TY - JOUR A1 - Schmidt, Andreas A1 - Rabsch, Wolfgang A1 - Broeker, Nina K. A1 - Barbirz, Stefanie T1 - Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens JF - BMC microbiology N2 - 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. KW - Salmonella Typhimurium KW - O-antigen KW - Tailspike protein KW - Bacteriophage KW - Phase variation KW - O-serotyping KW - Flow cytometry Y1 - 2016 U6 - https://doi.org/10.1186/s12866-016-0826-0 SN - 1471-2180 VL - 16 PB - BioMed Central CY - London ER - TY - GEN A1 - Schmidt, Andreas A1 - Rabsch, Wolfgang A1 - Broeker, Nina K. A1 - Barbirz, Stefanie T1 - Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 313 KW - Bacteriophage KW - Flow cytometry KW - O-antigen KW - O-serotyping KW - Phase variation KW - Salmonella Typhimurium KW - Tailspike protein Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-103769 ER - TY - JOUR A1 - Broeker, Nina K. A1 - Roske, Yvette A1 - Valleriani, Angelo A1 - Stephan, Mareike Sophia A1 - Andres, Dorothee A1 - Koetz, Joachim A1 - Heinemann, Udo A1 - Barbirz, Stefanie T1 - Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail JF - The journal of biological chemistry N2 - 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. KW - bacteriophage KW - lipopolysaccharide (YLPS) KW - structural biology KW - DNA viruses KW - glycobiology KW - fluorescence KW - Salmonella enterica KW - contractile tail KW - DNA ejection KW - O-antigen specificity KW - Salmonella myovirus KW - tailspike protein KW - molecular machine Y1 - 2019 U6 - https://doi.org/10.1074/jbc.RA119.008133 SN - 1083-351X VL - 294 IS - 31 SP - 11751 EP - 11761 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - GEN A1 - Broeker, Nina K. A1 - Kiele, Franziska A1 - Casjens, Sherwood R. A1 - Gilcrease, Eddie B. A1 - Thalhammer, Anja A1 - Koetz, Joachim T1 - In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620 T2 - Viruses N2 - Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 469 KW - O-antigen specific phage KW - podovirus KW - HK620 KW - lipopolysaccharide KW - in vitro particle opening KW - tailspike protein Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-417493 ER - TY - JOUR A1 - Broeker, Nina K. A1 - Kiele, Franziska A1 - Casjens, Sherwood R. A1 - Gilcrease, Eddie B. A1 - Thalhammer, Anja A1 - Koetz, Joachim T1 - In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620 JF - Viruses N2 - Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change. KW - O-antigen specific phage KW - podovirus KW - HK620 KW - lipopolysaccharide KW - in vitro particle opening KW - tailspike protein Y1 - 2018 U6 - https://doi.org/10.3390/v10060289 SN - 1999-4915 VL - 10 IS - 6 SP - 1 EP - 15 PB - Molecular Diversity Preservation International (MDPI) CY - Basel ER - TY - GEN A1 - Broeker, Nina K. A1 - Barbirz, Stefanie T1 - Not a barrier but a key: How bacteriophages exploit host's O‐antigen as an essential receptor to initiate infection T2 - Molecular microbiology N2 - Tailed bacteriophages specific for Gram‐negative bacteria encounter lipopolysaccharide (LPS) during the first infection steps. Yet, it is not well understood how biochemistry of these initial interactions relates to subsequent events that orchestrate phage adsorption and tail rearrangements to initiate cell entry. For many phages, long O‐antigen chains found on the LPS of smooth bacterial strains serve as essential receptor recognized by their tailspike proteins (TSP). Many TSP are depolymerases and O‐antigen cleavage was described as necessary step for subsequent orientation towards a secondary receptor. However, O‐antigen specific host attachment must not always come along with O‐antigen degradation. In this issue of Molecular Microbiology Prokhorov et al. report that coliphage G7C carries a TSP that deacetylates O‐antigen but does not degrade it, whereas rough strains or strains lacking O‐antigen acetylation remain unaffected. Bacteriophage G7C specifically functionalizes its tail by attaching the deacetylase TSP directly to a second TSP that is nonfunctional on the host's O‐antigen. This challenges the view that bacteriophages use their TSP only to clear their way to a secondary receptor. Rather, O‐antigen specific phages may employ enzymatically active TSP as a tool for irreversible LPS membrane binding to initiate subsequent infection steps. Y1 - 2017 U6 - https://doi.org/10.1111/mmi.13729 SN - 0950-382X SN - 1365-2958 VL - 105 SP - 353 EP - 357 PB - Wiley CY - Hoboken ER -