Host-Pathogen Interactions in Aspergillus Fumigatus Infection
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NEUTROPHILS, NUTRITIONAL IMMUNITY AND NETS: HOST-PATHOGEN INTERACTIONS IN ASPERGILLUS FUMIGATUS INFECTION by HEATHER L. CLARK Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology CASE WESTERN RESERVE UNIVERSTIY January 2017 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of Heather L. Clark Candidate for the degree of Doctor of Philosophy in Pathology Committee Chair: George Dubyak Committee Members: Clive Hamlin Amy Hise Eric Pearlman Theresa Pizarro Date of Defense: August 22nd, 2016 We also certify that written permission has been obtained for any proprietary material contained therein 2 Table of Contents List of Tables ...................................................................................................... 7 List of Figures ..................................................................................................... 8 Acknowledgements .......................................................................................... 11 List of Abbreviations ........................................................................................ 13 Abstract ............................................................................................................. 17 Chapter 1: Introduction .................................................................................... 19 Fungi and Human Disease .............................................................................. 20 Burden of Fungal Disease ........................................................................... 20 Fungal Keratitis ............................................................................................ 22 Current Anti-Fungal Therapy ....................................................................... 24 Immune Responses to Aspergillus fumigatus infection: A Critical Role for Neutrophils ...................................................................................................... 27 Neutrophil Recruitment ................................................................................ 27 The Neutrophil Anti-microbial Arsenal .......................................................... 29 Neutrophils and Nutritional Immunity ........................................................... 31 S100 Proteins in Nutritional Immunity .......................................................... 33 Neutrophil Extracellular Traps ...................................................................... 36 3 Neutrophil Recognition of Fungi ................................................................... 38 Intracellular Signaling in NETosis ................................................................ 42 NETs in Tissue Damage .............................................................................. 47 Other Immune Responses in Aspergillus Infection .......................................... 48 Aspergillus fumigatus Virulence Determinants ................................................ 49 The Fungal Cell Wall and Immune Evasion ................................................. 50 Antioxidant Mechanisms .............................................................................. 52 Nutrient Acquisition ...................................................................................... 53 Hypothesis and Goals ..................................................................................... 57 Chapter 2: Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection ...................................................................................... 59 Abstract ........................................................................................................... 60 Introduction ..................................................................................................... 61 Materials and Methods .................................................................................... 63 Results ............................................................................................................ 70 Discussion ....................................................................................................... 79 Figures ............................................................................................................ 87 Chapter 3: Atovaquone impairs mitochondrial function and metal homeostasis, and differentially inhibits growth of Aspergillus and Fusarium clinical isolates ............................................................................................... 95 Abstract ........................................................................................................... 96 Introduction ..................................................................................................... 97 4 Materials and Methods .................................................................................... 99 Results .......................................................................................................... 103 Discussion ..................................................................................................... 108 Figures .......................................................................................................... 115 Chapter 4: A Role for Neutrophil Extracellular Traps in Aspergillus fumigatus Infection ........................................................................................ 124 Abstract ......................................................................................................... 125 Introduction ................................................................................................... 127 Materials and Methods .................................................................................. 130 Results .......................................................................................................... 135 Discussion ..................................................................................................... 141 Figures .......................................................................................................... 148 Chapter 5: Data Summary and Discussion .................................................. 155 Calprotectin and nutritional immunity to A. fumigatus .................................... 156 Atovaquone as a novel anti-fungal agent ...................................................... 158 Neutrophil extracellular traps in A. fumigatus infection .................................. 160 Chapter 6: Future Directions and Preliminary Data ..................................... 165 NET-independent Calprotectin Release from Neutrophils ............................. 166 S100 Proteins in Fungal Infection.................................................................. 167 5 Novel Anti-microbial Peptides in Fungal Infection ......................................... 168 Copper Homeostasis in Aspergillus fumigatus virulence and Nutritional Immunity ........................................................................................................ 172 CR3 Signaling in Neutrophils......................................................................... 174 Dectin-2/Dectin-3 Signaling in NET formation ............................................... 175 Cytokine Regulation of Neutrophil Responses .............................................. 176 Antibodies, Pentraxins and Fc Receptors in Fungal Infection ....................... 179 Modulating NET Formation: Implications for Therapy ................................... 183 Concluding Remarks ...................................................................................... 186 Preliminary Figures ........................................................................................ 185 References ...................................................................................................... 190 6 List of Tables Table 1.1 Human fungal pathogens 20 Table 1.2 Spectrum of fungal species in fungal keratitis 22 Table 2.1 Aspergillus fumigatus strains used in this study 64 Table 3.1 Keratitis isolate strains and inhibitory concentrations 102 7 List of Figures Figure 1.1 Corneas of fungal keratitis patients 23 Figure 1.2 Anti-fungal drugs and their targets 25 Figure 1.3 Morphology of pathogenic fungi 26 Figure 1.4 Neutrophils in corneal ulcers of fungal keratitis patients 27 Figure 1.5 Microbicidal functions of neutrophils 30 Figure 1.6 Crystal structure of calprotectin 34 Figure 1.7 Neutrophil extracellular traps induced by IL-8 36 Figure 1.8 Pattern recognition receptors in fungal recognition 39 Figure 1.9 PAD4 mediates histone citrullination 45 Figure 1.10 Simplified view of NETosis 46 Figure 1.11 Cell wall components of pathogenic fungi 51 Figure 1.12 Model of A. fumigatus Zn transport 55 Figure 2.1 Effect of neutrophil calprotectin on A. fumigatus corneal infection 87 Figure 2.2 Effect of neutrophil calprotectin on A. fumigatus hyphal growth in vitro 88 Figure 2.3 Zn and Mn binding contribute to calprotectin Anti-Aspergillus activity 89 Figure 2.4 ZafA mediated zinc uptake in A. fumigatus virulence and susceptibility to calprotectin 90 Figure 2.5 Conidia killing by neutrophils is independent of calprotectin 91 Figure 2.S1 Supplementary Figure 1 92 8 Figure 2.S2 Supplementary Figure 2 93 Figure 2.S3 Supplementary Figure 3 94 Figure 3.1 Atovaquone inhibits growth of filamentous fungal keratitis isolates 115 Figure 3.2 Atovaquone