Characterization of Candida albicans genes involved in cell wall biogenesis and infection [Elektronische Ressource] / vorgelegt von Martin Zavrel

Characterization of Candida albicans genes involved in cell wall biogenesis and infection [Elektronische Ressource] / vorgelegt von Martin Zavrel

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Characterization of Candida albicans Genes Involved in Cell Wall Biogenesis and Infection Von der Fakultät Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung Vorgelegt von Martin Zavrel aus Usti nad Labem (CZ) Hauptberichter: Prof. Dr. H. Brunner Mitberichter: Prof. Dr. D. Wolf Tag der mündlichen Prüfung: 27. 10. 2010 Institut für Grenzflächenverfahrenstechnik der Universität Stuttgart 2010 I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person. Materials of work found by other researcher are mentioned by reference. This thesis, neither in whole nor in part, has been previously submitted for any degree. Stuttgart, June 2010 Martin Zavrel TABLE OF CONTENTS 1 SUMMARY V ZUSAMMENFASSUNG VII 2 LIST OF ABBREVIATIONS IX 2.1 Standard Abbreviations IX 2.2 Gene Abbreviations XI 3 INTRODUCTION 1 3.1 Introduction to Candida albicans 1 3.2 Candidal Infections 2 3.3 Hypha Formation 4 3.3.1 Transcription Factors Efg1 and Cph1 6 3.4 Cell Wall of Candida albicans 7 3.4.1 GPI Anchored Proteins - PGAs 9 3.5 Adhesion 10 3.5.

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Characterization of Candida
albicans Genes Involved in Cell Wall
Biogenesis and Infection



Von der Fakultät Energie-, Verfahrens- und Biotechnik
der Universität Stuttgart
zur Erlangung der Würde eines Doktors der
Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung

Vorgelegt von
Martin Zavrel
aus Usti nad Labem (CZ)


Hauptberichter: Prof. Dr. H. Brunner
Mitberichter: Prof. Dr. D. Wolf
Tag der mündlichen Prüfung: 27. 10. 2010


Institut für Grenzflächenverfahrenstechnik der Universität Stuttgart
2010



























































I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by
another person. Materials of work found by other researcher are mentioned by
reference. This thesis, neither in whole nor in part, has been previously submitted for
any degree.


