The exploitation of host iron sources by Candida albicans during oral infection [Elektronische Ressource] / von Ricardo Sergio Couto de Almeida

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• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2009
• Nombre de lectures: 20
• Langue: English
• Poids de l'ouvrage: 16 Mo

Extrait

The Exploitation of Host Iron Sources by
Candida albicans during Oral Infection






Dissertation
zur Erlangung des akademischen Grades doctor rerum naturalium
(Dr. rer. nat.)




vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller- Universität Jena




von
Master of Science Ricardo Sergio Couto de Almeida




geboren am 29. November 1976 in Lins, Brasilien









Jena, Juli 2008 Table of Contents
Table of Contents

1. Summary/Zusammenfassung 1
1.1. Summary 1
1.2. Zusammenfassung 3

5 2. Introduction
2.1. Iron and iron homeostasis in the host 5
2.1.1. The role of iron in biological systems 5
2.1.2. Iron proteins: haemoglobin, transferrin, lactoferrin and ferritin 5
2.1.3. Cellular iron uptake and storage 8
2.2. Iron and microbial infection 10
2.2.1. The importance of iron for infection: iron limitation and iron
10
overload
2.2.2. Iron sources and strategies used by pathogens 11
2.3. Candida albicans 12
2.3.1. The Genus Candida 12
2.3.2. The polymorphic fungus C. albicans 13
2.3.3. Growth and genetics of C. albicans 13
2.3.4. The pathogenic fungus C. albicans 14
2.4. C. albicans virulence factors 15
2.4.1. Hyphal formation 15
2.4.2. Adhesion 17
2.4.3. Secreted hydrolases 18
2.4.4. pH sensing 19
2.4.5. Iron uptake 19
2.5. The three known iron uptake systems of C. albicans 20
2.5.1. Iron reductive pathway 20
2.5.2.Siderophore uptake 21
2.5.3. Iron acquisition from haemoglobin 21
2.5.4. Host iron proteins used as iron sources by C. albicans 22
2.6. Aims of this study 23

3. Materials and Methods 24
3.1. Microorganisms 24
3.2. Preparation of low iron medium (LIM) 25
3.3. Fungal preculture conditions 26
3.4. Growth kinetics under iron limitation 26
3.5. Hyphal growth under iron limitation 27
3.6. Oral epithelial cells 27
3.7. Epithelial cell monolayer damage assay 28
3.8. High-affinity iron uptake assay 29
3.9. Iron quantification 29
3.10. Removal of free iron contamination from ferritin solution 29
3.11. Ferritin agar plates 30
3.12. Agar plates with pH indicator 30
3.13. Ferritin binding assay 30
3.14. Transmission electron microscopy 31
3.15. Biological function of ferritin binding 32
3.16. Immunofluorescence of infected epithelial cells 32
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Hans Knoell Institute Table of Contents
3.17. C. albicans transformation with CIp10 33
3.18. Sample preparation for RNA extraction 33
3.19. RNA extraction and labelling 34
3.20. Microarray hybridization and analysis 34

4. Results 35
4.1. Investigation of the influence of iron limitation on C. albicans growth 35
4.1.1. Iron limitation inhibits the growth of C. albicans in its yeast form 35
4.1.2. Iron limitation inhibits the hyphal growth form of C. albicans 36
4.1.3. C. albicans can store iron intracellularly 39
4.2. Analysis of C. albicans growth with ferritin as the sole source of iron 42
4.2.1. The ferritin content of epithelial cells influences the extent of cellular
42
damage caused by C. albicans
4.2.2. Development of a method to purify ferritin from free iron
44
contamination
4.2.3. C. albicans can use ferritin as the sole source of iron in vitro 47
4.2.4. Usage of ferritin as the sole source of iron in vitro requires
49
acidification of the medium
4.2.5. Functional mutant screening reveals that the reductive pathway is
50
essential for iron acquisition from ferritin
4.2.6. Iron uptake from haemoglobin is independent from the reductive
52
pathway
4.2.7. The pH sensing pathway is involved in iron acquisition from ferritin 53
4.3. Properties of ferritin binding by C. albicans 55
4.3.1. Hyphal, but not yeast cells of C. albicans can bind ferritin 55
4.3.2. Electron microscopy analysis of cells binding ferritin 57
4.3.3. Cell viability is not necessary for ferritin binding 57
4.3.4. Ferritin binding is not iron regulated 58
4.3.5. Binding is necessary for iron acquisition from ferritin 60
4.4. Identification of a ferritin receptor 60
4.4.1. The search for a ferritin receptor: in silico and genetic analysis 60
4.4.2. Transcription profiling of C. albicans cells binding ferritin identifies
62
a gene necessary for ferritin binding
4.4.3. Deletion of ALS3 precludes ferritin binding 64
4.4.4. Upstream regulators of ALS3 are required for ferritin binding 66
4.4.5. Als3 is a ferritin receptor 67
4.5. Analysis of a mutant lacking the ferritin receptor 69
69 4.5.1. The als3 mutant has no general iron uptake defect
4.5.2. C. albicans cells lacking Als3 lost their ability to grow with ferritin
69
as the sole source of iron
4.5.3. Invading C. albicans hyphae bind ferritin from epithelial cells during
70
infection
4.5.4. C. albicans mutants lacking genes essential for iron utilization from
72
ferritin are unable to damage epithelial cells
4.6. Cellular dissection of the interactions between C. albicans and oral
73
epithelial cells
4.6.1. Fluorescence microscopy analysis of C. albicans infecting epithelial
73
cells reveals a novel phenomenon termed the “glove effect”
4.6.2. The “glove effect” depends on the iron status of the epithelial cells 75

