Contents
INTRODUCTION
REVIEW OF LITERATURE
2.1. Clinical Manifestation of Candida a/bicans Infection
2.1.1. Systemic Candidiasis
2.1.2. Oral Candidiasis
2.1.3. Vulvovaginal Candidiasis
1-4
5-45
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2.2. Virulence Factors in C. albicans
2.2.1. Adhesion Factors
2.2.1.1. ALS (Agglutinin-Like Sequence) Protein Family
2.2.1.2. Transglutaminases (TGases) Substrate Protein Family
2.2.1.3. lntegrin-like Protein Family
2.2.1.4. Hydrophobic Proteins
2.2.2. Penetration and Dissemination Factors
2.2.3. Nutritional and Metabolic Factors
2.2.4. Necrotic Factors
2.2.4.1. Secreted Aspartyl Proteinases
2.2.4.2. Phospholipases
2.2.4.3. Lipases
2.2.5. Morphological Factors
2.2.5.1. Morphogenesis
2.2.5.2. Phenotypic Switching
2.2.6. Other Factors
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2.3. Host Defence against Candida albicans
2.3.1. Innate Immunity Mechanism
2.3.1.1. Polymorphonuclears
2.3.1.2. Macrophages
2.3.1 .3. Natural Killer Cells
2.3.1 .4. Epithelial Cells
2.3.1.5. Complement System and Mannose Binding Lectin
2.3.2. Acquired Immunity
2.3.2.1. Cell Mediated Immunity
2.3.2.2. Antibody Mediated Immunity
2.3.2.2.1. Mannan and Stress Mannoprotein-specific Monoclonal Antibodies
2.3.2.2.2. 13-D-Giucan-specific Antibodies
2.3.2.2.3. SAP-specific Vaginal Fluid Antibodies
2.3.2.2.4. Hsp90-specific Monoclonal Antibodies
2.3.2.2.5. Involvement of Candida-specific Antibodies in Human VVC
2.3.2.2.6. Involvement of Candida-specific Antibodies in OPC
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2.4. Antifungal Resistance: Epidemiology and Mechanism
2.4.1. Polyenes
2.4.2. Azoles
2.4.3. 5-Fiucytosine
2.4.4. Allylamines
2.4.5. Echinocandins
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2.5. Diagnosis of Candida albicans Infections
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2.6. Covalently Linked Proteins of Candida albicans Cell Wall
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2.6.1. Candida a/bicans GPI CWPs
2.6.2. Mild Alkali Cell Wall Proteins
2.6.3. Functions of Covalently Linked Cell Wall Proteins
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2.7. Role of Proteomics, Bioinformatics Tools in Target Identification
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MATERIAL AND METHODS
46-85
3.1. Fungal Cultures
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3.2. Culture Conditions for C. a/bicans
46
3.3. Generation and Characterization of Amphotericin B Resistant Strain
3.3.1. Experimental Induction of Drug Resistance
3.3.2. Confirmation of Resistance in Mouse Model of Candidiasis
3.3.3. Time Kill Assay
3.3.4. Characterization of Amphotericin B Resistant Strain
3.3.4.1. Morphological Characterization
3.3.4.2. Germ Tube Formation Assay
3.3.4.3. Dot Blot Assay for [3-1 ,6-glucan Synthase Activity
3.3.4.4. Spectrophotometric Semi-microdetermination of Ergosterol
3.3.4.5. Expression Analysis of lanosterol 14-a- demethylase (ERG11)
3.3.4.6. Extracellular Proteinase Activity Assay
3.3.4.7. Determination of Extracellular Phospholipase Activity
3.3.5. Exposure to H2 0 2 and Menadione
3.3.5.1. Preparation of Cell Lysate
3.3.5.2. Enzymatic Activity Assay
3.3.5.2.1. Catalase Activity
3.3.5.2.2. Glutathione peroxidase (GPx) activity
3.3.5.2.3. Glutathione-S-Transferase Activity Assay
3.3.6. Characterization of AMB-R at Genetic Level
3.3.6.1. DNA Isolation
3.3.6.1.1. Phenol: Chloroform Method
3.3.6.1.2. DNeasy Protocol
3.3.6.2. PCR Amplification and Sequencing
3.3.6.3. PCR Product Purification
3.3.6.4. Sequencing
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3.4. Isolation of C. albicans Yeast and Hyphal Cell Wall
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3.5. Isolation of Covalently Linked Proteins
3.5.1. HF-Pyridine Release of GPI Cell Wall Proteins
3.5.2. Enzymatic Release of GPI Cell Wall Proteins
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3.6. Protein Estimation Using 2D Quant Kit
59
3.7. Raising Polyclonal Sera in Mice against GPI Cell Wall Proteins of
C. a/bicans 60
3.8. SDS PAGE of Covalently Linked Cell Wall Proteins
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3.9. Electro-transfer of Proteins
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3.1 0. lmmunoblotting Using Pooled Patient Sera and Antiserum Raised in Mice
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3.11. Identification and Characterization of GPI CWPs Proteins by MALDI-TOF
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3.12. GPI CWPs and Macrophage Interaction
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3.12.1. Macrophage Culture
3.12.2. DNA Fragmentation Assay
3.12.3. Cytokine Level in Macrophages
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3.13. Generation of Hybridomas and Production of Monoclonal Antibodies
3.13.1. Cell Line
3.13.2. Mice
3.13.3. Immunization of Mice
3.13.4. Sub-culturing and Maintenance of Sp2/0
3.13.5. Preparation of Spleen Cells and Myeloma Cells for Fusion
3.13.6. Fusion of Splenocytes with Sp2/0 Cells
3.13.7. Screening of Positive Hybridomas
3.13.8. Single Cell Cloning of Positive Hybridoma Clones
