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S. Aureus Regulation of Virulence Factors

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S. Aureus Regulation of Virulence Factors The interaction of Staphylococcus aureus with macrophages Jamil Adib Jubrail Department of Infection and Immunity, University of Sheffield Medical School, UK Thesis submitted for the degree of Doctor of Philosophy, April 2014 Table of Contents Table of figures ...................................................................................................................................... vi Table of tables ...................................................................................................................................... viii Table of equations................................................................................................................................ viii Abbreviations .......................................................................................................................................... x Acknowledgements ............................................................................................................................ xxiii Declaration .......................................................................................................................................... xxv Abstract .............................................................................................................................................. xxvi Chapter 1 Introduction ..........................................................................................................................1 1.1. Staphylococcus aureus ................................................................................................................1 1.1.1. Brief history ...........................................................................................................................1 1.1.2. Summary of clinical manifestations ......................................................................................2 1.1.3. Antibiotic resistance ..............................................................................................................2 1.1.4. Genome sequence ...................................................................................................................5 1.1.5. Staphylococcus aureus virulence factors ...............................................................................6 1.1.5.1. Sortase A ............................................................................................................................6 1.1.5.2. Fibronectin binding proteins.............................................................................................8 1.1.5.5. Lipoteichoic acid .............................................................................................................. 10 1.1.5.6. Alpha toxin ...................................................................................................................... 10 1.1.5.7. Panton-Valentine Leukocidin ......................................................................................... 11 1.1.6. S. aureus regulation of virulence factors ........................................................................ 12 1.2. The innate immune system .................................................................................................. 18 1.2.1. The epithelial barrier ...................................................................................................... 18 1.2.2. Neutrophils ...................................................................................................................... 18 1.2.3. Eosinophils ....................................................................................................................... 20 1.2.4. Monocytes ........................................................................................................................ 21 1.2.5. Natural killer cells ........................................................................................................... 21 1.2.6. Macrophages.................................................................................................................... 21 1.2.6.1. Overview .......................................................................................................................... 21 1.2.6.4. Macrophage classification ............................................................................................... 25 1.2.6.5. Macrophage receptors ..................................................................................................... 29 1.2.6.5.1. Toll like receptors ................................................................................................... 29 1.2.6.5.2. Nod Like Receptor 2 ............................................................................................... 31 1.2.6.5.3. Cluster of differentiation 14 ................................................................................... 31 1.2.6.5.4. Macrophage Fc gamma receptors .......................................................................... 32 1.2.6.5.5. Macrophage complement receptors ....................................................................... 32 1.2.6.5.6. Scavenger receptors ................................................................................................ 33 1.3. Staphylococcus aureus evasion of the innate immune system ............................................ 46 1.3.1. Overview .......................................................................................................................... 46 ii 1.3.2. Chemotaxis inhibitory protein of Staphylococcus aureus and staphylococcal complement inhibitor ................................................................................................................... 46 1.3.3. Superantigen like protein 3 ............................................................................................. 47 1.3.4. Adenosine and staphyloxanthin ...................................................................................... 47 1.3.5. Catalase and superoxide dismutase ................................................................................ 48 1.4. Mathematical modelling of host: pathogen interactions .................................................... 49 1.4.1. Why use mathematical models? ...................................................................................... 49 1.4.2. Mathematical modelling methods ................................................................................... 51 1.4.2.1. Agent based models ......................................................................................................... 51 1.4.3. The Human Immunodeficiency Virus-1 mathematical model story ............................. 53 1.4.4. Bacterial mathematical models ....................................................................................... 58 1.5. Hypothesis, aims and objectives .......................................................................................... 59 Chapter 2 Materials and Methods .......................................................................................................61 2.1. Materials .............................................................................................................................. 61 2.2. Cell line maintenance........................................................................................................... 61 2.2.1. THP-1 cell maintenance .................................................................................................. 61 2.2.2. THP-1 cell differentiation ............................................................................................... 62 2.3. Monocyte derived macrophage isolation, differentiation and maintenance...................... 63 2.4. Bacterial preparation and maintenance ............................................................................. 63 2.4.1. Bacterial growth .............................................................................................................. 63 2.4.2. Preparation of nitrocellulose bead stocks ....................................................................... 63 2.4.3. Newman-green fluorescent protein transduction ........................................................... 65 2.5. Bacterial infection of cell cultures ....................................................................................... 65 2.5.1. Monocyte derived macrophage infection ........................................................................ 66 2.5.2. Infections with cytochalasin D ........................................................................................ 66 2.6. Determination of viable intracellular bacteria using a lysostaphin protection assay........ 66 2.7. Lysostaphin pulse chase killing assay ................................................................................. 67 2.7.1. Interferon gamma pulse chase killing assay ................................................................... 67 2.7.2. Trolox infections .............................................................................................................. 68 2.8. Microscopy ........................................................................................................................... 68 2.8.1. 4’6, diamidino-2-phenylindole and fluorescein isothiocyanate staining ........................ 68 2.8.2. Lysosomal associated membrane protein 1/2 staining ................................................... 69 2.8.3. Microscopic analysis of apoptosis ..................................................................................
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