The interaction of Staphylococcus aureus with macrophages Jamil Adib Jubrail

242 Pages·2014·3.58 MB·English

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
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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 ................................................................................... 69
2.8.4. Interferon gamma staining.............................................................................................. 69
2.8.5. pH rhodamine staining of intracellular bacteria ............................................................ 71
2.8.6. Live microscopy ............................................................................................................... 71
2.9. Bacterial co-infections ......................................................................................................... 71
2.9.1. Newman and Kanamycin resistant infection .................................................................. 71
2.10. Flow cytometry .................................................................................................................... 72
2.10.1. Live and dead flow cytometry ......................................................................................... 72
2.11. Mathematical modelling using R© ....................................................................................... 72
2.12. Statistics ............................................................................................................................... 73
Chapter 3 Macrophage control of Staphylococcus aureus infection ...................................................74
3.1. Introduction ......................................................................................................................... 74
3.2. Results .................................................................................................................................. 76
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3.2.1. Lysostaphin efficiently reduces extracellular Staphylococcus aureus ............................ 76
3.2.2. Dose dependent loss in the control of extracellular staphylococcal infection by
macrophages ................................................................................................................................. 78
3.2.3. Intracellular Staphylococcus aureus accumulate over time in macrophages ................ 80
3.2.4. S. aureus phagocytosis by macrophages is saturatable .................................................. 83
3.2.5. Macrophages demonstrate a time and dose dependent loss in the intracellular killing
of S. aureus ................................................................................................................................... 86
3.2.6. Interferon gamma only modestly enhances intracellular killing of S. aureus ............... 92
3.2.7. Macrophages still continue to phagocytose bacteria even after their capacity for
intracellular killing becomes exhausted ...................................................................................... 95
3.2.8. Macrophage inability to completely kill intracellular S. aureus is not merely the result
of exposure to high multiplicity of infection ................................................................................ 95
3.2.9. Macrophage inability to completely kill intracellular S. aureus is not cell line specific97
3.2.10. Macrophage inability to completely kill intracellular S. aureus is not a Newman
dependent phenotype ................................................................................................................... 97
3.2.11. Intracellular viable bacteria persist in macrophages for up to 40 hours post infection
..............................................................................................................................................100
3.3. Discussion ........................................................................................................................... 103
Chapter 4 A mathematical model to describe the interaction of Staphylococcus aureus with the
macrophage ......................................................................................................................................... 111
4.1. Introduction ....................................................................................................................... 111
4.2. The Model .......................................................................................................................... 114
4.2.1. Model structure ............................................................................................................. 114
4.2.2. Macrophage and bacterial populations ........................................................................ 114
4.2.3. Extracellular model equations and assumptions .......................................................... 116
4.2.4. Macrophage equations .................................................................................................. 117
4.2.5. Bacterial equations ........................................................................................................ 117
4.2.6. The initial interaction of S. aureus with macrophages ................................................. 119
4.3. Results ................................................................................................................................ 120
4.3.1. The effect of varying the extracellular bacterial replication rate ................................ 121
4.3.2. Modelling later events in the macrophage: S. aureus interaction ................................ 121
4.3.3. Modelling the intracellular phase of infection .............................................................. 126
4.4. Discussion ........................................................................................................................... 130
Chapter 5 Exploring the persistence of Staphylococcus aureus in macrophages ............................. 136
5.1. Introduction ....................................................................................................................... 136
5.2. Results ................................................................................................................................ 139
5.2.1. Macrophages phagocytose Staphylococcus aureus into lysosome associated membrane
protein 1 positive organelles ...................................................................................................... 139
5.2.2. Intracellular Staphylococcus aureus persist within lysosome associated membrane
protein 1 and 2 organelles .......................................................................................................... 142
5.2.3. The majority of intracellular S. aureus do not traffick to acidic compartments ........ 146
5.2.4. The failure to traffick to an intracellular compartment of high pH is S. aureus specific
..............................................................................................................................................147
5.2.5. Host mediated macrophage apoptosis is not engaged during S. aureus infection ....... 151
5.2.6. Macrophages retain viability for extended periods with intracellular S. aureus ........ 155
5.2.7. The increase in intracellular bacteria in macrophages over extended periods requires
repeated waves of cell lysis and re-uptake of released bacteria................................................ 159
5.2.8. The percentage of non-viable bacteria decreases with increasing dose ....................... 163
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5.2.9. Intracellular bacterial accumulation over the initial period of bacterial challenge is not
the result of intracellular replication ......................................................................................... 166
5.3. Discussion ........................................................................................................................... 171
Chapter 6 Discussion .......................................................................................................................... 176
6.1. Major findings ................................................................................................................... 176
6.2. S. aureus as an intracellular pathogen .............................................................................. 176
6.3. Mathematical models of host: pathogen interactions ....................................................... 180
6.4. Future directions ............................................................................................................... 183
6.4.1. Characterisation of the compartment of intracellular persistence .............................. 183
6.4.2. The mechanism of macrophage death by intracellular S. aureus ................................ 183
6.5. Conclusion .......................................................................................................................... 183
Chapter 7 References ......................................................................................................................... 185
Appendix 1 Script for extracellular mathematical model ................................................................. 211
Appendix 2 Script for intracellular mathematical model ................................................................. 212
Conference abstracts presented ......................................................................................................... 213

