Unveiling phenotypic differences among the genetically conserved Mycobacterium tuberculosis complex: Characterization of proteomes and post-translational modifications Alemayehu Godana Birhanu Thesis for the degree of Philosophiae Doctor (PhD) Institute of Clinical Medicine Department of Microbiology University of Oslo January 2019 Acknowledgements I want to express my gratitude to my supervisor Professor Tone Tønjum for letting me be part of her research group and for creating such a stimulating work environment. I am very grateful for her scientific inputs, encouragement and support throughout the whole study period. I would also like to thank my co-supervisor Dr. Markos Abebe for his role in establishing the collaboration and his valuable inputs to my papers and thesis. I wish to thank to all the co-authors for their invaluable contributions in my papers. My gratitude also goes to all the members of the Genome Dynamics group for being nice to me and for creating an excellent working environment. My sincere thanks to Dr. Solomon Abebe, Dr. Getachew Tesfaye and Dr. Ephrem Debebe for their feedback on the dissertation. It is a pleasure to thank my friends Mequannent, Meskerem, Addis and Hiwote for their nice friendship and making my stay in Oslo enjoyable. I would like to thank the Norwegian State Educational Loan Fund (Lånekassen) for the financial support to pursue my study. I am grateful to the Pasteur Legacy and EMBO for supporting my study. To my many friends and family, you should know that your support and encouragement worth more than I can express on paper. Finally, my deep and sincere gratitude to my wife, Beselam Tadesse, for her continuous and unparalleled love, unconditional support, understanding and never ending patience. To my son Haniel and daughter Yohanna, you have made me stronger, better and more fulfilled than I could have ever imagined. Alemayehu Godana Birhanu Oslo, January 2019 i ii TABLE OF CONTENTS LIST OF PAPERS 1 ABBREVIATIONS 3 SUMMARY 5 1. INTRODUCTION 7 1.1. The Mycobacterium tuberculosis complex 7 1.2. Epidemiology of tuberculosis 7 1.3. Genetic diversity and ancestral sequence inference of the MTBC species 8 1.4. Clinical consequences of strain variation in the MTBC 10 1.5. Pathophysiology of tuberculosis 11 1.5.1. Natural course of tuberculosis infection 11 1.5.2. Innate immune responses to tuberculosis 12 1.5.3. Adaptive immune responses to tuberculosis 14 1.6. The unique mycobacterial cell envelope 15 1.7. Virulence and pathogenicity in the MTBC 16 1.7.1. Defining virulence and pathogenicity in the MTBC 16 1.7.2. Virulence factors in the MTBC 16 1.8. MTBC genome characteristics 19 1.9. Mass spectrometry-based proteomics 20 1.9.1. Quantitative proteomics 23 1.9.2. Proteomic data analysis 23 1.10. MTBC proteomics 25 1.11. Post-translational modifications (PTMs) in the MTBC pathogenesis 26 1.11.1. Protein acetylation in the MTBC 27 1.11.2. Protein glycosylation in the MTBC 28 1.12. Comparative proteomics to characterize MTBC virulence factors 29 1.13. Conceptual framework of the study 32 2. AIM OF THE STUDY 33 3. SUMMARY OF PAPERS 34 4. GENERAL DISCUSSION 36 4.1. Comparative expression proteomics 37 4.1.1. Mapping the MTBC proteome by discovery mass spectrometry 37 4.1.2. Differential abundance of transport proteins 37 4.1.3. Differential abundance of proteins involved in cell wall and lipid biosynthesis 38 iii 4.1.4. Differential abundance of proteins involved in bioenergetics 38 4.2. Protein acetylation in the MTBC 39 4.2.1. First combined Nε- and O-acetylome map of the MTBC 39 4.2.2. Regulatory role of Nε- and O-acetylation 40 4.2.3. Gene Ontology analysis of acetylated proteins 41 4.2.4. Acetylated MTBC proteins with roles in antimicrobial resistance (AMR) 41 4.2.5. Quantitative acetylome analysis between MTBC lineage 7 and H37Rv strains 43 4.3. Protein glycosylation in the MTBC 44 4.3.1. MTBC clinical strains exhibit both O- and N-glycosylation 44 4.3.2. Gene Ontology analysis of glycoproteins 46 4.3.3. Glycoproteins involved in pathogen-host interaction 48 4.3.4. Glycoproteins involved in MTBC cell envelope biogenesis 49 4.3.5. Other clinically important glycoproteins identified 50 4.3.6. Quantitative glycoproteomic analysis in the MTBC 51 4.4. Methodological considerations 53 5. LIMITATIONS OF THE STUDY 55 6. CONCLUSIONS AND FUTURE PERSPECTIVES 56 REFERENCES 58 SUPPORTING INFORMATION APPENDIX: PAPERS I-III iv LIST OF PAPERS This dissertation is based on the following papers, which will be referred to by their Roman numbers. I. Yimer SA, Birhanu AG, Kalayou S, Riaz T, Zegeye ED, Beyene GT, Holm-Hansen C, Norheim G, Abebe M, Aseffa A, Tønjum T. Comparative Proteomic Analysis of Mycobacterium tuberculosis Lineage 7 and Lineage 4 Strains Reveals Differentially Abundant Proteins Linked to Slow Growth and Virulence. Frontiers in Microbiology. 