Isolation, molecular characterization and biomedical applications of an epipolythiodioxopiperazine from novel marine Aspergillus fumigatus AFK11
- Abstract
- Marine fungi are the most prolific producers of bioactive metabolites. The current study resulted in the isolation of a new strain of Aspergillus fumigatus showing 96% similarity to those reported and confirmed to be a marine strain through Scanning Electron Microscopy-Energy Dispersive Spectroscopy (EDS-SEM) analysis. Culture conditions were optimized using three physiological parameters (biomass production, hyphal density and radial extension) and two climatic factors (temperature and humidity). The isolated marine fungus was named as AFK11, which was tested for its ability to produce gliotoxin (160 mg/10 L culture) and it was characterized through 1H NMR, 13C NMR, DEPT, HMBC and HMQC aided by Mass spectroscopy analysis.
Biosynthesis of gliotoxin involves non-ribosomal peptide synthatase enzyme, which has been characterized in terrestrial Aspergillus sp. Molecular characterization of the gliotoxin biosynthetic gene cluster from AFK11 strain was performed and resulted in isolation of 12 genes which were translated into corresponding protein sequences and modeled thereafter. The models were compared based on available templates and their structural and functional aspects were determined. This is the first study on the molecular modeling of the gliotoxin biosynthetic gene cluster. Further, this study proposes a probable antifibrotic mechanism of gliotoxin in proximal tubule renal epithelial cells by inhibiting HIF-1 α, Ang-II and NF-B. Gliotoxin was seen to inhibit these mediators in a dose dependent manner.
Our study is aimed to extend the biomedical applications of gliotoxin from AFK11. Highly efficient gliotoxin loaded-functionalized Single Walled Carbon Nanotube- Chitooligosaccharide (f-SWNT-COS) mediated Folic Acid (FA), p53, Lysozyme coated delivery vehicles were fabricated as effective targeted drug delivery systems. Delivery systems have been chemically characterized with Fourier Transform Infrared Spectroscopy (FT-IR), Field Emission-Scanning (FE-SEM) and Transmission Electron Microscopy (TEM). Further, in vitro drug release was measured through MTT assay, Microscopic and Fluorescence Activated Cell Sorting (FACS) analyses. Our results suggest that the f-SWNTs-COS-GTX-p53 was the most effective delivery vehicle with a controlled release and enhanced cytotoxicity rendered through apoptosis in human cervical cancer (HeLa) cells (IC50=0.125µM). These systems can further be used for the delivery of other commercially available anti-cancer drugs as well.
The present piece of research has also tried to develop a simple though highly sensitive gliP gene based biosensor to detect the gliotoxin production in case of Invasive Aspergillosis (IA) by immobilization of 5'-thiol end labelled single stranded deoxyribonucleic acid probe (p-DNA-SH) onto gold (Au) coated glass electrode. This Au/AuNPs/p-DNA-SH electrode alone and hybridized with the genomic DNA (Au/AuNPs/p-DNA/t-DNA) has been characterized using XPS. The Au/AuNPs/p-DNA-SH electrode could specifically detect upto 10–60 ng/µl of t-dsDNA within 60 s of hybridization time at 25 oC by Cyclic Voltammetry (CV) using methylene blue (MB) as electro-active DNA hybridization indicator. The values of sensitivities of the Au/AuNPs/p-DNA/t-DNA electrodes have been determined as 0.0115 A/ngcm−2 with regression coefficient (R) as 0.999. This genosensor is stable for about 4 months when stored at 4 oC.
Under the light of obtained results, the present study holds immense promise for the implication of diverse biomedical applications of the fungal metabolite: Gliotoxin in the medical arena for treating clinical presentations of inflammation, renal fibrosis, various cancers and detection of gliotoxin production in IA patients.
- Issued Date
- 2013
- Awarded Date
- 2013. 2
- Type
- Dissertation
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Abstract i
Table of Contents iii
List of Figures vii
List of Tables xiii
List of Abbreviations xiv
Chapter 1. Introduction 1
1.1 Fungal secondary metabolism: saga continues 2
1.2 Epipolythiodioxopiperazines and gliotoxin: Fungal weapons 6
1.3 Drug delivery: Hitting the bull’s eye 9
1.4 Biosensors: Nano-probes 11
1.5 Specific aims and structure of the thesis 12
1.6 Scope of the thesis and plan of proposed work 14
Chapter 2. Isolation and characterization of marine fungi and its epipolythiodioxo- piperazine gliotoxin 15
2.1. Introduction 16
2.2. Materials and methods 18
2.2.1. Materials and Instrumentation 18
2.2.2. Fungus isolation and Culture optimization 19
2.2.3. Fungus identification 19
2.2.4. DNA extraction 19
2.2.5. rDNA ITS region amplification and Sequencing 20