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Table of figures

Figure 1.1: The sequence of events in a typical S. aureus infection. ............................................ 3
Figure 1.2: The structure of S. aureus. ......................................................................................... 7
Figure 1.3: A summary of the Agr system in S. aureus controlling virulence factor expression.
................................................................................................................................................... 15
Figure 1.4: An overview of the airway epithelium. .................................................................... 19
Figure 1.5: A summary of macrophage development from a universal progenitor cell. ............. 26
Figure 1.6: The product of z versus z ...................................................................................... 56
L U.
Figure 3.1: Extracellular S. aureus colony forming unit’s decreases with increasing
lysostaphin/gentamicin concentration. ....................................................................................... 77
Figure 3.2: Macrophages ability to control extracellular S. aureus replication is lost with
increasing multiplicity of infection. ........................................................................................... 79
Figure 3.3: Macrophages accumulate intracellular viable S. aureus at all multiplicities of
infection. .................................................................................................................................... 81
Figure 3.4: The numbers of extracellular adherent bacteria and internalised bacteria with
macrophages. ............................................................................................................................. 82
Figure 3.5: The percentage of macrophages ingesting bacteria. ................................................. 84
Figure 3.6: Macrophages phagocytose S. aureus at varying levels. ........................................... 85
Figure 3.7: Macrophage phagocytosis of S. aureus is saturatable. ............................................. 87
Figure 3.8: Macrophage killing of intracellular S .aureus occurs predominantly in the first 30
minutes after ingestion. .............................................................................................................. 89
Figure 3.9: Macrophages exhaust their intracellular killing abilities over time. ......................... 90
Figure 3.10: Intracellular bacteria are not killed by 16 hours post infection............................... 91
Figure 3.11: Absolute numbers of intracellular bacteria killed and percentage intracellular
bacteria killed in the first 30 minutes. ........................................................................................ 93
Figure 3.12: Interferon gamma stimulation only modestly enhances intracellular killing of S.
aureus. ....................................................................................................................................... 94
Figure 3.13: Macrophages phagocytose S. aureus when intracellular killing is exhausted. ....... 96
Figure 3.14: Intracellular bacteria persist at low MOIs. ............................................................. 98
Figure 3.15: Intracellular bacteria persist in MDMs. .................................................................. 99
Figure 3.16: SH1000 persists intracellularly. ........................................................................... 101
Figure 3.17: Intracellular bacteria persist for up to 40 hours post infection. ............................ 102
Figure 4.1: Summary of the interaction of macrophages with S.aureus during an extracellular
infection. .................................................................................................................................. 115
Figure 4.2: Extracellular model outputs. .................................................................................. 122
Figure 4.3: Increasing bacterial dose compromises macrophage control of bacterial replication.
................................................................................................................................................. 123
Figure 4.4: The effect of varying ‘r’ on Se and Si bacteria....................................................... 124
Figure 4.5: Two phase intracellular killing dynamics. ............................................................. 128
Figure 4.6: Intracellular model outputs. ................................................................................... 129
Figure 5.1: The majority of intracellular bacteria colocalise with LAMP-1. ............................ 140
Figure 5.2: Macrophages phagocytose S.aureus into LAMP-1 positive organelles. ................. 141
Figure 5.3: The majority of intracellular bacteria persist in a LAMP-1 positive compartment. 143
Figure 5.4: The majority of intracellular bacteria persist in a LAMP-2 positive compartment. 144
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Figure 5.5: The majority of intracellular bacteria persist in a LAMP-1/2 positive compartment.
................................................................................................................................................. 145
Figure 5.6: pH rhodamine labelled S. aureus fluoresces maximally at pH of 6 or below. ...... 148
Figure 5.7: The majority of S. aureus do not localise in a compartment of low pH. ................ 149
Figure 5.8: Failure to localise in a compartment of low pH is specific to S. aureus. ................ 150
Figure 5.9: Macrophage associated apoptosis is not engaged during S.aureus infection. ......... 152
Figure 5.10: Failure of macrophages to undergo apoptosis is not strain dependent. ................. 153
Figure 5.11: Representative images of macrophage apoptosis with S. aureus MOI 5. ............. 154
Figure 5.12: Without inhibition of extracellular replication, intracellular bacterial numbers
increase. ................................................................................................................................... 157
Figure 5.13: Macrophage viability gradually declines over time in the presence of S. aureus. 158
Figure 5.14: Representative images of macrophage lysis by S.aureus from video time lapse
microscopy. .............................................................................................................................. 160
Figure 5.15: The increase in intracellular viable bacteria is not the result of intracellular
replication. ............................................................................................................................... 162
Figure 5.16: Cytochalasin D does not affect macrophage viability. ......................................... 164
Figure 5.17: Macrophages accumulate viable intracellular S. aureus with increasing dose. .... 167
Figure 5.18: Intracellular replication is not apparent with S. aureus. ....................................... 169
Figure 5.19: Intracellular replication is not apparent with S. aureus as shown by video time
lapse microscopy. ..................................................................................................................... 170

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Table of tables

Table 1.1: List of several virulence factors produced by S. aureus. ........................................... 14
Table 1.2: Immune evasion molecules produced by S .aureus ................................................... 50
Table 2.1: List of bacterial strains used in this study. ................................................................. 64
Table 2.2: List of antibodies used in this study. ......................................................................... 70
Table 5.1: Macrophages have a finite capacity for intracellular bacterial killing which is
overwhelmed by increasing numbers of bacteria. .................................................................... 165

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Table of equations
Equations 4.1: Equations describing the extracellular model. .................................................. 118
Equations 4.2: Intracellular model equations. .......................................................................... 127

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