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A New Class of Molecules from Staphylococcus Species Affects Quorum Sensing and Growth of Gram-Negative Bacteria

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A New Class of Molecules from Staphylococcus Species Affects Quorum Sensing and Growth of Gram-Negative Bacteria A New Class of Molecules from Staphylococcus Species Affects Quorum Sensing and Growth of Gram-Negative Bacteria Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Ya-Yun Chu aus Taichung, Taiwan Tübingen 2014 Tag der mündlichen Qualifikation: 04.08.2014 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter Prof. Dr. Friedrich Götz 2. Berichterstatter PD Dr. Iris Maldener Table of Contents Table of contents 1 Abbreviations 2 Symbols 3 Summary 4 Zusammenfassung 5 Part I Structure and Function of Yayureas from Staphylococcus intermedius group Introduction 6 1. Quorum sensing (QS) 6 2. QS of Gram-negative bacteria 8 3. QS of Gram-positive bacteria 11 4. Interphylum interference 11 5. Aim of the study 13 Results 14 1. Structural elucidation of the QS-inhibiting compounds from S. delphini 14 2. Biological properties of yayureas 27 a) Suppression of respiratory toxins protects from killing by P. aeruginosa 27 b) Yayurea A and B are mainly produced in stationary growth phase 29 c) Yayurea A is more active in inhibiting QS-controlled traits in Gram-negative 30 d) Yayureas have antimicrobial but no cytotoxic effect 32 e) Yayurea A and B are perceived by the AHL-receptor LuxN of Vibrio harveyi 34 f) Phylogenetic position of yayureas producing species 37 3. Corresponding genes of yayureas 39 Discussion 42 Part II The role of protein A in Staphylococcus aureus adherence Introduction 48 Results and discussion 49 References 52 List of publications 61 Contribution to publications 62 Acknowledgements 63 Appendix: Publications incorporated in this study 64 1 Abbreviations 2D Two-dimensional ACN Acetonitrile Agr Accessory gene regulation AHL N-acyl homoserine lactone AI-2 Autoinducer-2 AIP Autoinducer peptide ATCC American Type Culture Collection BA Bovine serum BSA Bovine serum albumin CFU Colony forming unit DMSO Dimethyl sulfoxide DSMZ Deutsche sammlung von Mikroorganismen und Zellkulturen GmbH DTT Dithiothreitol ESI-MS Electrospray ionization mass spectrometry EtOH Ethanol FT-ICR-MS Fourier transform ion cyclotron resonance mass spectrometry g Gram GC-EI-MS Gas chromatography electrospray ionization mass spectrometry GC-MS Gas chromatography mass spectrometry h Hour HAI-1 N-(3-hydroxybutyryl)-homoserine lactone HCl Hydrogen chloride HPLC High performance liquid chromatography HSL Homoserine lactone HSQC Heteronuclear single quantum Coherence K2HPO4 Potassium hydrogen phosphate KCl potassium chloride LB Lysogenic broth M Molar (mol/l) m Milli m/z Mass-to-charge ratio MB Marine broth MeOH Methanol MgCl2 Magnesium chloride MHz Mega Hertz min Minute ml Milliliter MLST Multilocus sequence typing MS Mass spectrometry NaCl Sodium chloride nm Nanometer NMR Nuclear magnetic resonance OD Optical density 2 OEG Oligo-ethylene-glycol PBS Phosphate-buffered saline PEG Poly-ethylene-glycol Phe Phenylalanine QS Quorum sensing ROS Reactive oxygen species RP-HPLC Reversed phase HPLC rRNA Ribosomal RNA Rt Retention time Sar Staphylococcus accessory regulation SDS Sodium dodecyl sulphate SIG Staphylococcus intermedius group Trp Tryptophan TSB Tryptic Soy Broth Tyr Tyrosine UV Ultraviolet WT Wild type WTA Wall teichoic acid yayurea A N-[2-(1H-indol-3-yl)ethyl]-urea yayurea B N-(2-phenethyl)-urea Symbols µ Micro oC Degree(s) Celsius % Percentage Gamma ∆ Deletion 3 Summary This thesis addressed bacterial communication between Gram positive and Gram negative bacteria. The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. While studying the interaction of staphylococci with Gram-negative bacteria, we came across another communication system in a Staphylococcus intermedius group (SIG) species which can act as a zoonotic pathogen. This is one of the rare examples where Gram-positive bacteria excrete two compounds in comparatively high amounts that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. The excreted compounds, named yayurea A and B, were isolated from S. delphini and structurally characterized. In vitro studies with the N- acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds stimulated the LuxN-mediated phosphorylation of LuxU and caused the opposite effects with AHL. Furthermore, growth of yayurea A and B producing S. delphini is not suppressed by respiratory toxins when co-cultured with P. aeruginosa. Taken together, yayureas are presumably involved in self-protection and ensuring competitiveness in natural environments shared with Gram-negatives. In the second part of thesis, the role of protein A in Staphylococcus