xi
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
ABSTRACT (in English) v
ABSTRACT (in Thai) viii
LIST OF TABLES xvii
LIST OF FIGURES xviii
ABBREVIATIONS xxi
CHAPTER I INTRODUCTION 1
CHAPTER II LITERATURE REVIEWS 7
2.1 General information of Penicillium marneffei 7
2.2 Mycology 11
2.3 Antigenic proteins 16
2.3.1 Mannoprotein of Penicillium marneffei 16
2.3.2 Antigenic protein profile and cDNA screening-derived 16
clones
2.4 Putative virulence factors of Penicillium marneffei 18
2.4.1 Adhesins 19
2.4.2 Dimorphic switching 20
2.4.3 Stress response and adaptation proteins 21
2.4.4 Secreted enzymes from Penicillium marneffei 25
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2.4.5 Melanins 26
2.5 Molecular biology of Penicillium marneffei morphogenesis 27
2.5.1 Conidiation of Penicillium marneffei 27
2.5.2 Polar growth and dimorphism of Penicillium marneffei 31
2.6 Genome and mitochondrial genome of Penicillium marneffei 34
2.7 Molecular approaches to study Penicillium marneffei 36
2.7.1 Targeted gene deletion 36
2.7.2 Conditional expression method 40
2.7.3 RNA interference 42
2.7.4 Suppression subtractive hybridization 43
2.7.5 Proteomics 45
2.7.6 Random insertional mutagenesis 47
2.8 Polyamines in fungi 52
CHAPTER III OBJECTIVES 56
CHAPTER IV MATERIALS AND METHODS 57
4.1 Isolation and characterization of ura5 and ura3 genes from 57
Penicillium marneffei
4.1.1 Strains and growth conditions 57
4.1.2 Preparation of genomic DNA 58
4.1.3 Isolation of ura5 cDNA and ura5 genomic DNA of 59
Penicillium marneffei
4.1.4 Cloning the ura3 gene 63
4.1.5 Sequencing and sequence analysis 64
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Page
4.1.6 Total RNA extraction 65
4.1.7 Analysis of genes expression during phase transition 66
and stress conditions
4.2 Isolation and characterization of uracil auxotrophs of 67
Penicillium marneffei
4.2.1 Isolation of uracil auxotrophs of Penicillium marneffei 67
4.2.2 Measurement of enzymatic activity and the protein 68
content
4.3 Agrobacterium-mediated transformation (AMT) system 69
4.3.1 Strains and their maintenance 69
4.3.2 Antifungal susceptibility testing of Penicillium 70
marneffei and Penicillium citrinum
4.3.3 Agrobacterium-mediated transformation 70
4.3.4 PCR for identifying the integrative T-DNA 73
4.3.5 Southern blot analysis for determining the copy number 73
of T-DNA
4.3.6 Mitotic stability of AMT-derived transformants 74
4.4 Screening for mutants defective in morphogenesis, cell wall 74
production and melanin synthesis
4.5 Analysis of integrated genes or promoters 75
4.6 Characterization of selected mutants of interest 76
4.7 Functional analysis of �sadA mutant 77
4.7.1 Complementation of the Penicillium marneffei �sadA
mutation
77
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4.7.2 Phenotypic screening of Penicillium marneffei F4 78
(sadA+), I6 (�sadA), and C3 (�sadAsadA+) strains
CHAPTER V RESULTS 83
5.1 Isolation and sequencing of the ura5 gene 83
5.2 Isolation and sequencing of the ura3 gene 86
5.3 Differential gene expression during phase transition and 89
stress-induced conditions
5.4 Isolation and characterization of uracil auxotrophs of 93
Penicillium marneffei
5.5 Agrobacterium-mediated transformation in Penicillium
marneffei
97
5.6 Confirmation of ble gene integration 99
5.7 Inverse PCR analysis of the T-DNA insertion junctions 102
5.8 Characterization of mutant phenotypes and corresponding 105
mutations
5.9 Isolation of putative mutants defective in cell wall production, 107
melanin synthesis, and dimorphic switch of Penicillium
marneffei
5.10 Agrobacterium-mediated transformation in Penicillium 113
citrinum
5.11 Functional analysis of a mutant defective in conidiation, 115
�sadA mutant
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5.11.1 Isolation of a mutant defective in conidiation from 115
AMT-derived transformants
5.11.2 sadA is required for spermidine synthesis in Penicillium
marneffei
115
5.11.3 Spermidine is required for the conidiation of 118
Penicillium marneffei
5.11.4 Spermidine is essential for growth and conidial 121
germination of Penicillium marneffei
CHAPTER VI DISCUSSION
6.1 Isolation of ura5 and ura3 genes and generation of uracil 125
auxotrophic mutants in Penicillium marneffei
6.2 Agrobacterium mediated transformation in Penicillium 127
marneffei
6.3 Agrobacterium mediated transformation in Penicillium 134
citrinum
6.4 Functional analysis of �sadA mutant 135
CHAPTER VII SUMMARY 140
REFERENCES 144
APPENDICES 176
Appendix A 177
Appendix B 178
Appendix C 179
Appendix D 180
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Appendix E 181
Appendix F 182
Appendix G 183
Appendix H 187
CURRICULUM VITAE 198
xvii
LIST OF TABLES
Table Page
1 Heterologous probe for isolating Penicillium marneffei gene 36
2 Plasmids used for selectable markers in Penicillium marneffei
transformation
38
3 Primers used in gene isolation and sequencing study 80
4 Primers used in gene expression study 81
5 Primers used in Agrobacterium-mediated transformation and gene 82
function studies
6 Relative gene expression levels during phase transition and stress-
induced conditions
7 Colony diameter of uracil auxotrophs and Penicillium marneffei
wild type.
