@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}
}
@misc{BroekerKieleCasjensetal.2018,
  author    = {Broeker, Nina K. and Kiele, Franziska and Casjens, Sherwood R. and Gilcrease, Eddie B. and Thalhammer, Anja and Koetz, Joachim},
  title     = {In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620},
  series = {Viruses},
  journal   = {Viruses},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-417493},
  pages     = {15},
  year      = {2018},
  abstract  = {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.},
  language  = {en}
}
@article{BroekerKieleCasjensetal.2018,
  author    = {Broeker, Nina K. and Kiele, Franziska and Casjens, Sherwood R. and Gilcrease, Eddie B. and Thalhammer, Anja and Koetz, Joachim},
  title     = {In Vitro Studies of Lipopolysaccharide-Mediated DNA Release of Podovirus HK620},
  series = {Viruses},
  volume    = {10},
  journal   = {Viruses},
  number    = {6},
  publisher = {Molecular Diversity Preservation International (MDPI)},
  address   = {Basel},
  issn      = {1999-4915},
  doi       = {10.3390/v10060289},
  pages     = {1 -- 15},
  year      = {2018},
  abstract  = {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.},
  language  = {en}
}
@article{BroekerGohlkeMuelleretal.2013,
  author    = {Br{\"o}ker, Nina Kristin and Gohlke, Ulrich and M{\"u}ller, J{\"u}rgen J. and Uetrecht, Charlotte and Heinemann, Udo and Seckler, Robert and Barbirz, Stefanie},
  title     = {Single amino acid exchange in bacteriophage HK620 tailspike protein results in thousand-fold increase of its oligosaccharide affinity},
  series = {Glycobiology},
  volume    = {23},
  journal   = {Glycobiology},
  number    = {1},
  publisher = {Oxford Univ. Press},
  address   = {Cary},
  issn      = {0959-6658},
  doi       = {10.1093/glycob/cws126},
  pages     = {59 -- 68},
  year      = {2013},
  abstract  = {Bacteriophage HK620 recognizes and cleaves the O-antigen polysaccharide of Escherichia coli serogroup O18A1 with its tailspike protein (TSP). HK620TSP binds hexasaccharide fragments with low affinity, but single amino acid exchanges generated a set of high-affinity mutants with submicromolar dissociation constants. Isothermal titration calorimetry showed that only small amounts of heat were released upon complex formation via a large number of direct and solvent-mediated hydrogen bonds between carbohydrate and protein. At room temperature, association was both enthalpy- and entropy-driven emphasizing major solvent rearrangements upon complex formation. Crystal structure analysis showed identical protein and sugar conformers in the TSP complexes regardless of their hexasaccharide affinity. Only in one case, a TSP mutant bound a different hexasaccharide conformer. The extended sugar binding site could be dissected in two regions: first, a hydrophobic pocket at the reducing end with minor affinity contributions. Access to this site could be blocked by a single aspartate to asparagine exchange without major loss in hexasaccharide affinity. Second, a region where the specific exchange of glutamate for glutamine created a site for an additional water molecule. Side-chain rearrangements upon sugar binding led to desolvation and additional hydrogen bonding which define this region of the binding site as the high-affinity scaffold.},
  language  = {en}
}
@article{AndresGohlkeBroekeretal.2013,
  author    = {Andres, Dorothee and Gohlke, Ulrich and Br{\"o}ker, Nina Kristin and Schulze, Stefan and Rabsch, Wolfgang and Heinemann, Udo and Barbirz, Stefanie and Seckler, Robert},
  title     = {An essential serotype recognition pocket on phage P22 tailspike protein forces Salmonella enterica serovar Paratyphi A O-antigen fragments to bind as nonsolution conformers},
