Analysis of the interactions between Aspergillus fumigatus, Pseudomonas aeruginosa and a model of the alveolar surface A thesis submitted to Maynooth University for the degree of Doctor of Philosophy Anatte Margalit, B.Sc October 2019 Supervisor Co-supervisor Head of Department Prof. Kevin Kavanagh Dr. James C. Carolan Prof. Paul Moynagh Medical Mycology Applied Proteomics Dept. of Biology Laboratory Laboratory Maynooth University Department of Biology Department of Biology Maynooth University Maynooth University Chapter 1 Introduction .............................................................................................................. 1 1.1 Cystic fibrosis ..................................................................................................................... 2 1.1.1 The microbial environment of the CF airways ............................................................ 3 1.1.2 Impact of polymicrobial infections in CF .................................................................... 6 1.1.3 Advances in therapy for cystic fibrosis ........................................................................ 7 1.1.4 The microbial environment in non-cystic fibrosis-related conditions.......................... 9 1.2 Study of polymicrobial interactions .................................................................................. 11 1.3 In vitro models for the study of pathogen host interactions of the lung............................ 13 1.4 The A549 cell line as a model for infection at the alveolar surface .................................. 14 1.5 Aspergillus fumigatus; an opportunistic human pathogen ................................................ 15 1.5.1 A. fumigatus pathogenesis and host – from inhalation to germination ...................... 16 1.5.1.1 Host cell recognition of A. fumigatus ...................................................................... 17 1.5.1.2 The humoral response to A. fumigatus .................................................................... 18 1.5.1.3 Manipulation of host epithelial cells by A. fumigatus ............................................. 19 1.5.1.4 The innate immune cell response to A. fumigatus ................................................... 20 1.5.1.5 The adaptive immune response to A. fumigatus ...................................................... 22 1.5.3 Impact of A. fumigatus on cystic fibrosis patients ..................................................... 25 1.5.4 Microbial interactions involving A. fumigatus ........................................................... 27 1.6 Pseudomonas aeruginosa: an opportunistic human pathogen ...................................... 28 1.6.1 The host immune response to P. aeruginosa ............................................................. 30 1.6.1.1 The mucociliary elevator ........................................................................................ 30 1.6.1.3 Host cellular receptors to detect P. aeruginosa ...................................................... 32 1.6.1.4 Cellular response to P. aeruginosa ......................................................................... 33 1.6.2 P. aeruginosa virulence factors ................................................................................. 35 1.6.2.1 Flagella and Type IV Pili ........................................................................................ 35 1.6.2.2 Type III secretion systems ...................................................................................... 35 1.6.2.3 P. aeruginosa Biofilms and Quorum sensing ......................................................... 36 1.6.3 P. aeruginosa acute airway pathogenesis .................................................................. 37 1.6.4 Pathogenesis of P. aeruginosa in chronic airway conditions..................................... 37 1.6.5 Adaptation and colonization of P. aeruginosa in the CF lung ................................... 38 1.6.6 Microbial interactions involving P. aeruginosa ......................................................... 38 1.7 Interactions between A. fumigatus and P. aeruginosa ...................................................... 40 1.7.1 Antagonistic interactions between A. fumigatus and P. aeruginosa .......................... 41 1.7.2 Synergistic interactions between A. fumigatus and P. aeruginosa ............................ 42 1.8 Antimicrobial resistance in P. aeruginosa ....................................................................... 43 1.9 Novel antimicrobial drug discovery; approaches and challenges ..................................... 44 i 1.10 Choosing proteomics to investigate inter-species interactions ....................................... 45 1.10.1 Label-free quantitative proteomics .......................................................................... 46 1.10.2 The application of LFQ proteomics for investigating inter-species interactions ..... 47 1.11 Overview of thesis objectives ......................................................................................... 48 Chapter 2 Materials and Methods .................................................................................. 50 2.1 General Chemicals and Reagents ...................................................................................... 51 2.1.1 Phosphate Buffered Saline (PBS) .............................................................................. 51 2.1.1.2 PBS-Tween 80 (PBST) ........................................................................................... 51 2.1.2 Solutions for pH Adjustment ..................................................................................... 51 2.1.2.1 Hydrochloric Acid (HCl) (5 M) .............................................................................. 51 2.1.2.2 Sodium Hydroxide (NaOH) (5 M) .......................................................................... 51 2.2 Maintenance and preparation of P. aeruginosa ................................................................ 52 2.2.1 Bacterial culture media .............................................................................................. 52 2.2.2 Super Optimal Broth with catabolite repression (SOC medium); SOC agar (0.3%) . 52 2.2.3 Synthetic Cystic Fibrosis Medium (SCFM) ............................................................... 53 2.2.4 Maintenance of P. aeruginosa ................................................................................... 53 2.2.4.1 Preparation of P. aeruginosa in cell culture medium.............................................. 53 2.2.5 Bacterial toxicity assays ............................................................................................. 54 2.3 Maintenance and preparation of A. fumigatus................................................................... 54 2.3.1 Aspergillus culture media (Czapek-Dox) ................................................................... 54 2.3.2 Aspergillus culture media (Sabouraud) ...................................................................... 54 2.3.3 Minimal Medium Broth ............................................................................................. 55 2.3.4 Culture and maintenance of A. fumigatus .................................................................. 55 2.3.4.1 Preparation of A. fumigatus conidia in mammalian cell culture medium ............... 55 2.3.5 Culture and maintenance of A. flavus and A. nidulans............................................... 56 2.3.5.1 Media preparation ................................................................................................... 56 2.3.6 Culture and maintenance of A. flavus ........................................................................ 56 2.3.7 Culture and maintenance of A. nidulans .................................................................... 56 2.3.8 A. fumigatus culture filtrate and P. aeruginosa culture filtrates ................................ 56 2.3.8.1 A. fumigatus and P. aeruginosa co-culture culture filtrates .................................... 57 2.3.8.2 Exposure of A. fumigatus to culture filtrates ........................................................... 57 2.3.8.3 Exposure of P. aeruginosa to culture filtrates ........................................................ 57 2.4 A549 Cell culture medium and maintenance .................................................................... 58 2.4.1 Cell culture medium ................................................................................................... 58 2.4.2 Cryporeservation buffer ............................................................................................. 58 2.4.3 Maintenance and sub-culturing .................................................................................. 58 ii 2.4.4 Cryopreservation of A549 cells in Liquid Nitrogen .................................................. 59 2.4.5 Recovery of A549 cells from Liquid Nitrogen .......................................................... 59 2.4.6 Exposure of A549 cells to A. fumigatus and/or P. aeruginosa for 12 hours .............. 59 2.4.7 Sequential Exposure of A549 cells to A. fumigatus and P. aeruginosa ..................... 60 2.5 Protein extraction .............................................................................................................. 60 2.5.1 Lysis buffer/Resuspension buffer .............................................................................. 60 2.5.2 Protein quantification by Bradford protein assay ......................................................