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Cell Biology of the Interaction Between Listeria Monocytogenes and Colpoda Spp

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Cell Biology of the Interaction Between Listeria Monocytogenes and Colpoda Spp Cell Biology of the Interaction between Listeria monocytogenes and Colpoda spp. Rethish Raghu Nadhanan, B.Sc. (Hons) (Adelaide) A thesis submitted for the Degree of Doctor of Philosophy School of Molecular and Biomedical Science Faculty of Sciences, The University of Adelaide Adelaide, South Australia, Australia (December, 2012) i Table of Contents Chapter 1: Literature Review .......................................................................................... 1 1.1 Introduction to Listeria monocytogenes ....................................................................... 1 1.2 Listeriosis ..................................................................................................................... 2 1.2.1 Listeriosis in Humans ............................................................................................ 3 1.2.2 Listeriosis in Animals ............................................................................................ 4 1.3 Pathophysiology of L. monocytogenes ........................................................................ 5 1.3.1 Virulence Factors of L. monocytogenes ................................................................ 5 1.3.2 Invasion of Mammalian Cells by L. monocytogenes ............................................ 7 1.4 Is there an Environmental Reservoir for L. monocytogenes? ...................................... 7 1.5 Interactions between Bacteria and Protozoa ................................................................ 8 1.6 Protozoa as Model Organisms for Study of Pathogenesis ......................................... 10 1.7 Interactions between Pathogenic Bacteria and Protozoa ........................................... 11 1.7.1 Interactions between Gram negative Pathogens and Protozoa ............................ 12 1.7.2 Interactions between Gram positive Pathogens and Protozoa ............................ 19 1.8 Summary .................................................................................................................... 23 1.9 Hypotheses and Aims ................................................................................................ 24 1.9.1 Hypothesis 1 ........................................................................................................ 24 1.9.2 Hypothesis 2 ........................................................................................................ 25 1.9.3 Hypothesis 3 ........................................................................................................ 25 1.10 References ................................................................................................................ 26 Chapter 2: Co-culture of Colpoda spp. with planktonic and biofilm bacteria .......... 44 2.1 Abstract ...................................................................................................................... 44 2.2 Introduction ................................................................................................................ 45 2.3 Materials and Methods ............................................................................................... 48 2.3.1 Ciliates and Culture Conditions .......................................................................... 48 2.3.2 Isolation of Colpoda MLS-5 from a Food processing Environment .................. 48 2.3.3 Bacteria and Culture Conditions ......................................................................... 50 2.3.4 Co-culture of Colpoda and Bacteria in Liquid Medium ..................................... 51 2.3.5 Phagocytosis inhibition assay .............................................................................. 51 ii 2.3.6 Biofilm Grazing Experiments ............................................................................. 52 2.3.7 Haemolysis assay ................................................................................................ 53 2.3.8 Scanning Electron Microscopy (SEM) ............................................................... 53 2.3.9 Transmission Electron Microscopy (TEM) ......................................................... 53 2.3.10 Statistical analysis ............................................................................................. 54 2.4 Results ........................................................................................................................ 54 2.4.1 Isolation and identification of a ciliate isolate from a food processing environment ........................................................................................................ 54 2.4.2 Co-culture of Colpoda RR and MLS-5 with bacteria ......................................... 56 2.4.3 Inhibition of Colpoda Phagocytosis prevents Uptake of Bacterial Cells ............ 58 2.4.4 TEM of Co-cultures of Colpoda RR and MLS-5 with Bacteria. ........................ 59 2.4.5 Co-culture of Colpoda RR and activated charcoal-cultured L. monocytogenes DRDC8 ................................................................................................................ 61 2.4.6 Survival of L. monocytogenes DRDC8 within Colpoda RR Cysts ..................... 62 2.4.7 Biofilm grazing by Colpoda RR and MLS-5 ...................................................... 63 2.5 Discussion .................................................................................................................. 66 2.5.1 Colpoda kill Intra-ciliate L. monocytogenes ....................................................... 68 2.5.2 Differential processing of Gram positive and Gram negative Cells ................... 73 2.5.3 L. monocytogenes Cells associated with Colpoda Cysts ..................................... 74 2.5.4 Interaction of Colpoda with Biofilm Cultures of L. monocytogenes .................. 75 2.5.5 Conclusions ......................................................................................................... 76 2.6 Acknowledgements .................................................................................................... 77 2.7 References .................................................................................................................. 78 Chapter 3: Host Cell Processes Required for Inactivation of L. monocytogenes .... 113 3.1 Abstract .................................................................................................................... 113 3.2 Introduction .............................................................................................................. 114 3.3 Experimental Procedures ......................................................................................... 115 3.3.1 Organisms and Culture Conditions ................................................................... 115 3.3.2 Co-culture Assays ............................................................................................. 116 3.3.3 Enumeration of Intracellular Bacteria ............................................................... 116 iii 3.3.4 Inhibitors of Vacuolar Acidification, Phagosome-lysosome Fusion and Proteases ..................................................................................................... 116 3.3.5 Fluorescence microscopy .................................................................................. 118 3.3.6 Transmission electron microscopy (TEM) ........................................................ 118 3.3.7 Statistical Analysis ............................................................................................ 118 3.4 Results ...................................................................................................................... 119 3.4.1 Co-cultures of L. monocytogenes DRDC8 and Colpoda RR ............................ 119 3.4.2 Fluorescence Microscopy of Phagosome-lysosome Fusion and Vacuolar Acidification within Colpoda RR ..................................................................... 119 3.4.3 Inhibition of Phagosome-Lysosome Fusion inhibits killing of DRDC8 ........... 120 3.4.4 Bafilomycin and Monensin inhibit Vacuole Acidification in Colpoda RR ...... 121 3.4.5 Protease Inhibitors limit killing of DRDC8 within Phagolysosomes ............... 122 3.4.6 L-NMMA-induced inhibition of Ciliate Nitric Oxide Production .................... 123 3.4.7 Superoxide Production by Colpoda RR ............................................................ 123 3.5 Discussion ................................................................................................................ 124 3.5.1 Phagosome-lysosome Fusion mediates killing of L. monocytogenes ............... 124 3.5.2 Proteases assist killing of L. monocytogenes .................................................... 127 3.5.3 L. monocytogenes is insensitive to Nitric Oxide ............................................... 128 3.5.4 Superoxides ....................................................................................................... 129 3.5.5 Inhibitor mediated uptake of L. monocytogenes by Colpoda ............................ 130 3.5.6 Conclusions ....................................................................................................... 131 3.6 References ................................................................................................................ 133 Chapter 4: Secretion of L. monocytogenes within Ciliate-derived Faecal Pellets ... 151 4.1 Abstract .................................................................................................................... 151 4.2 Introduction .............................................................................................................
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