2.2.6. Sequence alignment and analysis 21
2.2.7. Solvent extraction 21
2.2.8. Isolation of Gliotoxin (GTX) from AFK11 ethyl acetate extract 21
2.3. Results and Discussion 21
2.3.1. Culture optimization 21
2.3.2. Morphological characteristics 26
2.3.3. Phylogenetic analysis 29
2.3.4. Chemical characterization of isolated compound as gliotoxin 33
2.4. Conclusion 37
Chapter 3. Molecular characterization of Gliotoxin biosynthetic gene cluster in AFK11 38
3.1. Introduction 39
3.2. Materials and methods 43
3.2.1. Culture of marine fungi 43
3.2.2. Harvesting fungal biomass 44
3.2.3. RNA isolation 44
3.2.4. cDNA synthesis and Polymerase Chain Reaction 45
3.2.5. Molecular modeling 45
3.3. Results and discussion 46
3.3.1. Culture of marine fungi 46
3.3.2. Isolation of gliotoxin biosynthetic gene cluster genes and their molecular modeling 46
3.4. Conclusion 55
Chapter 4. Biological activity of Gliotoxin isolated from AFK11 against renal fibrosis 56
4.1. Introduction 57
4.2. Materials and methods 60
4.2.1. Chemicals and Materials 60
4.2.2. Cell culture 61
4.2.3. Cytotoxicity assessment on HK-2 (Human kidney proximal tubule epithelial cells) 61
4.2.4. RNA isolation 62
4.2.5. Reverse Transcriptase-polymerase chain reaction 62
4.2.6. Routine PCR for renal fibrosis mediators in gliotoxin treated HK-2 cells 62
4.2.7. Protein isolation 63
4.2.8. Western blotting for renal fibrosis mediators in gliotoxin treated HK-2 cells 63
4.2.9. FACS analysis for the determination of ROS generation 64
4.2.10. Microscopic analysis 64
4.2.10.1. Hoechst staining for cell morphology 64
4.2.10.2. Fluorescence Microscopy to determine antifibrotic activity of gliotoxin 65
4.2.10.3. Scanning Electron Microscopy 65
4.3. Results and discussion 66
4.3.1. Gliotoxin at sub molar concentrations was found to be cyto- compatible to the human renal proximal tubular epithelial (HK-2) cells. 66
4.3.2. GTX inhibited fibrosis in TGF-β1 stimulated HK-2 cells. 68
4.3.3. GTX reduces TGF-β 1 levels by reducing Reactive Oxygen Species (ROS) 71
4.3.4. Gliotoxin decreases the mRNA and Protein expressions of inducers/mediators of renal fibrosis 73
4.4. Conclusion 77
Chapter 5. Biomedical Applications of Gliotoxin
5.1. Gliotoxin mediated carbon nanotube based targeted drug delivery system against cancer 78
5.1.1. Introduction 79
5.1.2. Experimental 84
5.1.2.1. Materials 84
5.1.2.2. Cell culture 84
5.1.2.3. Cytotoxicity Assay 84
5.1.2.4. Functionalization of carbon nanotubes 85
5.1.2.5. COS functionalization of f-SWNT 86
5.1.2.6. Characterization of f-SWNT 86
5.1.2.6.1. Transmission Electron Microscopy 86
5.1.2.6.2. Field Emission-Scanning Electron Microscopy 87
5.1.2.6.3. Fourier Transform –Infrared Spectroscopy 87
5.1.2.7. Drug Loading 87
5.1.2.8. Targeting molecule coating 87
5.1.2.9. in vitro drug release 88
5.1.2.10. Microscopy analysis 88
5.1.2.10.1. Hoechst nuclear staining 88
5.1.2.10.2. Annexin V-FITC/PI dual staining 89
5.1.2.10.3. Scanning Electron Microscopy (SEM) 90
5.1.2.11. Annexin-PI apoptosis assessment 90
5.1.2.12. DNA content analysis 91
5.1.3. Results and discussion 91
5.1.3.1. Characterization of functionalized SWNTs 91
5.1.3.2. Cytotoxicity profile 95
5.1.3.3. in vitro drug release 97
5.1.3.3.1. Hoechst nuclear staining 97
5.1.3.3.2. Annexin V- FITC/PI dual staining 99
5.1.3.3.3. Scanning Electron Microscopy 100
5.1.3.3.4. Degree of apoptosis 101
5.1.3.3.5. DNA content analysis 103
5.1.4. Conclusion 105
5.2. gliP genosensor for differential diagnosis of gliotoxin producing and nonproducing Aspergillus fumigatus sp. and implication in invasive aspergillosis 106
5.2.1. Introduction 107
5.2.2. Materials and methods 110
5.2.2.1. Fungal sample collection and characterization 110
5.2.2.2. DNA extraction, PCR and Gel electrophoresis 110
5.2.2.3. Preparation of thiolated DNA gliP Probe 111
5.2.2.4. Electrode preparation 111
5.2.2.5. Fabrication of DNA sensor 111
5.2.2.6. Characterization of thiolated DNA immobilized probe 112
5.2.2.6.1. X-Ray Photoelectron Spectroscopy (XPS) 112
5.2.2.7. Electrochemical analysis 112
5.2.2.8. Optimization of analytical performance 112
5.2.2.9. Clinical sample collection and Real sample analysis 113
5.2.3. Results and discussion 113
5.2.3.1. Fungal characterization 115
5.2.3.2. Fabrication of DNA sensor probe 115
5.2.3.3. Characterization of thiolated DNA immobilized probe 116
5.2.3.3.1. X-Ray Photoelectron Spectroscopy (XPS) 116
5.2.3.4. Electrochemical analysis 117
5.2.3.4.1. Optimization of analytical performance 118
5.2.3.4.2. Differential Pulse Voltammogram analysis for evaluation of analytical performance. 120
5.2.3.5. Clinical sample collection and Real sample analysis 121
5.2.4. Conclusion 123
Summary and Conclusion 124
Bibliography 127
Acknowledgements 142
- Degree
- Doctor
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