ROLE OF ACANTHAMOEBA SPP. IN THE ENVIRONMENTAL SURVIVAL OF LISTERIA MONOCYTOGENES Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by Yakubu Nale BSc, MSc (ABU Zaria) Department of Infection, Immunity and Inflammation University of Leicester August 2011 ABSTRACT Listeria monocytogenes causes a potentially deadly disease of man and is a major source of contamination in food industry. The mechanism of survival and persistence of L. monocytogenes in the environment is not fully known. The present study investigates the possible role of Acanthamoebae in the survival and persistence of L. monocytogenes in the environment. This was achieved through experiments that brings together the two organisms in a co-culture and then examined ability of bacteria to survive in the presence of amoeba, inside amoeba trophozoites and in their cysts. The effects of intracellular survival on L. monocytogenes’ morphology, ability to form biofilms and respond to biocides inside and outside the cysts were also examined. In summary, L. monocytogenes Scott A was found to survive and grow in Acanthamoeba over 72 h. In addition, exposure of bacteria to manganese enhanced intracellular growth and survival of L. monocytogenes within Acanthamoeba. While L. monocytogenes Scott A survived and replicated in A. castellanii, it barely survived in A. polyphaga and never survived in A. culbertsoni. None of the other strains of L. monocytogenes tested were able to survive in Acanthamoeba. Autophagy, which was previously shown to aid survival of L. monocytogenes in macrophages, was also found contribute to survival within Acanthamoeba. In addition to surviving within A. castellanii trophozoites, L. monocytogenes Scott A also survived encystment of the host amoeba. L. monocytogenes sequestered in cysts were protected from high level of chlorine that is lethal to free bacteria. In addition, L. monocytogenes recovered from cysts were predominantly filamentous and demonstrated enhanced ability to form biofilm and also exhibited increased resistance to a disinfectant and some antibiotics that are normally used in treatment of listerial infections. The observations suggest that A.castellanii could potentially contribute to the survival, dissemination, and persistence of bacteria in the environment. 2 ACKNOWLEDGEMENTS I give thanks to God almighty through Jesus Christ my redeemer, who saw me through this PhD research. I am grateful to the management of University of Leicester for given me the opportunity to study in this great university and for their financial assistance that enabled me complete this programme. I also thank my home university, Ahmadu Bello University Zaria for supporting me throughout the period of my research. I would like to thank my two supervisors Professor Peter Andrew and Dr Simon Kilvington for their supervision and advice in the course of my research and write up of my thesis. I thank Stefan Hyman and Natalie Allcock for processing my TEM samples. In addition, I also thank my wife Janet for standing by me through thick and thin and unrelenting in her support for me. My special thanks to my daughter Thelma and Ishina my son who have been a source of joy to me. I am greatly indebted to my older brother, Yusuf Nale for his sacrifice by giving me one of his kidneys thus, enabling me to live and do this PhD research. I am grateful to Clare Taylor, the Head of Welfare Department for her advice, and motherly care which gave me lots of impetus to forge on despite my predicaments. I would like to thank the pastor and all the members of the Knighton Evangelical Free Church, Leicester for their love, care, prayers and support during my research. I thank my colleagues in the laboratory, James Lonnen, Wayne Heaselgrave and Arine Ahmad for making life bearable for me and also for their numerous contributions that greatly enhanced my work. Other friends too numerous to mention, who supported me in one way or the other are greatly appreciated. 3 ABBREVIATIONS AGE- Amoebic granulomatous encephalitis ANOVA- Analysis of variance BSA- Bovine serum albumin BHI-Brain heart infusion Caco-2- Human colonic carcinoma cell line CCV- Clathrin-coated vesicle CFU- colony forming unit CM- conditioned medium CNS- central nervous system DMSO- Dimethyl sulphoxide DNA- Deoxyribonucleic acid DPD- N-N-Diethyl-P-phenylenediamine DPI- Diphenyleneiodonium ER- endoplasmic reticulum EV- endocytic vesicle FBS- Foetal bovine serum FLA- free-living amoeba hly- haemolysin H2O2- Hydrogen peroxide Hpt- Hexose phosphate transporter IFN-- Gamma interferon IL- Interleukins InlA- Internalins A InlB- Internalins B 4 kDa- Kilodalton LE- Late endosome LLO – Listeriolysin O LPS- Lipopolysaccharides K2HPO4- Dipotassium hydrogen phosphate MAC- Minimum amoebicidal concentration 3-MA- 