {1520-6106}, doi = {10.1021/jp4111713}, pages = {2523 -- 2534}, year = {2014}, language = {en} }