95
8 Comparison of the OPRTase activity of Penicillium marneffei F4 96
and its mutants
9 Average number of bleR transformants generated by 97
Agrobacterium-mediated transformation of Penicillium marneffei
10 Number of positive samples for inverse PCR using different 103
restriction enzymes
11 Genetic defects in AMT-generated mutants of Penicillium marneffei 106
12 Effect of different concentrations of spermidine on the growth and 120
conidiation of Penicillium marneffei
xviii
LIST OF FIGURES
Figure Page
1 Colony appearance and microscopic morphology 13
2 Diagrammatic representation of the Penicillium marneffei life cycle 15
3 Chemical reactions involving reactive oxygen species 22
4 Model describing the pathway proposed to operate in the signal 30
transduction of conidiation induction in Penicillium marneffei
5 Diagrammatic representation of the generation of saprobic mycelial 33
and parasitic yeast forms of Penicillium marneffei (in vitro model)
6 Schematic overview of the A. tumefaciens T-DNA transfer system 50
7 Polyamine biosynthetic pathway in Saccharomyces cerevisiae 53
8 Overview of Agrobacterium-mediated transformation system in 72
Penicillium marneffei
9 The nucleotide sequence and deduced amino acid sequence of the 84
ura5 gene from Penicillium marneffei
10 Alignment of the fungal Ura5 amino acid sequences 85
11 The nucleotide and deduced amino acid sequences of the ura3 gene 87
from Penicillium marneffei
12 Alignment of the fungal Ura3 amino acid sequences 88
13 Differential gene expression during phase transition and 90
stress-induced conditions
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Page
14 Expression of Penicillium marneffei actA and ura5 genes during 92
macrophage infection
15 Uracil requirement of Penicillium marneffei uracil auxotrophs 94
16 Colony appearance of Penicillium marneffei wild type strain and 94
uracil auxotrophs (5-FOAR1 and 5-FOAR8.1)
17 Selection of putative bleomycin-resistant transformants of 98
Penicillium marneffei
18 Determination of T-DNA integration by PCR 100
19 Southern blot analysis of selected AMT-generated transformants of 101
Penicillium marneffei
20 Identification of T-DNA insertion by inverse PCR 103
21 Sequence analysis of the T-DNA insertion junctions from AMT- 104
derived transformants of Penicillium marneffei.
22 A comparison of the colony appearance of the wild type 107
(P. marneffei F4) and the AMT-generated stuA and gasC mutants
23 Putative mutants defective in cell wall production 109
24 Putative mutants defective in melanin synthesis 110
25 Mutants defective in transition from mold to yeast 111
26 Mutants defective in transition from yeast to mold 112
27 Selection of putative bleomycin-resistant transformants of 114
Penicillium citrinum
28 Inverse PCR for identifying sadA gene and Southern blot analysis 116
for determining the copy number of T-DNA in sadA mutant
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Page
29 sadA is required for spermidine synthesis in Penicillium marneffei 117
30 Spermidine is required for the conidiation of Penicillium marneffei 119
31 Spotting assay 122
32 Mutation of sadA affects conidial germination at 27oC and 37o 123C
33 Spermidine is required for conidial germination at both 27o 124C and
37oC
xxi
ABBREVIATIONS
% PercentoC Degree Celsius
µg Microgram
µl Microliter
µm Micrometer
µM Micromolar
AIDS Acquired immunodeficiency syndrome
ATCC American Type Culture Collection
BLAST Basic local alignment search tools
BHI Brain heart infusion
bp Base pair
CBS Centraalbureau voor Schimmelcultures
cDNA Complementary deoxyribonucleic acid
CFU Colony forming unit
cm Centimeter
CMI Cell-mediated immunity
CO2
h hour(s)
Carbon dioxide
DEPC Diethylpyrocarbonate
DMEM Dulbecco’s modified Eagle’s medium
DNA Deoxyribonucleic acid
DNaseI Deoxyribonuclease I
EDTA Ethylenediamine tetraacetic acid
et al. And others
g Gram(s)
x g gravity
gDNA Genomic deoxyribonucleic acid
HIV Human immunodeficiency virus
xxii
H2O2
SDA Sabouraud dextrose agar
Hydrogen peroxide
kb Kilobase
kDa Kilodalton
l liter
lbs Pounds
LB Luria-Bertani media
M Molar
mg Milligram(s)
min Minute(s)
ml Milliliter(s)
mm Millimeter
mM Millimolar
mRNA Messenger ribonucleic acid
MW Molecular weight
NaOH Sodium hydroxide
NCBI National Center for Biological Information
ng Nanogram
nm Nanometer
no. Number
OD Optical density
ORF Open reading frame
PCR Polymerase chain reaction
pfu Plaque forming unit
pI Isoelectric point
RNA Ribonucleic acid
RNase Ribonuclease
ROS Reactive oxygen species
rpm Revolution per minute
rRNA Ribosomal ribonucleic acid
RST Random sequence tag
RT-PCR Reverse transcription-polymerase chain reaction
SD Standard deviation
xxiii
SDB Sabouraud dextrose broth
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
s Second
SOD Superoxide dismutase
SSH Suppression subtractive hybridization
Taq Thermus aquaticus
tRNA Transfer ribonucleic acid
U Unit(s)
UV Ultraviolet
V Volt
Wt Weight