3-Methyladenine MIC- Minimum inhibitory concentration MM- maintenance medium MnCl.4H2O- Manganese II chloride tetrahydrate MnSOD- Manganese superoxide dismutase MOI- Multiplicity of infection NADPH- Nicotinamide adenine dinucleotide hydrogen phosphate Na2HPO4- Disodium hydrogen phosphate Nf-kB- Nuclear factor kappa NOD- Nuclear oligodimerization domain - O2 -Superoxide OD- optical density OH - Hydroxyl radical P60- 60-Kilodalton protein PBS- Phosphate buffer saline PC-PLC- Phosphatidylcholine-specific phospholipase C PI-PLC- Phosphatidylinositol-specific phospholipase C PLC- Phospholipase C PrfA- Positive regulatory factor A 5 RER- Rough endoplasmic reticulum RM- Repeated measures RNA- Ribonucleic acid RNS- Reactive nitrogen species ROS- Reactive oxygen species rpm- Revolutions per minute SDS- Sodium dodecyl sulphate SE- sorting endosome SOD- Superoxide dismutase RS- ¼ strength Ringer‘s solution TEM- Transmission electron microscope TLR- Toll-like receptors TNF- Tumor necrotic factor TSA- Tryptone soya agar 6 CONTENTS 1 Chapter 1. General Introduction ............................................................................. 15 1.1 Free-living amoebae ......................................................................................... 15 1.1.1 Acanthamoeba .......................................................................................... 15 1.1.2 Biology and life-cycle of Acanthamoeba ................................................. 16 1.1.3 Ecology ..................................................................................................... 18 1.1.4 Feeding ...................................................................................................... 19 1.1.5 Phagocytosis ............................................................................................. 20 1.1.6 Phagosome maturation .............................................................................. 21 1.1.7 Respiratory burst of phagocytosis ............................................................. 23 1.1.8 Diseases caused by Acanthamoeba ........................................................... 25 1.2 Listeria monocytogenes ................................................................................... 25 1.2.1 Historical developments ........................................................................... 26 1.2.2 Listeriosis .................................................................................................. 27 1.2.3 Infection of mammalian cells ................................................................... 30 1.2.4 Listeria and biofilm formation .................................................................. 38 1.3 Autophagy ........................................................................................................ 39 1.3.1 Autophagy in mammalian cell infections ................................................. 40 1.4 Free-living amoebae as host for intracellular pathogens .................................. 43 1.4.1 Interactions of L. monocytogenes with Acanthamoeba ............................ 45 1.5 The aims of this study. ..................................................................................... 47 2 Chapter 2. Materials and Methods .......................................................................... 49 2.1 Media and reagents .......................................................................................... 49 2.2 Bacterial strains, assay and storage condition .................................................. 49 2.2.1 Determination of bacterial viability .......................................................... 52 2.3 Amoebae culture and culture condition ........................................................... 54 2.3.1 Determination of amoebae viability ......................................................... 55 2.4 Cryopreservation of cultures ............................................................................ 56 7 2.5 Screening of detergents for cell lyses ............................................................... 56 2.6 Co-culture experiments .................................................................................... 57 2.7 Viability of Listeria in Acanthamoeba-conditioned medium .......................... 58 2.8 Invasion assays ................................................................................................. 58 2.8.1 Uptake of L. monocytogenes by Acanthamoeba ...................................... 59 2.8.2 Light microscopy assay of bacteria uptake ............................................... 59 2.8.3 Intracellular growth kinetics of Listeria within Acanthamoeba ............... 60 2.8.4 Microplate assay of intracellular bacterial survival .................................