Identification of phagosomal escape relevant factors in Staphylococcus aureus infection DISSERTATION zur Erlangung des Akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Fakultät für Biologie der Julius‐Maximilians‐Universität Würzburg vorgelegt von Magdalena Urszula Grosz, M. Sc. geboren am 25.06.1985 in Kattowitz Würzburg, August 2015 Eingereicht am: Mitglieder der Prüfungskommission: Vorsitzender: Erstgutachter: Prof. Dr. Thomas Rudel Zweitgutachter: PD Dr. Knut Ohlsen Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am: 2 Table of contents Summary ................................................................................................................................................. 7 Zusammenfassung ................................................................................................................................... 8 1 Introduction ............................................................................................................................ 9 1.1 Staphylococcus aureus ............................................................................................................ 9 1.2 Virulence factors ................................................................................................................... 10 1.2.1 S. aureus toxins including exotoxins, enterotoxins and leukocidins ................................ 10 1.2.1.1 α‐haemolysin (α‐toxin) .............................................................................................. 10 1.2.1.2 β‐toxin ....................................................................................................................... 11 1.2.1.3 Bi‐component toxins: Leukotoxins ............................................................................ 12 1.2.1.4 Phenol soluble modulins ........................................................................................... 13 1.2.2 Staphylococcal enzymes, superantigens and protein A ................................................... 16 1.3 Pathogenesis and medical relevance of MRSA and S. aureus ............................................... 18 1.4 Invasion mechanisms of S. aureus in non‐professional phagocytes ..................................... 20 1.5 Phagosomal escape ............................................................................................................... 21 1.6 S. aureus induced host cell death .......................................................................................... 23 1.7 Aim of the study .................................................................................................................... 25 2 Material and Methods .......................................................................................................... 27 2.1 Material ................................................................................................................................. 27 2.1.1 Bacterial strains ................................................................................................................ 27 2.1.2 Cell lines ........................................................................................................................... 29 2.1.3 Plasmids ............................................................................................................................ 29 2.1.4 Antibodies ........................................................................................................................ 29 2.1.5 Oligonucleotides ............................................................................................................... 30 2.1.6 Kits .................................................................................................................................... 31 2.1.7 Antibiotics ......................................................................................................................... 31 2.1.8 Buffers, solutions and media ............................................................................................ 32 2.1.9 Chemicals ......................................................................................................................... 33 3 2.1.10 Equipment ........................................................................................................................ 34 2.1.11 Software programs and webpages................................................................................... 34 2.2 Methods ................................................................................................................................ 34 2.2.1 Bacterial culture techniques ............................................................................................ 34 2.2.1.1 S. aureus cultivation .................................................................................................. 34 2.2.1.2 Growing curves .......................................................................................................... 35 2.2.1.3 Generation electrocompetent S. aureus ................................................................... 35 2.2.1.4 Electroporation of S. aureus ...................................................................................... 35 2.2.1.5 Determination of the minimal inhibitory concentration of S. aureus ....................... 36 2.2.2 Cell culture methodes and infection ................................................................................ 36 2.2.2.1 Cell passage ............................................................................................................... 36 2.2.2.2 siRNA Transfection .................................................................................................... 36 2.2.2.3 S. aureus infection ..................................................................................................... 37 2.2.2.4 Flow cytometry analysis ............................................................................................ 37 2.2.2.5 Microscopic based escape marker recruitment assay .............................................. 40 2.2.2.6 Lysostaphin protection assay .................................................................................... 41 2.2.2.7 Crystal violet assay .................................................................................................... 41 2.2.2.8 Monitoring of the viable cell concentration with Trypan Blue ................................. 42 2.2.3 RNA methods ................................................................................................................... 42 2.2.3.1 RNA isolation ............................................................................................................. 42 2.2.3.2 Reverse transcription ................................................................................................ 42 2.2.3.3 Polymerase Chain Reaction (PCR) ............................................................................. 43 2.2.3.4 Gel electrophoresis .................................................................................................... 43 2.2.3.5 Quantitative real‐time PCR (qRT‐PCR) ....................................................................... 43 2.2.3.6 Dual‐RNA Seq............................................................................................................. 44 2.2.4 Protein biochemical methods .......................................................................................... 45 2.2.4.1 SDS‐PAGE ................................................................................................................... 45 2.2.4.2 Western blot .............................................................................................................. 46 4 2.2.5 Statistical analysis ............................................................................................................. 46 3 Results .................................................................................................................................. 47 3.1 Phagosomal escape of MSSA 6850 and CA‐MRSA strains LAC and MW2 ............................. 47 3.1.1 Phagosomal escape in epithelial and endothelial cell lines ............................................. 47 3.1.2 S. aureus escapes 2.5 h post infection ............................................................................. 49 3.2 S. aureus phagosomal escape is a synergistic process involving multiple toxins .................. 50 3.2.1 α‐toxin, β‐toxin, PIPLC and PVL not contribute to phagosomal escape in non‐ professional phagocytes ................................................................................................... 50 3.2.2 PSMα but not PSMβ or δ‐toxin (PSMγ) is required for phagosomal escape ................... 52 3.2.3 Expression changes of psmα, psmβ and agrA during infection ....................................... 56 3.2.4 Phagosomal escape in osteoblasts ................................................................................... 58 3.2.5 PSMα is not sufficient for phagosomal escape ................................................................ 59 3.2.6 LukAB and PSMα are required for phagosomal escape ................................................... 59 3.2.6.1 cd11b expression during S. aureus infection ............................................................. 61 3.2.6.2 fpr2 expression during S. aureus infection ................................................................ 62 3.2.7 Phagosomal escape in macrophages