Evaluation of bacterial ligands involved in receptor- mediated phagocytosis in Tetrahymena pyriformis Mara Adela Boboc A thesis submitted to Lancaster University in fulfilment of the requirements for the degree MSc by Research Biomedical Sciences January 2020 Declaration This dissertation is entirely my own work and has not been submitted in full or in part for the award of a higher degree at any other educational institution. Word count: 34,595 2 Acknowledgements I would like to thank my academic supervisor Dr Jackie Parry for her continuous support and guidance that led to completion of this project. I would also like to thank Janice Drinkall for all the help that she provided me with in the laboratory and my family, Ami, Ciprian and Luke, for all the support and motivation offered throughout all this time. 3 Table of Contents List of Figures ................................................................................................................................................ 8 List of Tables ............................................................................................................................................... 10 ABSTRACT .................................................................................................................................................... 11 CHAPTER 1: INTRODUCTION ....................................................................................................................... 13 1.1 General introduction to Protists ....................................................................................................... 13 1.2 Phagocytosis ..................................................................................................................................... 15 1.3 Pathogen-Associated Molecular Patterns (PAMPs) .......................................................................... 19 1.3.1 Lipopolysaccharides ................................................................................................................... 19 1.3.2 Peptidoglycan ............................................................................................................................. 21 1.3.3 Lipoteichoic acids (LTAs) ............................................................................................................ 22 1.3.4 Bacterial alteration of PAMPS .................................................................................................... 24 1.3.4.1 Modifications in LPS ............................................................................................................ 24 1.3.4.2 Modifications in peptidoglycan........................................................................................... 25 1.3.4.3 Modifications in Lipoteichoic acids ..................................................................................... 26 1.4 Pattern Recognition Receptors (PRRs) in immune cells.................................................................... 27 1.4.1 C-type lectins .............................................................................................................................. 28 1.4.1.1 Receptors that bind mannose and GlcNAc ......................................................................... 28 1.4.1.2 Receptors that bind GalNAc ................................................................................................ 30 1.4.2 Scavenger receptors (SRs) .......................................................................................................... 31 1.5 Pattern Recognition Receptors (PRRs) in protists ............................................................................. 33 1.5.1 C-type lectins .............................................................................................................................. 33 1.5.1.1 The mannose receptor ........................................................................................................ 34 1.5.1.2 The N-acetylglucosamine (GlcNAc) receptor ...................................................................... 35 1.5.1.3 The N-acetylgalactosamine (GalNAc) receptor ................................................................... 36 1.5.2 Scavenger Receptors .................................................................................................................. 37 1.6 Rationale for the current study and specific objectives ................................................................... 37 CHAPTER 2: MATERIALS AND METHODS .................................................................................................... 40 2.1 Maintenance of organisms and fluorescently-labelled microspheres .............................................. 40 2.1.1 Live heterotrophic bacteria ........................................................................................................ 40 2.1.2 Live autotrophic bacterium (‘Pico’) ............................................................................................ 40 4 2.1.3 Heat-killed-DTAF stained bacteria (‘Dead’)................................................................................ 40 2.1.4 Fluorescently labeled microspheres (‘Beads’) ........................................................................... 41 2.1.5 Tetrahymena pyriformis ............................................................................................................. 41 2.2 Counting cells and beads .................................................................................................................. 43 2.2.1 Counting non-fluorescent bacteria ............................................................................................ 43 2.2.2 Counting fluorescent bacteria and beads .................................................................................. 43 2.2.3 Counting Tetrahymena pyriformis ............................................................................................. 44 2.2.4 Counting prey within T. pyriformis cells ..................................................................................... 44 2.3 Description of a standard feeding experiment ................................................................................. 44 2.4 Specifics of the feeding experiments not involving blockers ............................................................ 45 2.4.1 Ingestion of monocultures ......................................................................................................... 45 2.4.2 Ingestion of mixed prey types .................................................................................................... 45 2.4.3 Effect of S-layer ‘stripping’ on ingestion .................................................................................... 45 2.4.4 Effect of proteolytic ‘shaving’ of surface-exposed proteins on ingestion ................................. 46 2.4.5 Effect of Formaldehyde fixing on ingestion ............................................................................... 46 2.5 Specifics of experiments involving blockers ..................................................................................... 46 2.5.1 Blocking sugar receptors ............................................................................................................ 47 2.5.2 Blocking scavenger receptors .................................................................................................... 47 2.6 Statistics ............................................................................................................................................ 47 CHAPTER 3: RESULTS ................................................................................................................................... 48 3.1 Feeding experiments ......................................................................................................................... 48 3.1.1 General Overview ...................................................................................................................... 48 3.1.2 Processing of Beads and inherent variation in ciliate feeding ................................................... 50 3.1.3 Processing of Bacteria ................................................................................................................ 55 3.1.3.1 Variation between Live strains – monoculture experiments .............................................. 55 3.1.3.2 Live vs Heat-Killed Dead cells .............................................................................................. 58 3.1.3.2.1 Vacuole formation factor (VFF) .................................................................................... 59 3.1.3.2.2 Prey uptake factor(s) (PUF) .......................................................................................... 60 3.1.3.3 Summary ............................................................................................................................. 63 3.1.4 Effect of specific treatments of Live cells on their uptake and vacuole production ................. 64 3.1.4.1 Effect of the removal of the S-layer .................................................................................... 64 3.1.4.2 Effect of formaldehyde fixation and trypsin treatment ...................................................... 65 5 3.2 Receptor blocking experiments ........................................................................................................ 66 3.2.1 Sugar blocking experiments ......................................................................................................