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3.14. Western Blotting Using GPI CWPs
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3.15. Production of Monoclonal Antibodies under Serum-free Conditions
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3.16. Cryopreservation of Positive Clones
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3.17. lso-typing ofthe Monoclonal Antibodies
73
3.18. Cross Reactivity of MAbs with Other Fungi and Different Strains of C. a/bicans 74
3.19. Cross Reactivity of MAbs with Bacteria
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3.20. Monoclonal Antibody Production by Ascites Growth
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3.21. Purification of Monoclonal Antibody
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3.22. Inhibition of Attachment of C. albicans with Composite Material
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3.23. Inhibition of Germination of Candida Cells
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3.24. Effect of MAbs on Colony Forming Units (cfu)
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3.25. MTT Assay
77
3.26. Fluorescence Activated Cell Sorting (FACS analysis)
78
3.27. Phagocytosis Assay
3.27.1. Treatment of Yeast and Germ Tubes with MAb
3.27.2. Phagocytosis and Geimsa Staining
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3.28. Epitope Localization on the Surface of C. a/bicans
80
3.29. Protection against Systemic Challenge by Passive Transfer of
Antibodies
Monoclonal
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3.30. Assessment of in vivo Diagnostic Potential of Monoclonal Antibody
3.30.1. Biotin Labelling of Monoclonal Antibody
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3.30.2. Diagnosis Potential of Monoclonal Antibody in Mouse Model of C. albicans Infection
81
3.31. Detection of Antigens Recognized by MAb in Candida Positive Patient Sera
Using ELISA
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3.32. RNA Isolation from Hybridoma Cells
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3.33. Reverse Transcriptase PCR
83
3.34. Development of Biologically Active Antibody Derived Peptides
3.34.1. RT PCR Product Sequencing
3.34.2. Peptide Designing
3.34.3. Peptide Synthesis
3.34.4. In vitro Antifungal Activity of Paratope Derived Peptides
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RESULTS
86-120
4.1. Acquired Amphotericin B Resistance in Candida a/bicans
86
4.2 Time Kill Assay
86
4.3. Morphological Characterization
87
4.4. Germ Tube Formation Assay
87
4.5. Dot Blot Assay for (3-1 ,6-glucan synthase Activity
88
4.6. Spectrophotometric Semi-Microdetermination of Ergosterol
90
4.7. Extracellular Proteinase Activity
91
4.8. Extracellular Phospholipase Activity
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4.9. Expression Analysis of ERG11
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4.1 0. DNA Isolation
93
4.11. PCR Amplification, Sequencing and CLUSTALW Analysis
93
4.12. Exposure to H 2 0 2 and Menadione
96
4.13. Cell Wall Preparation and Protein Extraction
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4.14. Isolation of Covalently Linked Cell Wall Proteins
99
4.15. Protein Estimation Using 2D Quant Kit
99
4.16. lmmunoblotting of GPI Cell Wall Proteins with Pooled Patient Sera and Antiserum
Raised in Mice
100
4.17. Identification and Characterization of GPI Cell Wall Proteins by MALDI-TOF
100
4.18. DNA Fragmentation and Cytokine Assay
105
4.19. Generation and Selection of Monoclonal Antibodies
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4.20. Determination of Monoclonal Antibodies lsotype
106
4.21. Production of Monoclonal Antibodies Under Serum-Free Conditions
108
4.22. Cross Reactivity of MAb 2A11, 10D2 and 2C1 with Other Fungi and Different
Strains of C. albicans
108
4.23. Cross Reactivity of MAbs with Bacteria
108
4.24. Monoclonal Antibody Production by Ascites Growth
110
4.25. Purification of Monoclonal Antibody
4.26.1. Effect of Monoclonal Antibodies on Colony Forming Units (cfu)
4.26.2. Inhibition of Attachment of C. a/bicans with Composite Material
4.26.3. Inhibition of Germination of Candida Cells
4.26.4. MTT Assay
4.26.5. Cell-Viability Assay by FAGS
4.26.6. Epitope Localization on the Surface of C. a/bicans
4.26.7. Phagocytic Assay
4.26.8. Protection Against Experimental Murine Candidiasis by MAb 2A 11 and 1002
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4.27. Assessment of In Vivo Diagnostic Potential of Monoclonal antibody
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4.28. Detection of Antigens Recognized by MAb 2C1 in Candida Positive
Using ELISA
4.29. Peptide Synthesis and Its Evaluation In Vitro
DISCUSSION
Patient Sera
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121-144
5.1. Generation and Characterization of Amphotericin B Resistant Strain
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5.2. Identification and Characterization of Covalently Linked Cell Wall
129
Proteins
129
5.3. Generation of Monoclonal Antibodies
132
5.5. Conclusion
143
REFERENCES
APPENDIX
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