aureus adhesion to ethylene glycol coated surfaces was investigated. It was found that the coating materials, HS- (CH2)11EG3OMe (EG3OMe) and poly-ethylene-glycol (PEG) prevented bacterial adhesion of S. aureus cells. However, pretreatment of EG3OMe and PEG coatings with -globulins or serum strongly promoted adherence of S. aureus cell. Furthermore, the spa-deletion mutant S. aureus, lacking the IgG binding protein A, showed reduced adherence and pretreatment of S. aureus with serum significantly decreased adherence. These results suggested that -globulins play a crucial role in promoting S. aureus cells adhesion by its IgG binding proteins. Particularly γ- globulins bound to the coated surfaces thus mediating adherence of S. aureus via its protein A. Intercepting this adherence should prove to be useful in preventing biofilm formation. 4 Zusammenfassung Diese Arbeit behandelt bakterielle Kommunikation zwischen Gram-positiven und Gram-negativen Bakterien. Mit dem Wissen, dass viele Krankheitserreger auf Zell- zu-Zell-Kommunikation setzen (Quorum Sensing), öffnet sich eine neue Strategie der Krankheitsbekämpfung: Quorum quenching. Während Interaktionsstudien von Staphylokokken mit Gram-negativen Bakterien sind wir auf ein neuartiges Kommunikationssystem gestoßen in dem zwei Substanzen in vergleichsweise hohen Mengen von Bakterien der Staphylococcus intermedius Gruppe ausgeschieden werden, welche Quorum Sensing-Signale und das Wachstum eines breiten Spektrums von Gram-negativen beta-und gamma-Proteobakterien unterdrücken. Die ausgeschiedenen Verbindungen, yayurea A und B, wurden von S. delphini isoliert und strukturell charakterisiert. In vitro-Studien mit dem N-Acylhomoserinlacton (AHL)-Rezeptor LuxN von V. harveyi zeigten, dass beide Verbindungen die LuxN- vermittelte Phosphorylierung von LuxU stimulieren und den entgegengesetzten Effekt zu AHL verursachen. Darüber hinaus wird das Wachstum des Yayurea A und B produzierenden S. delphini nicht durch die von P. aeruginosa produzierten Atemgifte während der Kokultivierung gehemmt. Zusammengefasst ist zu vermuten, dass Yayureas beim Selbstschutz und der Sicherung der Wettbewerbsfähigkeit der Produzenten in der natürlichen Umgebung mit Gram-negativen Bakterien beteiligt sind. Im zweiten Teil der Arbeit wurde die Rolle von Protein A bei der Adhäsion von S. aureus an Ethylenglykol beschichteten Oberflächen untersucht. Wir konnten zeigen, dass die Stoffe HS-(CH2) 11EG3Ome (EG3OMe) und Polyethylenglykol (PEG) die bakterielle Adhäsion von S. aureus-Zellen verhindern können. Die Vorbehandlung der EG3OMe und PEG Beschichtungen mit -Globulinen oder Serum führte jedoch zu einer starken Erhöhung der Adhäsion von S. aureus Zellen. Sowohl die spa-Deletionsmutante, als auch die Vorbehandlung von S. aureus mit Serum bewirken verminderte Adhäsion. Diese Ergebnisse legen nahe, dass die an die an die beschichteten Oberflächen gebundenen -Globuline die Adhäsion von S. aureus mittels Protein A vermitteln. Diese Adhäsion zu unterbinden könnte sich bei der Präventionen von Biofilmbildung als nützlich erweisen. 5 Part I Structure and Function of Yayureas from Staphylococcus intermedius group Introduction 1. Quorum sensing (QS) Quorum sensing (QS) is a process of bacterial communication, first discovered in the marine photobacteria Vibrio fischeri (Nealson et al., 1970). The signal transduction of QS is mediated through the synthesis and secretion of small membrane-diffusible hormone-like molecules termed autoinducers. When they accumulate and reach a critical threshold, autoinducers bind to cognate receptors and trigger QS-regulated gene expression. The signal-activated receptor directly or indirectly controls the expression of target genes. Since the concentration of signaling molecules in liquid culture is proportional to cell density, gene expression is regulated in response to bacterial population densities (Atkinson and Williams, 2009; Waters and Bassler, 2005). QS empowers bacteria to coordinate pathogenicity, cell division (Swift et al., 1996), sporulation (Jabbari et al., 2011), conjugation (White and Winans, 2007), biofilm formation, swarming, toxin production (Falcao et al., 2004), bioluminescence (Miyamoto et al., 2000) and production of secondary metabolites (Swift et al., 1996) to rapidly respond to the environment. There is growing interest in utilizing QS as a new prospective therapy against bacterial infection, as the evolutionary pressure to develop drug resistance with QS inhibitor treatment is less strong. In contrast to classic antibiotics, quorum-quenching compounds are inhibitors of bacterial virulence, rather than of bacterial growth (Cegelski et al., 2008), and therefore might serve as promising anti-virulence therapies
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