Stuttgart, June 2010 Martin Zavrel










TABLE OF CONTENTS

1 SUMMARY V
ZUSAMMENFASSUNG VII
2 LIST OF ABBREVIATIONS IX
2.1 Standard Abbreviations IX
2.2 Gene Abbreviations XI
3 INTRODUCTION 1
3.1 Introduction to Candida albicans 1
3.2 Candidal Infections 2
3.3 Hypha Formation 4
3.3.1 Transcription Factors Efg1 and Cph1 6
3.4 Cell Wall of Candida albicans 7
3.4.1 GPI Anchored Proteins - PGAs 9
3.5 Adhesion 10
3.5.1 Proteins Involved in Adhesion Process 11
3.5.2 Identification of Genes Relevant for Adhesion 15
3.5.2.1 PGA7 15
3.5.2.2 PGA23 16
3.5.2.3 PRA1 16
3.5.2.4 AUF8 18
3.5.3 Aims of Work 18
4 MATERIAL 19
4.1 Lab Equipment 19
4.2 Lab Consumables 20
4.3 Chemicals 20
4.4 Enzymes 21
4.5 Other Material (Reaction Kits) 22
4.6 Tissue Cell-lines, Bacterial and Yeast Strains 22
4.7 Plasmids 25
4.8 Buffers and Solutions 27
4.9 Media 29
4.10 Oligonucleotides 30
4.11 Computational Analysis and Software 33
5 METHODS 34
5.1 Sterilization of Media and Instruments 34
5.2 Cultivation and Storage of C. albicans and S. cerevisiae Strains 34
5.3 Cultivation and Storage of E. coli Strains 34
5.4 Measuring of the Cell Optical Density 34
I
5.5 Isolation of Plasmid DNA from E. coli 35
5.6 Isolation of Genomic DNA from C. albicans 35
5.7 DNA Digestion Using Restriction Endonucleases 35
5.8 Purification DNA Fragments after Enzymatic Reactions 36
5.9 Dephosphorylation 36
5.10 Ligation 36
5.11 Electrophoretic Analysis of DNA 36
5.12 Isolation of DNA Fragments from Agarose Gel 37
5.13 Verification of DNA Constructs 37
5.14 RNA Isolation 37
5.15 Determination of Nucleic Acid Concentration 38
5.16 Northern Blot 38
5.17 Southern Blot 39
5.18 Radioactive Probe Labeling and Blot Hybridization 39
5.18.1 Probe Labelling 39
5.18.2 Blot Hybridization 40
5.19 Isolation of Taq Polymerase 40
5.20 PCR 41
5.21 Quantitative Real Time PCR 41
5.22 Transformation of E. coli Cells 43
5.22.1 Preparation of Competent Cells 43
5.22.2 Heat Shock Transformation 43
5.23 Transformation of C. albicans Cells by Electroporation 44
5.24 Transformation of S. cerevisiae Cells by LiAc Method 44
5.25 Flipping Out of the SAT1 Cassette 45
5.26 Preparation of Plasmids 45
5.26.1 Plasmids Used for AUF Gene Studies 45
5.26.2 Plasmids for S. cerevisiae AUF8 Heterologous Expression 47
5.26.3 pXFP-SAT1 Plasmids 47
5.26.4 Plasmids Used for Overexpression Studies 48
5.27 Drop Tests 48
5.28 Wash Assay 49
5.29 Biofilm Formation Assay 49
5.30 Competition Assays 50
5.31 Cell Wall Stability Assay 50
5.32 Fungal Cell Wall Composition Analysis 51
5.32.1 Relative Concentrations of Polysaccharides 51
5.32.2 Absolute Concentration of Glucans Related to Biomass 51
5.33 Fluorescent β-1,3-glucan Staining 52
5.34 Cell Culture Techniques 53
5.34.1 Cell Culture 53
5.34.2 Preparation of Cryocultures 53
II
5.34.3 Thawing of Stored Cryotubes 54
5.35 Protein Coating of 24-Well Plates 54
5.36 Adhesion Assays 54
5.37 Invasion Assays 55
5.37.1 Isolation of Rat Tail Tendon Collagen 55
5.37.2 Construction of Human Reconstituted Epithelia 55
5.37.3 Histological Processing of Invasion Assays 56
5.37.4 Staining 56
5.38 LDH Assay 57
5.39 End-Point Dilution Survival Assays 58
5.40 ROS Assay 58
5.40.1 Using Bone Marrow Derived Macrophages 58
5.40.2 Using RAW264.7 Cell Line 59
5.41 Interaction of DCs with Candida 60
5.42 Microscopy and Fluorescent Microscopy 60
5.43 Transmission Electron Microscopy 60
6 RESULTS 62
6.1 Effect of Efg1 and Cph1 Transcriptional Factors on C. albicans Cell Wall 62
6.1.1 Determination of Cell Wall Changes 62
6.1.2 Determination of Cell Wall Polysaccharide Composition 65
6.1.3 β-1,3-Glucan Exposure 69
6.1.4 Interaction with Immune System Components 70
6.1.4.1 Interaction with Macrophages and ROS Release 72
6.1.4.2 Transcriptional Response of Dendritic Cells 74
6.1.5 Effects of EFG1 Haploinsufficiency 75
6.2 Investigation of Function of Genes Induced During Adhesion 79
6.2.1 Identification and Characteristics of PGA7, PGA23 and PRA1 79
6.2.2 Transcriptional Profile 80
6.2.3 Characterization of Strains Deleted for pga7, pga23 and pra1 81
6.2.4 An Adhesion Deficient Strain Model to Identify Adhesins by Ectopic
Expression 85
6.2.5 Phenotypical Characterization 89
6.2.5.1 Growth 89
6.2.5.2 Cell Size and Filamentation 90
6.2.5.3 Adhesive and Invasive Properties 92
6.2.5.4 PRA1 and Adhesion to Fibrinogen 98
6.2.5.5 PGA7 and Iron Metabolism 98
6.2.5.6 Cell Wall Biosynthesis 99
6.2.5.7 Cell Wall Stability 100
6.2.5.8 Cell Wall Thickness 101
6.2.5.9 Cell Wall Composition 103
6.2.5.10 Interaction with Macrophages and Dendritic Cells 105
III
6.3 AUF Genes 109
6.3.1 Identification and Characterization 109
6.3.2 Construction of Deletion and Revertant Strains 113
6.3.3 Adhesive and Invasive Properties of Mutant Strains 117
6.3.4 AUF8 GFP Tagging 120
6.3.4.1 Construction of pXFP-SAT1 Plasmids 120
6.3.4.2 Tagging of AUF8 in C. albicans Wild-Type 121
6.3.4.3 Expression of AUF8GFP Construct in S. cerevisiae 122
6.3.5 Additional Phenotypical Characterization 123
6.3.6 Metabolism of Stationary Cells and AUF8 Regulation 128
6.3.7 Interaction with Macrophages 130
7 DISCUSSION 132
7.1 Cell Wall Effects of efg1 and cph1 Deletion 132
7.2 Characterization of the Genes PGA7, PGA23 and PRA1 136
7.3 AUF Genes 142
8 LITERATURE 146
THANKS 158
CURRICULUM VITAE 159


