International PhD Program in Biomolecular Sciences Centre for Integrative Biology 29 th Cycle “PERSISTENCE AND ADAPTATION OF PSEUDOMONAS AERUGINOSA IN CYSTIC FIBROSIS AIRWAY” Tutor Olivier JOUSSON Centre for Integrative Biology (CIBIO) Advisor Irene BIANCONI Centre for Integrative Biology (CIBIO) Ph.D. Thesis of Silvia D’ARCANGELO Centre for Integrative Biology (CIBIO) Academic Year 2015/2016 1 DECLARATION I (Silvia D’Arcangelo) confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged. CONTRIBUTIONS My PhD project was focused on the understanding of dynamics that Pseudomonas aeruginosa undergoes during the adaptation in cystic fibrosis patients airway. Professor Olivier Jousson, Dr Irene Bianconi and I designed the experimental workflow, with the valuable contributions of Dr Kate Bailey at CIBIO – Centre for Integrative Biology, University of Trento, Trento, Italy. Experiments involving Artificial Sputum Medium were performed in collaboration with Prof. Craig Winstanley and Dr Jo Fothergill at the Institute of Infection and Global Health, University of Liverpool, Liverpool, UK. Mutagenesis experimental procedures were carried out in collaboration with Prof. Alain Filloux and Dr Thomas Wood at the Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK. Experiments on Galleria mellonella were performed with the help of Prof. Giovanni Di Bonaventura and Dr Arianna Pompilio at the Laboratory of Clinical Microbiology, Department of Medical, Oral and Biotechnological Sciences, School of Medicine and Health Sciences, University of Chieti-Pescara “Gabriele D’Annunzio”, Chieti, Italy. Phenotypic assays were performed with the precious help of Elena Piffer, Postgraduate student, CIBIO, University of Trento, Trento, Italy. Internalization assay was carried out with the contribution of Wojtek Berkowicz, Master student from University of Warsaw, Warsaw, Poland. Antibiograms were performed in collaboration with Dr Paola Gualdi, Dr Marco Vizzini and Loredana Barberi, at Laboratorio di Patologia Clinica, Ospedale “Santa Maria del Carmine”, Rovereto, Italy. Bioinformatic analyses were performed with the contributions of Dr Alfonso Esposito and Dr Mattia Benedet, CIBIO, University of Trento, Trento, Italy. 2 Publications • “Draft Genome Sequences of 40 Pseudomonas aeruginosa Clinical Strains Isolated from the Sputum of a Single Cystic Fibrosis Patient Over an 8-Year Period.” Irene Bianconi, Silvia D’Arcangelo, Mattia Benedet, Kate E. Bailey, Alfonso Esposito, Elena Piffer, Alex Mariotto, Ermanno Baldo, Grazia Dinnella, Paola Gualdi, Michele Schinella, Claudio Donati and Olivier Jousson. Genome Announcements , 2016 Nov-Dec; 4(6): e01205-16. • “Persistence and microevolution of Pseudomonas aeruginosa in the Cystic Fibrosis lung: a single-patient longitudinal genomic study.” Irene Bianconi, Silvia D'Arcangelo, Alfonso Esposito, Mattia Benedet, Elena Piffer, Grazia Dinnella, Paola Gualdi, Michele Schinella, Ermanno Baldo, Claudio Donati and Olivier Jousson, submitted to BMC Microbiology. Conference abstracts • “The role of Type VI Secretion System on the competitiveness of P. aeruginosa clinical isolates.” Silvia D’Arcangelo et al., 16 th International Conference on Pseudomonas, 5-9th September 2017, Liverpool, UK • “A population genomics longitudinal study of P. aeruginosa reveals adaptive changes related to the acquisition of multidrug-resistant phenotypes during persistence in cystic fibrosis airways.” Irene Bianconi, Silvia D’Arcangelo et al., 30 th Annual North American Cystic Fibrosis Conference, 27-29 th October 2016, Orlando, FL, USA • “A longitudinal study focusing on population genomics and phenotypic factors leading to adaptation of Pseudomonas aeruginosa in a Cystic Fibrosis patient.” Silvia D’Arcangelo et al., Cortona Procarioti 2016, 12-14 th May 2016, Cortona, Italy • “A longitudinal study focusing on population genomics and phenotypic factors leading to adaptation of Pseudomonas aeruginosa in a cystic fibrosis patient.” Silvia D’Arcangelo et al., 31 st Meeting of SIMGBM – Società Italiana di Microbiologia Generale e Biotecnologie Microbiche, 23-26 th September 2015, Ravenna, Italy • “A longitudinal investigation of genomic and phenotypic factors leading to adaptation of Pseudomonas aeruginosa in a cystic fibrosis patient.” Silvia D’Arcangelo et al., 38 th European Cystic Fibrosis Conference, 10-13 th June 2015, Brussels, Belgium. 3 CONTENTS 1. ABSTRACT ................................................................................................................................................... 6 2. INTRODUCTION ....................................................................................................................................... 8 2.1. Pathogenesis of Cystic fibrosis ........................................................................................................... 8 2.1.1. Impact of defective CFTR on the airway .................................................................................... 9 2.1.2 Microbiology of CF in the airway .............................................................................................. 11 2.2. Genomic studies on Pseudomonas aeruginosa ............................................................................. 11 2.3. Adaptation of Pseudomonas aeruginosa to the CF airway .......................................................... 13 2.4. Antibiotic resistance ......................................................................................................................... 14 2.5. Pseudomonas aeruginosa virulence factors ................................................................................... 15 2.5.1. Role of the T6SS in the progression of infection .................................................................... 19 2.6. Aim of the thesis ................................................................................................................................ 22 3. POPULATION STRUCTURE AND MICROEVOLUTION .................................................................. 23 3.1. Materials and methods ..................................................................................................................... 23 3.1.1. Strains ........................................................................................................................................... 23 3.1.2. Genome sequencing ................................................................................................................... 25 3.1.3. MLST ............................................................................................................................................ 26 3.1.4 Comparative sequence analysis ................................................................................................. 26 3.1.5. Phenotypic assays ....................................................................................................................... 26 3.1.6. Antibiotic susceptibility ............................................................................................................. 27 3.1.7. Statistical analysis ....................................................................................................................... 27 3.2. Results ................................................................................................................................................ 28 3.2.1. Whole Genome Sequencing ...................................................................................................... 28 3.2.2. Phenotypic and genotypic characterization ........................................................................... 41 4. FUNCTIONAL STUDIES ON SPECIFIC STRAINS ............................................................................. 53 4.1. Materials and methods ..................................................................................................................... 53 4.1.1. Cell cultures and invasion assay ............................................................................................... 53 4.1.2. IL-8 secretion .............................................................................................................................. 53 4.1.3. Deletion of tssK3 in P.aeruginosa and complementation ..................................................... 53 4.1.4. Artificial Sputum Medium ........................................................................................................ 59 4.1.5. T6SS-mediated bacterial competition ..................................................................................... 60 4.1.6. Caenorhabditis elegans slow killing assay .............................................................................. 62 4.1.7. Galleria mellonella killing assay ............................................................................................... 63 4.1.8. Statistical analysis ...................................................................................................................... 63 4.2. Results ................................................................................................................................................ 64 4 4.2.1. Phenotypic characterization ..................................................................................................... 64 4.2.3. Artificial Sputum Medium (ASM) assays ............................................................................... 66 4.2.4. T6SS-mediated