Strukturanalyse von Virulenzfaktoren und essentiellen Proteinen aus Clostridium difficile

Clostridium difficile is a Gram-positive, anaerobic, endospore-forming bacterium that produces several virulence factors, most prominently the secreted protein toxins Toxin A (TcdA) and Toxin B (TcdB). Clostridium difficile infections (CDI) are often hospital acquired and antibiotic-associated. Treatment of CDI currently involves taking broad-spectrum antibiotics, e.g. vancomycin. Due to the extremely high relapse rate of CDI after antibiotic treatment, the emergence of new highly virulent C. difficile strains and the threatening antibiotic resistance, the need for new therapeutic treatment methods for CDI is more urgent than ever before. To develop new therapeutics, a detailed knowledge of the molecular processes inside the pathogen as well as a comprehensive structural and functional knowledge of its virulence factors and proteins involved in infection is essential. Aim of this thesis was therefore the structural characterization of several important proteins of Clostridium difficile: its main virulence factor TcdB, proteins that are involved in basic cellular processes (i.e. growth and sporulation: CD1219 and CD1823) and the so-called diffocin proteins CD1363 and CD1364, bacteriophage tail-like proteins that act as bacteriocins. Full-length genes of the respective proteins (and truncated fragments of TcdB) were cloned in expression vectors, the proteins were expressed in E. coli or Bacillus megaterium and purified by affinity chromatography, ion exchange chromatography and size exclusion chromatography. Pure protein samples were used for structural analysis by small angle X-ray scattering and X-ray crystallography. SAXS envelopes were calculated for all proteins in this thesis, crystal structures were determined for CD1219, CD1823, CD1363 and CD1364. Based on the crystal structures of the proteins hypotheses about their molecular function could be derived.Das grampositive, anaerobe, endosporenbildende Bakterium Clostridium difficile produziert mehrere Virulenzfaktoren, allen voran die beiden sekretierten homologen Proteintoxine Toxin A (TcdA) und Toxin B (TcdB). Die von Clostridium difficile verursachten Infektionen werden häufig im Krankenhaus und nach Antibiotika-Behandlung erworben. Die Behandlung erfolgt häufig mit der Einnahme von Breitband-Antibiotika, z.B. Vancomycin. Aufgrund der hohen Rate an Reinfektionen nach Absetzung der Antibiotika-Therapie, der Entdeckung neuer höchst virulenter C. difficile Stämme und deren Antibiotikaresistenz ist der Bedarf an neuen Therapiemöglichkeiten für C. difficile Infektionen dringender denn je. Dafür ist eine detaillierte Kenntnis der molekularen Prozesse im Bakterium, sowie eine umfassende strukturelle und funktionelle Analyse seiner Virulenzfaktoren und anderer Proteine, die an essentiellen Stoffwechselvorgängen und der Infektion beteiligt sind, unerlässlich. Ziel dieser Arbeit war daher die Strukturanalyse verschiedener Proteine aus Clostridium difficile: eines seiner wichtigsten Virulenzfaktoren (TcdB), verschiedener Proteine, die essentiell für das Wachstum bzw. die Sporulation des Bakteriums sind (CD1219 und CD1823) und der Diffocin-Proteine CD1363 und CD1364, die strukturelle Ähnlichkeit zu Bacteriophagen-Schwänzen zeigen und als Bacteriocine agieren. Die Gene der Volllängen-Proteine (sowie Fragmente von TcdB) wurden in Expressionsvektoren kloniert, in E. coli oder Bacillus megaterium exprimiert und über Affinitätschromatographie, Ionenaustauschchromatographie und Gelfiltration gereinigt. Proteine in ausreichender Reinheit wurden für SAXS (Kleinwinkelröntgenstreuung) und Röntgenkristallographie-Experimente verwendet. In dieser Arbeit konnten SAXS-Hüllen für alle Proteine ermittelt und zusätzlich Röntgenkristallstrukturen für CD1219, CD1823, CD1363 und CD1364 bestimmt werden. Basierend auf den Kristallstrukturen der jeweiligen Proteine konnten Hypothesen über deren molekulare Funktion abgeleitet werden

Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From in the Pre-assembled State.

iffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin

Presentation_1_Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From Clostridium difficile in the Pre-assembled State.pdf

<p>Diffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.</p