IV 1 Summary
1 SUMMARY
Candida albicans is a commensal organism living on skin and mucosal surfaces of
humans and warm blooded animals. Its presence becomes a problem in
immunocompromised patients where it may turn into an opportunistic pathogen.
Candida colonizes various host niches including skin, gastrointestinal and the
urogenital tract, which offer various environments in terms of pH and nutrient
availability. The cell surface of the fungus is the site of direct interaction of Candida
with the host, mediating environmental sensing, adhesion and also interaction with
the host immune system. Since the cell wall is not present in humans its components
are a prime target for drug development.
One part of this work is focused on characterization of cell wall changes
induced by deletion of genes efg1 and cph1. These genes encoding for transcription
factors were previously described to be involved in many cellular functions including
filamentation and pathogenicity. Deletion of efg1 affects mostly the glucan
polysaccharide part of the fungal cell wall. In contact with macrophages and dendritic
cells, deletion of efg1 itself or together with cph1 alters significantly the
immunogenicity of the strains, while deletion of cph1 alone plays only a minor role.
In order to reveal whether C. albicans is able to specifically respond to
different host niches, transcriptional profiling was used in order to identify C. albicans
genes differentially regulated during adhesion on polystyrene, vaginal and intestinal
tissue models. The second part of this work is following up on the results of the
transcriptional profiling. It is focused on functional characterization of several genes
differentially regulated under the tested conditions and encoding for putative cell
surface proteins. Among the genes characterized in detail are two encoding for
predicted GPI-proteins, Pga7 and Pga23 and one for a protein secreted to the
medium, Pra1. Functional studies of PGA7, PGA23 and PRA1 with regard to
adhesion and invasion as well as cell wall structure and stability were performed
using deletion and overexpression strains. These studies qualify Pga7, Pga23 and
Pra1 as structural elements of the cell wall rather than adhesins. The proper function
of Pra1 seems to be also in interaction with host components.
Finally, a family of genes with one member induced during adhesion (AUF8
Adhesion Upregulated Factor 8) was analyzed. These genes were predicted to be
localized in the plasma membrane, carrying four transmembrane domains. There are
totally seven homologues in the C. albicans genome including AUF8, and of which
V 1 Summary
six are located on chromosome 5 in a 10 kb gene-cluster. When heterologously
expressed in S. cerevisiae, one of the proteins, Auf8GFP localizes to the plasma
membrane. Deletion studies did not show any direct involvement of the AUF genes in
the adhesion or invasion process. However, there are some indications about their
importance during stationary phase of growth.


VI