@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{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{SchmidtRabschBroekeretal.2016,
  author    = {Schmidt, Andreas and Rabsch, Wolfgang and Broeker, Nina K. 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     = {11},
  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}
}