Variability of biofilm formation in Candida glabrata and Candida parapsilosis and its consequences on the infection process Dissertation In partial fulfillment of the requirements for the degree “Doctor of Philosophy (PhD)” at the Georg-August-Universität Göttingen within the “Biology” Doctoral program at the Georg-August University School of Science (GAUSS) submitted by Emilia Gómez Molero born in Albacete, Spain Göttingen, 2019 To my Family “Todo hombre puede ser, si se lo propone, el escultor de su propio cerebro” Dr. Santiago Ramón y Cajal (1852-1934) I Supervisors: Dr. rer. nat. Oliver Bader (Main Supervisor) (Institute for Medical Microbiology, Department of Medical Microbiology, University Medical Center Göttingen) Prof. Dr. med. Michael Weig (Second Supervisor) (Institute for Medical Microbiology, Department of Medical Microbiology, University Medical Center Göttingen) Members of the Thesis Committee: Prof. Dr. med. Uwe Groß (Institute for Medical Microbiology, Department of Medical Microbiology, University Medical Center Göttingen) Prof. Dr. Gerhard Braus (Institute for Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University Göttingen) Dr. rer. nat. Oliver Bader (Supervisor) (Institute for Medical Microbiology, Department of Medical Microbiology, University Medical Center Göttingen) Members of the Examination Board: Prof. Dr. med. Uwe Groß (1st Reviewer) (Institute for Medical Microbiology, Department of Medical Microbiology, University Medical Center Göttingen) Prof. Dr. Gerhard Braus (2nd Reviewer) (Institute for Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University Göttingen) Further Members of the Examination Board: Prof. Dr. rer. nat. Carsten G. K. Lüder (Institute for Medical Microbiology, Department of Medical Microbiology, University Medical Center Göttingen) PD Dr. Michael Hoppert (Institute for Microbiology and Genetics, Department of General Microbiology, University Göttingen) Prof. Dr. Fabian Commichau (Institute for Microbiology and Genetics, Department of General Microbiology, University Göttingen) Prof. Dr. Kai Heimel (Institute for Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University Göttingen) Date of Disputation: II TABLE OF CONTENTS TABLE OF CONTENTS ........................................................................................................ III ACKNOWLEDGEMENTS ................................................................................................... VII LIST OF TABLES ................................................................................................................ XI LIST OF FIGURES .............................................................................................................. XII LIST OF ABREVIATIONS ................................................................................................... XV ABSTRACT ....................................................................................................................... XX 1. INTRODUCTION ................................................................................................... 1 1.1 Epidemiology of Candida spp. .................................................................................... 1 1.2 Features and phylogeny of the genus Candida ......................................................... 5 1.3 C. albicans infections and host-pathogen interactions ............................................. 7 1.4 Candida biofilms ....................................................................................................... 10 1.5 The fungal cell wall ................................................................................................... 13 1.6 Candida spp. cell wall proteins (CWP) ..................................................................... 14 1.7 GPI-anchored Cell Wall proteins (adhesins) ............................................................ 15 1.8 Candida glabrata ...................................................................................................... 18 1.8.1 Candida glabrata cell wall proteins ......................................................................... 20 1.9 Candida parapsilosis ................................................................................................ 21 1.9.1 Candida parapsilosis cell wall proteins .................................................................... 22 1.10 Candida species interaction in host invasion ........................................................... 23 1.11 Aims of the study ..................................................................................................... 25 2. MATERIALS AND METHODS ............................................................................... 26 2.1 Materials .................................................................................................................. 26 2.1.1 Synthetic oligonucleotides ....................................................................................... 30 2.1.2 Candida spp. clinical isolates .................................................................................... 30 2.1.3 Candida spp. reference strains ................................................................................ 31 2.2 Methods ................................................................................................................... 32 2.2.1 Routine diagnostic procedures ................................................................................ 32 2.2.2 Candida spp. strain maintenance and growth conditions ....................................... 32 2.2.3 MALDI-TOF analyses species-identification ............................................................. 33 2.2.4 C. glabrata biofilm formation capacity to polystyrol ............................................... 33 2.2.5 C. glabrata biofilm formation capacity to silicone elastomers ................................ 35 III 2.2.6 C. parapsilosis biofilm formation capacity to polystyrol .......................................... 36 2.2.7 Antifungal susceptibility test .................................................................................... 38 2.2.8 Agar invasion capacity .............................................................................................. 39 2.2.9 Optical and phase-contrast microscopy .................................................................. 39 2.2.10 Sedimentation assay of clinical morphotypes ......................................................... 40 2.2.11 Electrophoretic karyotyping of colony morphotypes .............................................. 40 2.2.12 Genomic DNA isolation ............................................................................................ 41 2.2.13 Polymerase chain reaction analyses of adhesin-encoding genes in clinical morphotypes ............................................................................................................ 42 2.2.14 DNA Sanger sequencing ........................................................................................... 43 2.2.15 Candida cell wall isolation ........................................................................................ 43 2.2.16 Candida spp. genome sequencing analyses ............................................................. 44 2.2.17 In vivo infection analyses. Galleria mellonella animal model .................................. 44 2.2.18 C. albicans and C. glabrata identification from patient blood cultures .................. 45 2.2.19 C. albicans and C. glabrata interaction under phase-contrast microscopy ............. 45 3. RESULTS ............................................................................................................ 47 3.1 Genomic plasticity enhances phenotypic diversity in Candida glabrata clinical isolates from different clades .................................................................................. 47 3.1.1 Changes in the genome of hyper-biofilm-forming C. glabrata isolates .................. 47 3.1.2 Genomic changes between time-resolved C. glabrata isolates .............................. 53 3.2 Phenotypic variability in C. glabrata clinical isolates correlates with variations in cell wall proteome and infection-relevant parameters ........................................... 55 3.2.1 Biofilm formation capacity to polystyrol of a large C. glabrata clinical strain collection .................................................................................................................. 58 3.2.2 Virulence of C. glabrata isolates in the Galleria mellonella model ......................... 59 3.2.3 Variations in C. glabrata biofilm formation capacity to silicone elastomers .......... 62 3.2.4 Different adhesins are present in hyper biofilm-forming clinical isolates ............... 64 3.2.5 Hyper biofilm-forming C. glabrata clinical isolates’ adherence to C. albicans. ....... 69 3.2.6 C. albicans and C. glabrata mixed culture analysis .................................................. 71 3.3 Pheno- and genotypic analyses of Candida parapsilosis clinical isolates ................ 76 3.3.1 Establishing a classification reference ..................................................................... 76 IV 3.4 Candida parapsilosis polymorphism drives differences at morphological and genotypic level in isolates from a single patient ...................................................... 80 3.4.1 The five clinical isolates are distributed into