2017;8. II. Birhanu AG, Yimer SA, Holm-Hansen C, Norheim G, Aseffa A, Abebe M, Tønjum T. Nε- and O-Acetylation in Mycobacterium tuberculosis lineage 7 and lineage 4 Strains: Proteins Involved in Bioenergetics, Virulence, and Antimicrobial Resistance Are Acetylated. Journal of proteome research. 2017 Oct 4;16(11):4045-59. III. Birhanu AG, Yimer SA, Kalayou S, Riaz T, Zegeye ED, Holm-Hansen C, Norheim G, Aseffa A, Abebe M, Tønjum T. Ample glycosylation in membrane and cell envelope proteins may explain the phenotypic diversity and virulence in the Mycobacterium tuberculosis complex. Scientific Reports 2019, 9(1):2927. 1 2 ABBREVIATIONS AMR antimicrobial resistance BCG bacilli Calmette-Guérin strain CR complement receptors CTLs cytotoxic CD8+ T lymphocytes DC dendritic cell DC-SIGN DC-specific intercellular adhesion molecule (ICAM)-3-grabbing non- integrin DR direct repeat GlcNAc N-acetylglucosamine IFN-γ interferon-gamma iNOS inducible nitric oxide synthase IS insertion sequence IS6110-RFLP insertion sequence (IS) 6110 restriction fragment length polymorphism LC-MS/MS liquid chromatography-tandem mass spectrometry LAM lipoarabinomannan LM lipomannan mAGP mycolyl arabinogalactan-peptidoglycan ManLAM mannose-capped lipoarabinomannan Mce mammalian cell entry protein MDR multidrug-resistance MHC major histocompatibility complex MIRU-VNTR mycobacterial interspersed repetitive-unit-variable-number tandem repeat MR mannose receptor MØ macrophage Mtb Mycobacterium tuberculosis MTBC Mycobacterium tuberculosis complex MurNAc N-acylated muramic acid NK natural killer NLRs nucleotide binding and oligomerization domain (NOD-) like receptors NOD nucleotide binding and oligomerization domain NOX2 NADPH oxidase PAMPs pathogen-associated molecular patterns PDIM phthiocerol dimycocerosate 3 PG peptidoglycan PGL phenolglycolipid PIM phosphatidyl-myo-inositol mannosides PRRs pattern recognition receptors PTMs post-translational modifications RD region of difference ROI reactive oxygen intermediates RNI reactive nitrogen intermediates SNP single nucleotide polymorphism TB tuberculosis TDM trehalose-6, 6-dimycolate (cord factor) TLRs toll-like receptors TNF-α tumour necrosis factor-alpha WGS whole genome sequencing XDR extensively drug-resistance 4 SUMMARY The Mycobacterium tuberculosis complex (MTBC) is the causative agent of tuberculosis (TB), one of the world's deadliest communicable diseases with 10 million new cases and 1.6 million deaths in 2017. Despite the high degree of genomic conservation among members of the MTBC, TB infections exhibit vast discrepancies in clinical outcome and epidemiological behaviour. Bacterial factors that contribute to these phenotypic variability remain elusive and cannot be explained by MTBC genomics alone. We hypothesize that qualitative and quantitative differences in the abundance of proteins and post-translational modifications (PTMs) may explain the phenotypic diversity across members of the MTBC. This study analyzed the proteomic and PTM (acetylation and glycosylation) profiles among members of the MTBC. These two PTMs are reported to be important determinants of MTBC virulence and pathogenicity. Shotgun-based proteomics yielded the identification of 2867 MTBC proteins. Quantitative proteomic analysis of the MTBC reference strain H37Rv and slow-growing lineage 7 strains demonstrated a differential abundance of proteins involved in virulence growth and bioenergetics. The acetylome analysis identified 2490 class-I acetylation sites on 953 proteins. The MTBC proteins found to be acetylated are involved in core metabolic processes, bioenergetics, virulence, and drug resistance. Notably, O-acetylation of MTBC was described for the first time. Quantitative PTM analysis revealed reduced acetylation on 97.5% of the differentially acetylated virulence factors in MTBC lineage 7 strains as compared to H37Rv. The glycoproteomic analysis of clinical MTBC strains representing lineages 3, 4, 5 and 7 identified 2944 glycosylation events on 1325 proteins. These proteins are involved in MTBC cell envelope biogenesis, pathogen-host interaction, membrane transport, and pathogenesis. The study also provides the first report on N- linked glycosylation in MTBC and in Gram-positive bacteria. Quantitative analysis revealed differential glycosylation of 67 proteins involved in MTBC fitness and survival. Identification of glycoproteins and their function contributes to a better understanding of the pathogenesis and survival strategies adopted by MTBC, which is fundamental to diseases management. The data represents the highest number of acetylated and glycosylated proteins in MTBC recorded to date. The presence of significant differences in the proteome