Role of lysX gene from Mycobacterium avium hominissuis in metabolism and host-cell interaction Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat) Submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by GREANA KIRUBAKAR from Chennai, India Berlin, 2019 This work was accomplished between October 2015 and July 2019 at the Robert Koch Institute under the supervision of Dr. Astrid Lewin. 1st Reviewer: Dr. Astrid Lewin 2nd Reviewer: Prof. Dr. Rupert Mutzel Date of Defense: 21.10.2019 Acknowledgement I express my deepest gratitude to my doctoral guide, Dr. Astrid Lewin for giving me the opportunity to work in this prestigious Robert Koch Institute (RKI). Her constant guidance and encouragement has enabled me to successfully pursue this project. I appreciate all her contributions of time, ideas, and funding recommendations to make my PhD. experience productive and stimulating. I would also like to thank Prof. Dr. Lothar Wieler, the President of the Robert Koch-Institute, for providing me permission to carry out my PhD research work. My sincere thanks to my second supervisor, Prof. Dr. Rupert Mutzel, for his insightful comments and valuable suggestions throughout the period of my thesis. I am also very thankful to Elisabeth Kamal, for her excellent technical assistance and taking care of both my experimental and personal issues. I also acknowledge Dr. Toni Aebischer for his valuable advice in my research project and his hard questions which incented me to widen my research from various perspectives. In regards to the Proteomics experiments, I like to thank Dr. Jayaseelan Murugaiyan for collaborating with our institute. The research group of Dr. Uwe Roesler at the Institute for Animal Hygiene and Environmental Health and Dr. Murat Eravci, Dr. Christoph Weise from the Institute of Chemistry and Biochemistry, Free University Berlin have also contributed to the proteomics study. The BIOLOG data analysis which is discussed in this dissertation would not have been possible without the contribution of Dr. Flavia Dematheis at the Institute of Microbiology and Epizootics, Free University Berlin. I appreciate this collaboration, as the metabolic analysis was conducted using the BIOLOG facility available the University lab. For the microscopic examinations, Gudrun Holland and Dr. Christoph Schaudinn, from the ZBS4 division, Advanced Light and Electron microscopy, RKI, Berlin have made significant contributions and the inspirational discussions with Dr. Schaudinn regarding the experiments gave me newer perspectives in the research project. I also appreciate the help of Dr. Hubert Schäfer and Barbara Kropp in the GPL experiments. I am also indebted for his motivational support, local expertise and sharing of knowledge during my research work. I am also grateful to Dr. Volker Rickerts and the animal facility of RKI (MF3 division: Dr. Petra Kirsch, Annette Dietrich and Alice Stern) for enabling me to conduct in vivo experiment using the Galleria mellonella larval infection model. I gratefully acknowledge the funding sources that made my PhD. work possible. I was funded by the Rosa Luxemburg fellowship for my first 3 years and was honored to receive the Georg & Agnes Blumenthal Stiftung and the Robert Winter Stiftung in my completion phase. My time at RKI, Berlin, was made enjoyable in large part due to the many friends and groups that became a part of my life. I am thankful to the FG16 division group members at the RKI, who backed me up and were always ready to help. Especially, the lab members of our research group, Dr. Andrea Sanchini, Suriya Akter, Linus Fiedler, Anna Maria Oschmann and Alexander Lüders made my stay memorable by creating a friendly atmosphere. I truly appreciate the funtimes spent with them during the long working hours and some delightful trips together. My time in Germany was also enriched by the warm affections extended by the elders and fellow friends from the ODMC Church and the Berlin International Community church. Lastly, I would like to thank my family for all their love, prayers and encouragement. For my parents, Kirubakar and Mercy Paulkani who raised me with a love to achieve greater heights in my education and supported me in all my pursuits. For the presence of my sister, Selina Kirubakar here in Berlin for the three of my years here. And most of all I would never forget the grace of God which enabled me to survive through every difficulty and obstacles that I needed to cross in this journey of my life. GREANA KIRUBAKAR Table of Contents 1 Introduction .................................................................................................................. 1 1.1 Genus Mycobacterium ........................................................................................... 2 1.2 Mycobacterium avium hominissuis (MAH) ........................................................... 4 1.3 Pathogenesis of MAH ............................................................................................ 5 1.4 Cell envelope of MAH ........................................................................................... 6 1.5 The lysX gene ......................................................................................................... 7 2 Aim and justification of the study ................................................................................ 9 3 Materials and Methods ............................................................................................... 10 3.1 Bacterial strains and growth conditions ............................................................... 10 3.2 Mass spectrometry - proteomic analysis .............................................................. 11 3.2.1 Mapping of differentially expressed proteins onto metabolic pathways ...... 11 3.3 BIOLOG metabolic phenotype microarray ......................................................... 12 3.4 Bacterial pyruvate quantification ......................................................................... 12 3.5 Electron microscopy ............................................................................................ 13 3.6 Fluorescence microscopy ..................................................................................... 13 3.7 Antibiotic susceptibility test ................................................................................ 14 3.8 Intracellular growth measurement ....................................................................... 14 3.9 Stress resistance tests ........................................................................................... 15 3.10 Cytokine measurement in infected human PBMC ........................................... 16 3.11 Evaluation of macrophage fusion during MAH infection ................................ 16 3.12 In vivo study using Galleria mellonella ........................................................... 17 3.13 Extraction of glycopeptidolipids (GPL) ........................................................... 18 3.14 Measurement of GPL-Antigenic reactivity using ELISA assay ...................... 19 4 Results ........................................................................................................................ 20 4.1 Proteomic analysis of the MAH strains ............................................................... 20 4.2 Metabolic phenotype characterization ................................................................. 24 4.3 Structural examination of MAH strains ............................................................... 26 4.4 Antibiotic susceptibility testing of the MAH strains: .......................................... 29 4.5 Intracellular survival of MAH strains in human blood monocytes ...................... 31 4.6 Effect of H2O2, NO and defensins on the growth and viability of MAH strains . 32 4.7 Cytokine responses of human PBMCs infected with M. avium strains .............. 33 4.8 Multinucleated giant cell formation on macrophage infection with MAH strains 35 4.9 Virulence of MAH in Galleria mellonella larvae ................................................ 37 4.10 MAH GPL expression and its antigenic reactivity........................................... 38 5 Discussion .................................................................................................................. 41 6 Summary .................................................................................................................... 52 7 Zusammenfassung ...................................................................................................... 54 8 References .................................................................................................................. 56 9 Supplementary Material ............................................................................................. 66 9.1 Supplementary Table S1: Complete list of proteins identified in the M. avium strains (W: wild-type, M: lysXmut and C: lysXcomp) by Proteome analysis and comparative statistical differential analysis .................................................................... 66 9.2 Supplementary Table S2a: Pathway analysis via DAVID of differentially regulated genes of M. avium hominissuis lysX mutant in comparison to wild type .... 100 9.2.1 Supplementary Table S2b: Functional enrichment analysis of differentially regulated genes of M. avium hominissuis lysX mutant in comparison to wild type strain 104 using STRING ........................................................................................