  series = {Glycobiology},
  volume    = {23},
  journal   = {Glycobiology},
  number    = {4},
  publisher = {Oxford Univ. Press},
  address   = {Cary},
  issn      = {0959-6658},
  doi       = {10.1093/glycob/cws224},
  pages     = {486 -- 494},
  year      = {2013},
  abstract  = {Bacteriophage P22 recognizes O-antigen polysaccharides of Salmonella enterica subsp. enterica (S.) with its tailspike protein (TSP). In the serovars S. Typhimurium, S. Enteritidis, and S. Paratyphi A, the tetrasaccharide repeat units of the respective O-antigens consist of an identical main chain trisaccharide but different 3,6-dideoxyhexose substituents. Here, the epimers abequose, tyvelose and paratose determine the specific serotype. P22 TSP recognizes O-antigen octasaccharides in an extended binding site with a single 3,6-dideoxyhexose binding pocket. We have isolated S. Paratyphi A octasaccharides which were not available previously and determined the crystal structure of their complex with P22 TSP. We discuss our data together with crystal structures of complexes with S. Typhimurium and S. Enteritidis octasaccharides determined earlier. Isothermal titration calorimetry showed that S. Paratyphi A octasaccharide binds P22 TSP less tightly, with a difference in binding free energy of similar to 7 kJ mol(-1) at 20 degrees C compared with S. Typhimurium and S. Enteritidis octasaccharides. Individual protein-carbohydrate contacts were probed by amino acid replacements showing that the dideoxyhexose pocket contributes to binding of all three serotypes. However, S. Paratyphi A octasaccharides bind in a conformation with an energetically unfavorable phi/epsilon glycosidic bond angle combination. In contrast, octasaccharides from the other serotypes bind as solution-like conformers. Two water molecules are conserved in all P22 TSP complexes with octasaccharides of different serotypes. They line the dideoxyhexose binding pocket and force the S. Paratyphi A octasaccharides to bind as nonsolution conformers. This emphasizes the role of solvent as part of carbohydrate binding sites.},
  language  = {en}
}
@phdthesis{Broeker2012,
  author    = {Br{\"o}ker, Nina Kristin},
  title     = {Die Erkennung komplexer Kohlenhydrate durch das Tailspike Protein aus dem Bakteriophagen HK620},
  url       = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-60366},
  school      = {Universit{\"a}t Potsdam},
  year      = {2012},
  abstract  = {Kohlenhydrate stellen aufgrund der strukturellen Vielfalt und ihrer oft exponierten Lage auf Zelloberfl{\"a}chen wichtige Erkennungsstrukturen dar. Die Wechselwirkungen von Proteinen mit diesen Kohlenhydraten vermitteln einen spezifischen Informationsaustausch. Protein-Kohlenhydrat-Interaktionen und ihre Triebkr{\"a}fte sind bislang nur teilweise verstanden, da nur wenig strukturelle Daten von Proteinen im Komplex mit vorwiegend kleinen Kohlenhydraten erh{\"a}ltlich sind. Mit der vorliegenden Promotionsarbeit soll ein Beitrag zum Verst{\"a}ndnis von Protein-Kohlenhydrat-Wechselwirkungen durch Analysen struktureller Thermodynamik geleistet werden, um zuk{\"u}nftig Vorhersagen mit zuverl{\"a}ssigen Algorithmen zu erlauben. Als Modellsystem zur Erkennung komplexer Kohlenhydrate diente dabei das Tailspike Protein (TSP) aus dem Bakteriophagen HK620. Dieser Phage erkennt spezifisch seinen E. coli-Wirt anhand der Oberfl{\"a}chenzucker, der sogenannten O-Antigene. Dabei binden die TSP des Phagen das O-Antigen des Lipopolysaccharids (LPS) und weisen zudem eine hydrolytische Aktivit{\"a}t gegen{\"u}ber dem Polysaccharid (PS) auf. Anhand von isolierten Oligosacchariden des Antigens (Typ O18A1) wurde die Bindung an HK620TSP und verschiedener Varianten davon systematisch analysiert. Die Bindung der komplexen Kohlenhydrate durch HK620TSP zeichnet sich durch große Interaktionsfl{\"a}chen aus. Durch einzelne Aminos{\"a}ureaustausche im aktiven Zentrum wurden Varianten generiert, die eine tausendfach erh{\"o}hte Affinit{\"a}t (KD ~ 100 nM) im Vergleich zum Wildtyp-Protein (KD ~ 130 μM) aufweisen. Dabei zeichnet sich das System dadurch aus, dass die Bindung bei Raumtemperatur nicht nur enthalpisch, sondern auch entropisch getrieben wird. Ursache f{\"u}r den g{\"u}nstigen Entropiebeitrag ist die große Anzahl an Wassermolek{\"u}len, die bei der Bindung des Hexasaccharids verdr{\"a}ngt werden. R{\"o}ntgenstrukturanalysen zeigten f{\"u}r alle TSP-Komplexe außer f{\"u}r Variante D339N unabh{\"a}ngig von der Hexasaccharid-Affinit{\"a}t analoge Protein- und Kohlenhydrat-Konformationen. Dabei kann die Bindestelle in zwei Regionen unterteilt werden: Zum einen befindet sich am reduzierenden Ende eine hydrophobe Tasche mit geringen Beitr{\"a}gen zur Affinit{\"a}tsgenerierung. Der Zugang zu dieser Tasche kann ohne große Affinit{\"a}tseinbuße durch einen einzelnen Aminos{\"a}ureaustausch (D339N) blockiert werden. In der zweiten Region kann durch den Austausch eines Glutamats durch ein Glutamin (E372Q) eine Bindestelle f{\"u}r ein zus{\"a}tzliches Wassermolek{\"u}l generiert werden. Die Rotation einiger Aminos{\"a}uren bei Kohlenhydratbindung f{\"u}hrt zur Desolvatisierung und zur Ausbildung von zus{\"a}tzlichen Wasserstoffbr{\"u}cken, wodurch ein starker Affinit{\"a}tsgewinn erzielt wird. HK620TSP ist nicht nur spezifisch f{\"u}r das O18A1-Antigen, sondern erkennt zudem das um eine Glucose verk{\"u}rzte Oligosaccharid des Typs O18A und hydrolysiert polymere Strukturen davon. Studien zur Bindung von O18A-Pentasaccharid zeigten, dass sich die Triebkr{\"a}fte der Bindung im Vergleich zu dem zuvor beschriebenen O18A1-Hexasaccharid verschoben haben. Durch Fehlen der Seitenkettenglucose ist die Bindung im Vergleich zu dem O18A1-Hexasaccharid weniger stark entropisch getrieben (Δ(-TΔS) ~ 10 kJ/mol), w{\"a}hrend der Enthalpiebeitrag zu der Bindung g{\"u}nstiger ist (ΔΔH ~ -10 kJ/mol). Insgesamt gleichen sich diese Effekte aus, wodurch sehr {\"a}hnliche Affinit{\"a}ten der TSP-Varianten zu O18A1-Hexasaccharid und O18A-Pentasaccharid gemessen wurden. Durch die Bindung der Glucose werden aus einer hydrophoben Tasche vier Wassermolek{\"u}le verdr{\"a}ngt, was entropisch stark beg{\"u}nstigt ist. Unter enthalpischen Aspekten ist dies ebenso wie einige Kontakte zwischen der Glucose und einigen Resten in der Tasche eher ung{\"u}nstig. Die Bindung der Glucose in die hydrophobe Tasche an HK620TSP tr{\"a}gt somit nicht zur Affinit{\"a}tsgenerierung bei und es bleibt zu vermuten, dass sich das O18A1-Antigen-bindende HK620TSP aus einem O18A-Antigen-bindenden TSP evolution{\"a}r herleitet. In dem dritten Teilprojekt der Dissertation wurde der Infektionsmechanismus des Phagen HK620 untersucht. Es konnte gezeigt werden, dass analog zu dem verwandten Phagen P22 die Ejektion der DNA aus HK620 allein durch das Lipopolysaccharid (LPS) des Wirts in vitro induziert werden kann. Die Morphologie und Kettenl{\"a}nge des LPS sowie die Aktivit{\"a}t von HK620TSP gegen{\"u}ber dem LPS erwiesen sich dabei als essentiell. So konnte die DNA-Ejektion in vitro auch durch LPS aus Bakterien der Serogruppe O18A induziert werden, welches ebenfalls von dem TSP des Phagen gebunden und hydrolysiert wird. Diese Ergebnisse betonen die Rolle von TSP f{\"u}r die Erkennung der LPS-Rezeptoren als wichtigen Schritt f{\"u}r die Infektion durch die Podoviren HK620 und P22.},
  language  = {de}
}