Effects of oral streptococci and selected probiotic bacteria on the pathogen Streptococcus pyogenes: viability, biofilms, molecular functions, and virulence traits Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat) der Mathematisch-Naturwissenschaftlichen Fakultät der Univesität Rostock vorgelegt von Catur Riani geb. am 13.08.1976 auf Pulau Sambu aus Indonesien Rostock, Januar 2009 urn:nbn:de:gbv:28-diss2009-0087-1 Prof. Johannes Knobloch (Gutachter / Reviewer) Universitätsklinikum Schleswig-Holstein Campus Lübeck Ratzeburger Allee 160 23538 Lübeck Prof. Dr. Hubert Bahl (Gutachter / Reviewer) Uni Rostock Institut für Biologie Albert Einstein Str. 3 18059 Rostock Prof. Dr. Regine Hakenbeck (Gutachter / Reviewer) Technische Universität Kaiserslautern FB Biologie P.-Ehrlich-Str. 67663 Kaiserslautern Prof. Dr. Andreas Podbielski (Gutachter & Betreuer / Reviewer & Supervisor) Uni Rostock Medizin Abt. für Medizinische Mikrobiologie, Virologie und Hygiene Schillingalle 70 18057 Rostock Abgabedatum / date of submission: 30 Januar 2009 Verteidigungsdatum / date of defence: 4 Mai 2009 “Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes” Table of content Table of content Abbreviations I. Introduction ........................................................................................................... 1 I.1 Streptococcus pyogenes as a human pathogen ............................................. 1 I.2 The physiological microflora of the upper respiratory tract ........................ 4 I.3 Streptococci and their value as upper airways probiotics ............................ 7 I.4 Aims of the present study ............................................................................. 10 II. Material and Methods ............................................................................................ 12 II.1 Material ........................................................................................................ 12 II.1.1 Bacterial strains ................................................................................ 12 II.1.2 Culture media for bacteria ................................................................ 12 II.1.3 Eukaryotic cells and media for cell culture ...................................... 13 II.1.4 Plasmid ............................................................................................. 14 II.1.5 Antibodies ........................................................................................ 14 II.1.6 Reagents and buffers ........................................................................ 14 II.1.7 Instruments ....................................................................................... 14 II.2 Methods ........................................................................................................ 15 II.2.1 Bacterial culture condition ............................................................... 15 II.2.2 Culture condition and preparation of eukaryotic cell culture ........... 16 II.2.3 DNA/RNA methods and manipulation ............................................ 16 II.2.3.1 S. pyogenes DNA preparation ........................................... 16 II.2.3.2 Plasmid isolation from E. coli ........................................... 17 II.2.3.3 HEp-2 cells RNA isolation ................................................ 17 II.2.3.4 DNA/RNA concentration measurement ............................ 18 II.2.3.5 Polymerase Chain Reaction (Mullis et al., 1986) ............. 18 II.2.3.6 DNA restriction digest ...................................................... 19 II.2.3.7 DNA ligation reaction ....................................................... 19 II.2.3.8 Agarose electrophoresis for DNA (Sambrook et al., 1989) 19 II.2.3.9 Construction of the S. pyogenes M6 sagA-luc reporter gene strain ......................................................................... 20 II.2.4 Preparation and transformation of E. coli DH5 competent cells ... 21 II.2.5 Preparation and transformation of S. pyogenes competent cells ...... 21 II.2.6 Quantitative co-culture and transwell system .................................. 22 II.2.7 Bacteriocin assay .............................................................................. 23 II.2.8 Growth curve measurement ............................................................. 23 i Table of content II.2.9 Quantitative assays for sagA-luciferase activity .............................. 23 II.2.10 Hemolysis assay ............................................................................... 24 II.2.11 Coaggregation assay (modified from Cisar et al., 1979) ................. 24 II.2.12 Biofilm quantification with safranin assay ....................................... 25 II.2.13 Microscopic observation and documentation of biofilms (Fluorescence, SEM, CLSM) ........................................................... 25 II.2.13.1 Fluorescence microscopy ............................................... 25 II.2.13.2 Scanning Electron Microscopy (SEM) .......................... 25 II.2.13.3 Confocal Laser Scanning Microscope (CLSM) ............. 26 II.2.14 Adherence and internalization assay ................................................ 26 II.2.15 Eukaryotic cell viability assay .......................................................... 27 II.2.16 Double-immunofluorescence assay .................................................. 28 II.2.17 HEp-2 cells microarray .................................................................... 29 III. Results ................................................................................................................... 31 III.1 S. pyogenes co-culture experiments: direct and indirect contact ................. 31 III.2 Bacteriocin assay .......................................................................................... 35 III.3 Effect on S. pyogenes sagA transcription ..................................................... 35 III.3.1 Construction of an S. pyogenes serotype M6 sagA-luc reporter gene strain ........................................................................................ 36 III.3.2 sagA-luc activity measurement in the presence of selected oral bacteria and E. coli Nissle ................................................................ 39 III.4 Effect of spent medium on S. pyogenes hemolytic activity ......................... 42 III.5 Coaggregation of S. pyogenes with oral bacteria and E. coli Nissle ............ 42 III.6 Effect of oral bacteria and E. coli Nissle on S. pyogenes biofilms ............... 43 III.6.1 Evaluation of growth medium and monospecies biofilm behaviour .......................................................................................... 44 III.6.2 Investigation of mixed-species biofilms .......................................... 46 III.6.3 The effect of artificial saliva on the species interaction ................... 50 III.7 Effect of oral bacteria and E. coli Nissle on S. pyogenes adherence to and internalization into host cells ........................................................................ 53 III.7.1 Quantitative assay ............................................................................ 53 III.7.2 Double immunofluorescence ............................................................ 59 III.8 Effect of oral bacteria and E. coli Nissle on S. pyogenes cytotoxicity ......... 61 III.9 Transcriptional response of HEp-2 cells in the presence of S. salivarius and S. oralis .................................................................................................. 63 ii Table of content IV. Discussion ............................................................................................................. 68 IV.1 General considerations ................................................................................. 68 IV.2 Changes of S. pyogenes numbers and viability in co-culture experiments 69 IV.3 Co-culture effects on S. pyogenes virulence factor expression .................... 71 IV.4 Co-culture effects on S. pyogenes biofilms .................................................. 73 IV.5 Co-culture effects on S. pyogenes interactions with eukaryotic cells .......... 75 IV.6 Co-culture effects on the integrity and metabolism of eukaryotic cells ....... 78 V. Conclusion ............................................................................................................. 82 VI. Reference ............................................................................................................... 84 VII. Appendix ............................................................................................................... 95 iii Abbreviations Abbreviations aad9 resistance gene for spectinomycin ATCC American Type Culture Collection aqua dest. aqua destillata bar pressure unit BHI Brain Heart Infusion bp base pair BSA bovine serume albumin C Coulomb, international unit for electric charge °C Celcius centigrade CaCl2 calciumchloride cfu colony forming unit CLSM Confocal Laser Scanning Microscopy CO2 carbondioxide Col collagen cpa gene encoding collagen binding protein DMEM Dulbecco’s modified Eagle’s medium DNA deoxyribonucleic acid dNTP dideoxynucleosidetriphosphate DSMZ Deutsche Sammlung für Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) E. coli Escherichia coli E. faecalis Enterococcus faecalis EDTA ethylene diamine tetraacetic acid emm gene encoding M protein EPS exopolysaccharide EtBr ethidiumbromide fbp gene encoding