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DTable of Contents
5. Discussion 77
5.1. C. albicans needs iron for growth and has intracellular iron storage 77
5.2. Ferritin, a novel iron source used by C. albicans during oral infections 78
5.3. The molecular mechanisms of ferritin exploitation by C. albicans 79
5.4. The ferritin receptor Als3 82
5.5. The ability to exploit iron from ferritin during interaction with host cells 83
5.6. Iron availability and virulence factors 84
5.7. Environmental challenges and hyphal-associated proteins 84
5.8. Multiple functions for Als3 86
5.9. The iron permease Ftr1 is required for iron acquisition from two different
87
host iron proteins
5.10. The “glove effect” phenomenon and immune cell evasion 87
5.11. Conclusions and Outlook 88

91 6. References

7. List of Abbreviations 99

8. Acknowledgments 100

Leibniz Institute for Natural Product Research and Infection Biology iii
Hans Knoell Institute 1. Summary/ Zusammenfassung
1. Summary/ Zusammenfassung

1.1. Summary
Iron is an essential element for almost all organisms, from microbes to multicellular
animals. Using high affinity iron-binding molecules, higher organisms can sequester
virtually all free iron, causing a natural resistance to infection known as “nutritional
immunity”. Therefore, pathogenic microbes are forced to utilise iron from host
molecules during infection. For example, within the oral cavity, extracellular iron is
mostly bound to lactoferrin found in saliva whilst intracellular iron is stored in
association with ferritin. Although ferritin is abundant in epithelial cells, only one
bacterial species (Neisseria meningitidis) has yet been shown to use ferritin indirectly as
an iron source within infected epithelial cells by manipulating the cellular machinery.
Candida albicans is a polymorphic yeast which belongs to the normal microbial flora of
human beings. The fungus exists as a harmless commensal on mucosal surfaces in
healthy individuals but can cause several types of infections in predisposed patients,
ranging from superficial to life-threatening disease. C. albicans possesses three known
iron uptake systems: (i) uptake and usage of iron from haemoglobin, mediated by a
haemoglobin receptor (Rbt5) and a haem oxygenase (Hmx1); (ii) siderophore iron
utilisation mediated by the receptor Sit1 and (iii) free iron and transferrin iron uptake,
mediated by the reductive iron uptake system.
During oral infections, C. albicans must be able to exploit the host iron resources. Since
it was observed that the ferritin concentration within oral epithelial cells was directly
related to their susceptibility to damage by C. albicans, it was hypothesized that host
ferritin may be used as an iron source by this organism. In agreement with this,
C. albicans was shown to grow on agar at physiological pH with ferritin as the sole iron
source. In contrast, the baker’s yeast Saccharomyces cerevisiae was unable to grow
under these conditions. A screen of mutants lacking components of each of the three
iron acquisition systems showed that only the reductive pathway is involved in ferritin
iron utilization. The ftr1 mutant - which lacks a high affinity iron permease - grew
with free iron, haemoglobin, but not with ferritin as the sole source of iron. The fact that
growth with ferritin was enhanced when the initial pH of the medium was low,
suggested that pH plays a crucial role in the release of iron from ferritin. Indeed,
C. albicans was only able to use ferritin as an iron source under conditions which
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