Development of highly sensitive tools to investigate the Salmonella Type III Secretion System Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Sibel Westerhausen, geb. Şeker aus Uşak/Türkei Tübingen 2020 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 28.01.2021 Stellvertretender Dekan: Prof. Dr. József Fortágh 1. Berichterstatter: Prof. Ph.D. Samuel Wagner 2. Berichterstatter: Prof. Dr. Ana J. Garcia-Saéz Table of Contents List of abbreviations .............................................................................................................................. IV Deutsche Zusammenfassung ................................................................................................................. VI Abstract ................................................................................................................................................ VII 1 Introduction ..................................................................................................................................... 1 1.1 General protein transport pathways through the inner membrane........................................... 1 1.1.1 Protein degradation system .............................................................................................. 3 1.2 The type III secretion system .................................................................................................. 5 1.2.1 The two-virulence associated type III secretion systems of Salmonella ......................... 6 1.3 SPI-1 T3SS - The molecular syringe ....................................................................................... 8 1.3.1 Assembly of the injectisome ......................................................................................... 10 1.3.2 Energy source of the injectisome .................................................................................. 12 1.3.3 Needle length regulation and substrate specificity switch ............................................. 14 1.3.4 Effectors of the T3SS-1 ................................................................................................. 20 1.4 Tools for investigating T3SS secretion and injection ............................................................ 23 1.4.1 Fractionation based assays ............................................................................................ 24 1.4.2 Enzymatic assays ........................................................................................................... 24 1.4.3 Fluorescence protein-based assays ................................................................................ 26 1.4.4 Bioluminescence and bioluminescence-based assays ................................................... 28 1.5 The development of the engineered luciferase Nanoluc ....................................................... 29 2 Aim of this thesis ........................................................................................................................... 33 3 Material and Methods .................................................................................................................... 35 3.1 Chemical and Materials ......................................................................................................... 35 3.2 Bacterial strains and growth conditions ................................................................................ 35 3.3 Molecular cloning and plasmid construction......................................................................... 35 3.4 Allelic exchange .................................................................................................................... 35 3.5 Protein biochemical and immunological methods................................................................. 36 3.5.1 Western blot-based secretion assay ............................................................................... 36 3.5.2 Spheroplasting ............................................................................................................... 36 3.5.3 SDS PAGE, Western blotting and immunodetection .................................................... 37 3.5.4 MBP-NLuc and MBP-HiBiT purification ..................................................................... 37 3.6 Luciferase based assays ......................................................................................................... 37 3.6.1 Luciferase secretion assays ............................................................................................ 37 3.6.2 NLuc assay for wall-bound protein ............................................................................... 38 3.6.3 Test of NLuc stability .................................................................................................... 38 3.6.4 Kinetic measurements ................................................................................................... 38 I 3.6.5 Luminescence detection of whole cells ......................................................................... 39 3.7 Cell culture ............................................................................................................................... 40 3.7.1 Injection assay and injection kinetics with host cells .................................................... 40 4 Results ........................................................................................................................................... 48 4.1 Development of a quick and highly sensitive assay for the analysis of protein secretion and injection by bacterial type III secretion systems ....................................................................... 48 4.1.1 Translational effector-luciferase fusion proteins provide a quick readout for type III secretion ......................................................................................................................... 48 4.1.2 Proving the sensitivity and suitability of the Nanoluc based secretion assay for kinetic measurements ................................................................................................................ 53 4.1.3 Assessing the SipA-NLuc secretion assay for high troughput screening ...................... 56 4.1.4 PMF-dependence of type III secretion and assessment of effects of different type of inhibitors by SipA-NLuc secretion assay ...................................................................... 61 4.2 Development of NLuc-based host cell injection assays ........................................................... 63 4.3 Development of a periplasmic secretion assay for investigating substrate specificity switching ............................................................................................................................................... 67 4.3.1 Co-translational targeting of MBP fusion proteins ........................................................ 71 4.3.2 Further optimization of co-translational targeting of the MBP fusion proteins ............ 72 4.3.3 Overcoming periplasmic degradation of MBPs fusion proteins.................................... 75 4.3.4 Salmonella periplasmic protease deficient mutants: Tsp targets MBP-fusion constructs in periplasm ................................................................................................................... 82 4.3.5 Biological perspective of the periplasmic secretion assay: investigation of needle deficient mutants provides insights of assembly process .............................................. 85 5 Summary and Discussion .............................................................................................................. 93 5.1 Development of the NLuc based secretion assay into extracellular environment ................. 93 5.2 Development of SipA-NLuc injection assay: secretion into host cell ................................... 95 5.3 Development of a periplasmic secretion assay for investigating substrate specificity switch .. ............................................................................................................................................... 96 5.3.1 MBP is not an optimal fusion partner for the periplasmic secretion assay.................... 98 5.3.2 Tail specific protease deficient mutants would positively affect the periplasmic secretion assay by enhancing the signal ...................................................................... 100 5.3.3 Evaluation of the different NanoGlo-Kits in context of periplasmic secretion assay . 101 5.3.4 Is the secretion of early substrates dependent on various T3SS components ensuring the hierarchical secretion? ................................................................................................. 102 6 Conclusion and outlook ............................................................................................................... 105 7 Literature ..................................................................................................................................... 106 8 Appendix ..................................................................................................................................... 125 8.1 Additional data ...................................................................................................................