The role of coagulation factor XIII in the early innate immune response against streptococcal infections Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo - Wilhelmina zu Braunschweig zur Erlangung des Grades eine r Doktor in der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Christin Deicke aus Wernigerode 1. Referent: Professor Dr. Manfred R o hde 2. Referent in : Professor Dr. Susanne Engelmann eingereicht am: 07.10.2015 mündliche Prüfung (Disputation) am: 14.12.2015 Druckjahr 2016 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebens - wissenschaften, vertreten durch den Mentor der Arbeit in folgenden Beiträgen vorab veröffentlicht: Publikationen Loof T. G., Deicke C., and Medina E. (2014): The role of coagulation/fibrinolysis during Streptococcus pyogenes infection. Frontiers in Cellular and Infection Microbiology 4 : 128 . Deicke C., Chakrakodi B., Pils M. C, Dickneite G., Johansson L., Medina E., and Loof T. G. (2015): Local activation of Coagulation Factor XIII reduces systemic complications and improves the survival of mice after Streptococcus pyogenes M1 skin infection . International Journal of Medical Microbiology ( under revision ). Tagungsbeiträge Deicke C., Rohde M., Chhatwal G.S., Nitsche - Schmitz D.P., Loof T.G. (2013): Fibrin formation as a mechanism of innate immune defense is counteracted by streptokinase. 65. Jahrestagung der Deutsche n Gesellschaft für Hygiene und Mikrobiologie (DGHM) , Rostock (Poster). Deicke C., Rohde M., Dickneite G., Medina E., and Loof T.G (2014): Protective role of coagulation factor XIII in Streptococcus pyogenes skin infections. 66. Jahrestagung der Deutsche n Gesellschaft für Hygiene und Mikrobiologie , Dresden (Poster). “ Learning is the only thing the mind never exhausts, never fears, and never regrets. ” Leonardo da Vinci T ABLE OF CONTENTS Table of contents Table of contents ................................ ................................ ...................... I List of figures ................................ ................................ .......................... VI List of tables ................................ ................................ ......................... VIII Abbreviations ................................ ................................ .......................... IX 1 ABSTRACT ................................ ................................ .................... 1 2 INTRODUCTION ................................ ................................ ............ 2 2.1 The genus Streptococcus ................................ ................................ ... 2 2.2 Streptococcus pyogenes ................................ ................................ .... 3 2.2.1 Pathogenic potential of Streptococcus pyogenes ................................ ........... 4 2.2.1.1 Superficial infections ................................ ................................ .................. 4 2.2.1.2 Invasive infections ................................ ................................ ...................... 5 2.2.1.3 Toxin - mediated infections ................................ ................................ .......... 6 2.2.1.4 Post - infection sequelae ................................ ................................ .............. 6 2.2.2 Treatment and prevention of streptococcal mediated diseases ..................... 7 2.2.3 Virulence of Streptococcus pyogenes ................................ .............................. 8 2.2.3.1 Surface - associated virulence factors ................................ ........................ 10 2.2.3.1.1 Streptococcal M protein ................................ ................................ ...... 10 2.2.3.1.2 M like Proteins ................................ ................................ ................... 12 2.2.3.1.3 Hyaluronic acid capsule ................................ ................................ ..... 12 2.2.3.1.4 Lipoteichoic acid ................................ ................................ ................. 13 2.2.3.1.5 Fibronectin binding proteins ................................ ............................... 13 2.2.3.1.6 C5a peptidase ................................ ................................ .................... 13 2.2.3.2 Secreted virulence factors ................................ ................................ ........ 14 2.2.3.2.1 Haemolysins ................................ ................................ ...................... 14 2.2.3.2.2 Streptococcal pyogenic exotoxins ................................ ...................... 14 2.2.3.2.3 SPE - B ................................ ................................ ................................ 14 2.2.3.2.4 Streptokinase ................................ ................................ ..................... 15 I T ABLE OF CONTENTS 2.2.3.2.5 Streptococcal inhibitor of complement - mediated lysis ........................ 15 2.3 The human coagulation system ................................ ....................... 15 2.3.1 Coagulation during hemostasis ................................ ................................ ...... 15 2.3.1.1 Extrinsic coagulation pathway ................................ ................................ .. 17 2.3.1.2 Intrinsic coagulation pathway ................................ ................................ ... 17 2.3.1.3 Fibrinogen and Fibrin ................................ ................................ ............... 17 2.3.1.4 Fibrinolysis ................................ ................................ ............................... 19 2.3.2 Coagulation as a part of innate immunity against streptococcal infections 19 2.3.2.1 Innate immune response against Streptococcus pyogenes ...................... 19 2.3.2.2 Bacterial entrapment within fibrin networks ................................ .............. 21 2.3.2.1 Importance of coagulation factor XIII ................................ ........................ 23 2.4 Aims of the study ................................ ................................ ............... 26 3 MATERIAL AND METHODS ................................ ....................... 27 3.1 Material ................................ ................................ ............................... 27 3.1.1 Chemicals ................................ ................................ ................................ ......... 27 3.1.2 Expendable material and instruments ................................ ............................ 29 3.1.3 Proteins and enzymes ................................ ................................ ..................... 31 3.1.4 Antibodies and plasma ................................ ................................ .................... 32 3.1.5 Kits ................................ ................................ ................................ .................... 33 3.1.6 Antibiotics ................................ ................................ ................................ ........ 33 3.1.7 Plasmid vectors ................................ ................................ ................................ 34 3.1.8 Oligonucleotides ................................ ................................ .............................. 34 3.1.9 Bact erial strains ................................ ................................ ............................... 35 3.1.9.1 Streptococcus pyogenes strains (Group A Streptococci, GAS) ................ 35 3.1.9.2 Group G Streptococci ................................ ................................ ............... 36 3.1. 9.3 Other streptococci strains ................................ ................................ ......... 36 3.1.9.4 Escherichia coli strains ................................ ................................ ............. 36 3.1.9.5 Lactococcus lactis strains ................................ ................................ ......... 37 3.2 Microbiological methods ................................ ................................ ... 38 3.2.1 Cultivation of bacterial strains ................................ ................................ ........ 38 3.2.2 Monitoring of bacterial growth and adjusting of bacterial suspensions ...... 39 3.2.3 Preparation and transformation of competent bacteria ................................ 39 II T ABLE OF CONTENTS 3.2.3.1 Preparation of chemical competent E. coli cells ................................ ........ 39 3.2.3.2 Transformation of chemically competent E. coli cells ................................ 40 3.2.3.3 Preparation of electro competent E. coli cells ................................ ........... 40 3.2.3.4 Transformation of electrocompetent E. coli cells ................................ ...... 40 3.2.3.5 Preparation of competent L. lactis cells ................................ .................... 40 3.2.3.6 Transformation of competent L. lactis cells ................................ ............... 41 3.2.4 Immobilization of S. pyogenes within the fibrin network .............................. 41 3.3 Molecular biology methods ................................ .............................. 42 3.3.1 Isolation of chromosomal DNA from S. pyogenes ................................ ......... 42 3.3.2 Isolation of plasmid DNA ................................ ................................ ................. 42 3. 3.3 Agarose gel electrophoresis ................................ ................................ ........... 42 3.3.4 Polymerase chain