INVESTIGATIONS TO IRON LIMITATION IN STREPTOCOCCUS PNEUMONIAE I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Juliane Hoyer geboren am 18.08.1988 in Potsdam Greifswald, den 18.12.2018 Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Dörte Becher 2. Gutachter: Prof. Dr. Jan Maarten van Dijl Tag der Promotion: 25.04.2019 Table of contents TABLE OF CONTENTS Table of contents ...................................................................................................................................................... I Abbreviations ........................................................................................................................................................... V 1. Summary ............................................................................................................................................................. 1 2. Zusammenfassung .......................................................................................................................................... 3 3. Introduction ...................................................................................................................................................... 7 3.1. Streptococcus pneumoniae ................................................................................................................... 7 3.1.1. Historical and general aspects ..................................................................................................... 7 3.1.2. Carriage and diseases ...................................................................................................................... 7 3.1.3. Treatment and prevention ............................................................................................................ 8 3.2. Proteomics ................................................................................................................................................ 10 3.2.1. Mass spectrometry-based proteomic workflow ................................................................ 10 3.2.2. Mass spectrometry-based protein quantification ............................................................. 12 3.3. Iron .............................................................................................................................................................. 14 3.3.1. Role of iron and its acquisition by bacteria .......................................................................... 14 3.3.2. Iron acquisition by pneumococci .............................................................................................. 22 3.3.3. Establishment of in vitro iron starvation ............................................................................... 28 3.4. Objective of the thesis .......................................................................................................................... 30 4. Materials and methods ............................................................................................................................... 31 4.1. Bacterial strain ....................................................................................................................................... 31 4.2. Chemicals .................................................................................................................................................. 31 4.3. Water and media .................................................................................................................................... 33 4.3.1. Water .................................................................................................................................................... 33 4.3.2. Chemically defined medium ....................................................................................................... 33 4.3.3. Stable Isotope Labeling by Amino Acids in Cell Culture - CDM .................................... 34 4.3.4. Todd-Hewitt broth with yeast extract medium .................................................................. 34 4.4. Consumables............................................................................................................................................ 34 I Table of contents 4.5. Instruments ............................................................................................................................................. 35 4.6. Computer software ............................................................................................................................... 36 4.7. Experimental design and cultivation ............................................................................................ 37 4.7.1. Strain maintenance ........................................................................................................................ 37 4.7.2. Cultivation and sampling ............................................................................................................. 37 4.7.3. Cell harvest and disruption ........................................................................................................ 42 4.7.4. Gram-staining ................................................................................................................................... 43 4.7.5. Electron microscopy ...................................................................................................................... 43 4.8. Protein analysis ..................................................................................................................................... 46 4.8.1. Determination of protein concentration ............................................................................... 46 4.8.2. Preparation of heavy labeled internal standard ................................................................ 46 4.8.3. In-solution protein digestion ..................................................................................................... 47 4.8.4. Peptide purification ....................................................................................................................... 47 4.9. Mass spectrometric analyses ........................................................................................................... 48 4.9.1. Liquid chromatography coupled to tandem-mass spectrometry (LC-MS/MS) .... 48 4.9.2. Inductively coupled plasma-mass spectrometry (ICP-MS) ........................................... 49 4.10. Data analyses .......................................................................................................................................... 49 4.10.1. Determination of incorporation rate ...................................................................................... 49 4.10.2. Proteome analysis .......................................................................................................................... 49 5. Results ............................................................................................................................................................... 51 5.1. Prerequisites for proteome analyses ............................................................................................ 51 5.1.1. Modified cultivation workflow .................................................................................................. 51 5.1.2. Generation of Voronoi treemap layout for S. pneumoniae ............................................. 53 5.2. Pneumococcal adaptation to iron limitation in CDM ............................................................. 56 5.2.1. Determination of suitable BIP concentration and BIP toxicity test ........................... 56 5.2.2. Determination of incorporation rate for SILAC quantification .................................... 58 5.2.3. Proteome analysis .......................................................................................................................... 60 5.2.4. Cell morphology .............................................................................................................................. 63 II Table of contents 5.2.5. Iron concentrations of CDM and THY ..................................................................................... 65 5.3. Pneumococcal adaptation to iron limitation in THY ............................................................... 65 5.3.1. Determination of suitable BIP concentration ...................................................................... 65 5.3.2. Proteome analysis ........................................................................................................................... 66 5.4. Comparison of CDM vs. THY ............................................................................................................. 71 5.4.1. Proteomic adaptation to media ................................................................................................. 71 5.4.2. Comparative analysis of cell morphology ............................................................................. 73 6. Discussion ........................................................................................................................................................ 77 6.1. Functional categorization of pneumococcal proteins ............................................................ 77 6.2. Comparison of